Dev Bio

Cortical rotation initiates formation of the Nieuwkoop center

-at grey crescent

Epigenetic Modification

A heritable change in gene expression that does not involve a change in the DNA sequence of the gene. ex) Methylating a promotor will prevent transcription of a gene

In-situ Hybridization

A technique using nucleic acid hybridization with a labeled probe to detect the location of a specific mRNA in an intact organism. hybridization-not quantitative but tells you where mRNA for gene is expressed This technique uses dyes to allow researchers to look at entire embryos (or their organs) without sectioning them, thereby observing large regions of gene expression next to regions devoid of expression. labels w die the region in the embryo/organism that gets expressed First an mRNA detection probe— the in situ probe—had to be created. The probe is an antisense RNA molecule that can typically vary in length from 200bp to 2000bp. More important is that the uridine triphosphate (UTP) nucleosides in this RNA strand are conjugated with digoxigenin Digoxigenin—a compound made by particular groups of plants and not found in animal cells—does not interfere with the coding properties of the result- ing mRNA, but it does make it recognizably different from any other RNA in the cell. During the procedure, the embryo is permeabilized by lipid solvents and proteinases so that the probe can get in and out of its cells. Once in the cells, hybridization occurs between the probe anti-sense RNA and the targeted mRNA. To visualize the cells in which hybridization has occurred, researchers apply an antibody that specifically recognizes digoxigenin. This antibody, however, has been artificially conjugated to an enzyme, such as alkaline phosphatase. After incubation in the antibody and repeated washes to remove all unbound antibodies, the embryo is bathed in a solution containing a substrate for the enzyme (traditionally NTB/BCIP for alkaline phosphatase) that Differential Gene expression 85 can be converted into a colored product by the enzyme. The enzyme should be present only where the digoxigenin is present, and the digoxigenin should be present only where the specific complementary mRNA is found. Thus, in FiGUrE 3.35c, the dark blue precipitate formed by the enzyme indicates the presence of the target mRNA. RNA made complimentary to specific mRNA with a Digoxigenin label on Uradine, poke hole in cell membrane and let new RNA bind to mRNA. Add antibody w alkanine phosphatase that conjugayes to the digoxigenin. Wash away excess antibodies, then add substrate that when phosphate gets removed it becomes colored w a die, this will happen when mRNA is expressed and thus can se where specific mRNA gets expressed

Hox Genes

Class of homeotic genes. Changes in these genes can have a profound impact on morphology--segmental development in flies

mesenchyme

a loosely organized, mainly mesodermal embryonic tissue that develops into connective and skeletal tissues, including blood and lymph.

hermaphrodite

Combination of both male and female organs

Blocks to polyspermy: Sea urchant

-Fast block-change in membrane potential mediated by influx of Na + ions: ---Sodium channels are closed, thereby preventing the entry of sodium ions (Na+) into the egg, and the egg cell membrane maintains an electrical voltage gap between the interior of the egg and its environment. This resting membrane potential is generally about 70 mV, which is expressed as -70 mV because the inside of the cell is negatively charged with respect to the exterior. Within 1-3 seconds after the binding of the first sperm, the membrane potential shifts to a positive level—about +20 mV—with respect to the exterior (FIGuRE 7.15A; Jaffe 1980; Longo et al. 1986). The shift from negative to positive is the result of a small influx of Na+ into the egg through newly opened sodium channels. Sperm cannot fuse with egg cell membranes that have a positive resting potential, so the shift means that no more sperm can fuse to the egg. The importance of Na+ and the change in resting potential from negative to posi- tive was demonstrated by Laurinda Jaffe and colleagues. They found that polyspermy can be induced if an electric current is applied to artificially keep the sea urchin egg membrane potential negative. Conversely, fertilization can be prevented entirely by artificially keeping the membrane potential of eggs positive (Jaffe 1976). The fast block to polyspermy can also be circumvented by lowering the concentration of Na+ in the surrounding water (FIGuRE 7.15b). If the supply of sodium ions is not sufficient to cause the positive shift in membrane potential, polyspermy occurs (Gould-Somero et al. 1979; Jaffe 1980). An electric block to polyspermy also occurs in frogs (Cross and Elin- son 1980; Iwao et al. 2014), but probably not in most mammals. --positive shift only happens for about a minute -Slow block-enzymes in cortical granules: Protease cleaves bindin receptor, vitelline link Mucopolysacharide released, takes up water, expands Enzymes-hardens envelope Research article-ZP2 cleavage ---polyspermy can still occur if the sperm bound to the vitelline envelope are not somehow removed (Carroll and Epel 1975). This sperm removal is accomplished by the cortical granule reaction, also known as the slow block to polyspermy. This slower, mechanical block to polyspermy. becomes active about a minute after the first successful sperm- egg fusion (Just 1919). This reaction is found in many animal species, including sea urchins and most mammals. Cortical granule exo- cytosis and formation of the sea urchin fertilization envelope. (A) Schematic diagram of events leading to the forma- tion of the fertilization envelope. As corti- cal granules undergo exocytosis, they release cortical granule serine protease (CGSP), an enzyme that cleaves the pro- teins linking the vitelline envelope to the cell membrane. Mucopolysaccharides released by the cortical granules form an osmotic gradient, causing water to enter and swell the space between the vitelline envelope and the cell membrane. The enzyme Udx1 in the former cortical gran- ule membrane catalyzes the formation of hydrogen peroxide (H2O2), the substrate for soluble ovoperoxidase (OVOP). OVOP and transglutaminases (TG) harden the vitelline envelope, now called the fer- tilization envelope. (B,C) Transmission electron micrographs of the cortex of an unfertilized sea urchin egg and the same region of a recently fertilized egg. The raised fertilization envelope and the points at which the cortical granules have fused with the egg cell membrane of the egg (arrows) are visible in (C). (A after Wong et al. 2008; B,C from Chandler and Heuser 1979, courtesy of D. E. Chandler.) Directly beneath the sea urchin egg cell membrane are about 15,000 cortical granules, each about 1 μm in diameter (see Figure 7.4B). Upon sperm entry, cortical granules fuse with the egg cell membrane and release their contents into the space between the cell membrane and the fibrous mat of vitelline envelope proteins. Several proteins are released by cortical granule exocytosis. One of these, the enzyme cortical granule serine protease, cleaves the protein posts that connect the vitelline envelope proteins to the egg cell membrane; it also clips off the bindin receptors and any sperm attached to them (Vacquier et al. 1973; Glabe and Vacquier 1978; Haley and Wessel 1999, 2004). (C) (D) The components of the cortical granules bind to the vitelline envelope to form a fertilization envelope. The fertilization envelope starts to form at the site of sperm entry and continues its expansion around the egg. This process starts about 20 seconds after sperm attachment and is complete by the end of the first minute of fertilization (FIGuRE 7.16; Wong and Wessel 2004, 2008). extra info: The fertilization envelope is elevated from the cell membrane by mucopolysac- Gilbert the envelope moves radially away from the egg. The fertilization envelope is then sta- bilized by crosslinking adjacent proteins through egg-specific peroxidase enzymes and a transglutaminase released from the cortical granules (FIGuRE 7.17; Foerder and Sha- piro 1977; Wong et al. 2004; Wong and Wessel 2009). This crosslinking allows the egg and early embryo to resist the shear forces of the ocean's intertidal waves. As this is happening, a fourth set of cortical granule proteins, including hyalin, forms a coating around the egg (Hylander and Summers 1982). The egg extends elongated microvilli whose tips attach to this hyaline layer, which provides support for the blastomeres during cleavage.

Genetic Based Lineage Tracing

-Fluorescent reporter (GFP) downstream from lineage-specific promoter -Cre-lox system to generate reporter cells in tissue- and time-dependent way-(Brainbow) -Bar coding approach -Single cell transcriptome analysis allow you to follow all the cells in an embryo simultaneously

Differences with internal fertilization in mammals

-Hard to study, reproduce environment -Issue of species specificity not as significant -Capacitation--does not occur in mammals until the sperm has been inside the female reproductive tract for a certain period of time. -Sperm plasma membrane, not acrosome membrane binds in mammals

Properties of the organizer

-Self differentiate dorsal mesoderm: prechordal plate, notochord, etc -Dorsalize mesoderm, from ventral mesoderm to axial mesoderm -Dorsalize ectoderm-neural tube -Initiate gastrulation movements

Recognition of sperm and egg

1. Chemoattraction of the sperm to the egg by soluble molecules secreted by the egg 2. Exocytosis of the sperm acrosomal vesicle and release of its enzymes 3. Binding of the sperm to the extracellular matrix (vitelline envelope or zona pellucida) of the egg 4. Passage of the sperm through this extracellular matrix 5. Fusion of the egg and sperm cell membranes

GFP and Fate Mapping

1. GFP+ primitive endoderm cell injected into early mouse embryo 2. Donor cells are genetically marked to GFP or LacZ 3. Only see the cells in extraembryotic tissue--no endoderm

CRISPR/cas9 Genome Editing

1. Nuclease (Cas9) Directed to site in DNA by guide RNA (sgRNA) 2. CRISPR is a stretch of DNA containing short regions that when transcribed into RNA serve as guides (short-guide RNAs or sgRNAs) for recognizing segments of DNA (or a specific gene). The RNA also binds to an endonuclease called Cas9 (CRISPR associated enzyme 9). When the sgRNA binds to viral DNA, the RNA brings Cas9 with it, which catalyzes a double-strand break in DNA. When CRISPR sgRNAs specific for a gene are introduced into cells along with Cas9, the Cas9 protein is guided by the CRISPR to the gene of interest and causes a double-strand break in the DNA. This technique is highly successful at creating gene mutations. Cells will naturally try to repair double-strand breaks through a process called non-homologous end joining (NHEJ). In an effort to reconnect the DNA rapidly and avoid catastrophic DNA damage, however, NHEJ is often imperfect in its repairs, resulting in indels (an insertion or deletion of DNA bases). Whether the indel is an insertion or a deletion, there is a signifi- cant chance that it will cause a frameshift in the gene and conse- quently create a premature stop codon somewhere downstream of the mutation; hence, there will be a loss of gene function--so, can be dangerous In addition, the system can be used to precisely edit a genome by including short DNA fragments with the CRISPR/Cas9. These DNA pieces are engineered to have sequence homology on their 5′ and 3′ ends to encourage homologous recombination flanking the double- stranded breaks. Plasmid insertions with homology to regions surrounding the sgRNA target sites are used to foster the insertion of known sequences used to try to fix gene mutations in diseases such as muscular dystrophy

Mouse Acrosome reaction

1. Sperm activated by female reproductive tract 2. sperm bonds to zona pellucida 3. acrosome rxn 4. sperm lyses hole in zona pellucida 5. sperm and cell membrane fuse Izumo protein and membrane fusion in mouse fertilization. (A) Localization of Izumo to the inner and outer acrosomal membrane. Izumo is stained red, acrosomal proteins green. (B) Diagram of sperm-egg cell membrane fusion. During the acrosome reaction, Izumo localized on the acrosomal becomes translo- cated to the sperm cell membrane. There it meets the complex of Juno and CD9 proteins on the egg microvil- li, initiating membrane fusion and the entry of the sperm into the egg. (After Satouh et al. 2012; photographs courtesy of M. Okabe.) differences from sea urchant: rxn starts on contact w zona pellucida not jelly/cumulous layer. Also, no actin polymerization

Frog Gastrulation

1. Vegetal rotation 2. Bottle cells 3. Involution 4. Intercalation/Convergent extension of Involuting marginal zone (IMZ) 5. Epiboly forming archenteron displaces blastcoel Vegetal rotation pushes endoderm up posterior side displacing bastocoel. Then blastopore lip (boundary between mesoderm and endoderm) form from bottle cells. converging extension of mesoderm (IMZ) moves with extension of archenteron and region at former blastopore lip undergoes involution, displaces blastocoel ventrally, superficial cells (ectoderm) epiboly. Then, more convergent extension of IMZ, and epiboly of ectoderm which now surrounds embryo, yolk plug (endoderm) forms where blastopore was Epiboly of the Ectoderm Is Accomplished by Cell Division and Intercalation --epiboly involves the assembly of fibronectin into fibrils by the blastocoel roof. This fibrillar fibronectin is criti- cal in allowing the vegetal migration of the animal cap cells and enclosure of the embryo (Rozario et al. 2009). In Xenopus and many other amphibians, it appears that the involuting mesodermal precursors migrate toward the animal pole by traveling on an extracellular lattice of fibronectin secreted by the presumptive ectoderm cells of the blastocoel roof (Figure 11.9A,B). Confirmation of fibronectin's importance for the involuting mesoderm came from experiments with a chemically synthesized peptide fragment that was able to compete with fibronectin for the binding sites of embryonic cells (Boucaut et al. 1984). If fibronectin were essential for cell migration, then cells binding this synthesized peptide fragment instead of extracellular fibronectin should stop migrating. Unable to find their "road," these prospective mesodermal cells should cease involution. That is precisely what happened, and the mesodermal precursors remained outside the embryo, forming a convoluted cell mass (Figure 11.9c,D). Thus, the fibronectin-containing extracellu- lar matrix appears to provide both a substrate for adhe- sion as well as cues for the direction of cell migration. Vegetal rotation (arrows) pushes the prospective pharyngeal endoderm (orange; speci- fied by hhex and cerberus expression) to the side of the blastocoel. (C,D) The vegetal endoderm (yellow) movements push the pharyngeal endoderm forward, driving the mesoderm passively into the embryo and toward the animal pole The bottle cells of the margin move inward to form the dorsal lip of the blastopore, and the mesodermal precursors involute under the roof of the blastocoel. AP marks the position of the animal pole, which will change as gastrulation continues. (C,D) Mid-gastrulation. The archenteron forms and displaces the blastocoel, and cells migrate from the lateral and ventral lips of the blastopore into the embryo. The cells of the animal hemisphere migrate down toward the vegetal region, moving the blastopore to the region near the vegetal pole. (E,F) Toward the end of gastrulation, the blastocoel is obliterated, the embryo becomes surrounded by ectoderm, the endoderm has been internalized, and the mesodermal cells have been positioned between the ectoderm and endoderm. Amphibian blastulae are faced with the same tasks as the invertebrate blastulae we followed in Chapters 8 through 10—namely, to bring inside the embryo those areas destined to form the endodermal organs; to surround the embryo with cells capable of forming the ectoderm; and to place the mesodermal cells in the proper positions between the ectoderm and the endoderm. The cell movements of gastrulation that will accomplish this are initiated on the future dorsal side of the embryo, just below the equator, in the region of the gray crescent (i.e., the region opposite the point of sperm. Here the cells invaginate to form the slitlike blastopore. These bottle cells change their shape dramatically. The main body of each cell is displaced toward the inside of the embryo while maintaining contact with the outside surface by way of a slender neck. As in sea urchins, the bottle cells will initiate the formation of the archenteron (primitive gut).1 However, unlike sea urchins, gastrulation in the frog begins not in the most vegetal region but in the marginal zone—the region sur- rounding the equator of the blastula, where the animal and vegetal hemispheres meet (Figure 11.4A,B). Here the endodermal cells are not as large or as yolky as the most vegetal blastomeres. But cell involution is not a passive event. At least 2 hours before the bottle cells form, internal cell rearrangements propel the cells of the dorsal floor of the blastocoel toward the animal cap. This vegetal rotation places the prospective pharyngeal endoderm cells adjacent to the blastocoel and immediately above the involuting mesoderm (see Figure 11.5D). These cells then migrate along the basal surface of the blastocoel roof, traveling toward the future anterior of the embryo (Figure 11.4c-e; Nieuwkoop and Florschütz 1950; Winklbauer and Schürfeld 1999; Ibrahim and Winklbauer 2001). The superficial layer of marginal cells is pulled inward to form the endodermal lining of the archenteron, merely because it is attached to the actively migrating deep cells. Although experimentally removing the bottle cells does not affect the involution of the deep or superficial marginal zone cells into the embryo, removal of the deep involuting mar- ginal zone (imZ) cells stops archenteron formation.

Rotational Cleavage in Mouse

16 cell satge cell undergoes compaction, forms inner cell mass and blastocyst

Nurse Cells

???

Androgen insensitivity syndrome:

A condition caused by a congenital lack of functioning androgen receptors; XY, normal Sry testosterone normal, receptor mutant Wolfian can't develop Mullerian degenerates Androgen> estrogen>female traits Sterile in a person with XY sex chromosomes, causes the development of a female with testes but no internal sex organs

RT-PCR

A technique in which RNA is first converted to cDNA by the use of the enzyme reverse transcriptase, then the cDNA is amplified by the polymerase chain reaction. This thus detects RNA expression ex) FGF5 mRNA expression during neural differentiation of mouse embryonic stem cells--shows bar graph of expression levels of the FGF5 mRNA gene over 7 days--CAN SEE WHATS HAPPENING IN REAL TIME/TEMPORALLY but only seeing how much DNA used? RNA?

Acrosome/ZP2 example

Acrosome-reacted mouse sperm bind to the zona and are successful at fertilizing the egg. (A) The acrosomes of mouse sperm were labeled with GFP such that intact acrosomes fluoresced green; sperm tails were labeled with red fluorescent markers. When the labeled sperm were allowed to interact with a mouse egg and cumulus, the resulting video revealed that the fertilizing sperm (arrowhead at 4.30 seconds) showed no green fluorescence when it reached the surface of the zona pellucida at 6.20 seconds—indicating that it had undergone the acrosome reaction before that time. An adjacent sperm did fluoresce green, meaning its acro- some remained intact. Such acrosome-intact sperm remain bound to the zona without undergoing the acrosome reaction or progressing to the egg cell membrane. (B) Gain-of-function experiment demonstrating that human sperm bind to ZP2. Of the four human zona pellucida proteins, only ZP4 is not found in mouse zona. Transgenic mouse oocytes were constructed that expressed the three normal mouse zona proteins and also one of the four human zona proteins. When human sperm were added to the mouse oocytes, they bound only to those transgenic oocytes that expressed human ZP2. Human sperm did not bind to cells expressing human ZP1, ZP3, or ZP4. (A from Jin et al. 2011, courtesy of N. Hirohashi; B from Baibakov et al. 2012.)

8 cell stage blastomere

Animal pole = ectoderm + nervous system (anterior nervous system) vegetal pole = notochord, endoderm, mesenchyme, and muscle (anterior notochord, posterior muscle) 8 cell blastomeres know their fate, thus isolating a certain cell gives rise to that specific function ex) anterior vegetal pole cell makes notochord and endoderm

anterior-posterior axis mammal

Anterior-posterior patterning in the mouse embryo. (A) Concentration gradi- ents of BMPs, Wnts, and FGFs in the late-gas- trula mouse embryo (depicted as a flattened disc). The primitive streak and other posterior tissues are the sources of Wnt and BMP pro- teins, whereas the organizer and node and its derivatives (such as the notochord) produce antagonists. Fgf8 is expressed in the posterior tip of the gastrula and continues to be made in the tailbud. Its mRNA decays, creating a gradient across the posterior portion of the embryo

Cadherin-Mediated Cell Adhesion-homophilic interaction

As their name suggests, cadherins are calcium-dependent adhesion molecules. They are critical for establishing and maintaining intercellu- lar connections, and they appear to be crucial to the spatial segregation of cell types and to the organization of animal form (Takeichi 1987). Cadherins are transmembrane proteins that interact with other cad- herins on adjacent cells. The cadherins are anchored inside the cell by a complex of proteins called catenins (fiGure 4.6), and the cadherin- catenin complex forms the classic adherens junctions that help hold epithelial cells together. Moreover, because the cadherins and the catenins bind to the actin (microfilament) cytoskeleton of the cell, they integrate the epithelial cells into a mechanical unit. Blocking cadherin function (by antibodies that bind and inactivate cadherin) or blocking cadherin synthesis (with antisense RNA that binds cadherin mes- sages and prevents their translation) can prevent the formation of epithelial tissues and cause the cells to disaggregate (Takeichi et al. 1979). Cadherins perform several related functions. First, their external domains serve to adhere cells together. Second, cadherins link to and help assemble the actin cytoskel- eton, thereby providing the mechanical forces for forming sheets and tubes. Third, cadherins can serve to initiate and transduce signals that can lead to changes in a cell's gene expression.

Primitive streak formation of endoderm and mesoderm

At the anterior end of the primitive streak is a regional thickening of cells called hensen's node (also known as the primitive knot; see Figure 12.4C). The center of Hensen's node contains a funnel-shaped depression (sometimes called the primitive pit) through which cells can enter the embryo to form the notochord and prechordal plate. Hensen's node is the functional equivalent of the dorsal lip of the amphib- ian blastopore (i.e., the organizer)1 and the fish embryonic shield (Boettger et al. 2001). The primitive streak defines the major body axes of the avian embryo. It extends from posterior to anterior; migrat- ing cells enter through its dorsal side and move to its ventral side; and it separates the left portion of the embryo from the right. The axis of the streak is equivalent to the dorsal- ventral axis of amphibians. The anterior end of the streak— Hensen's node—gives rise to the prechordal mesoderm, notochord, and medial part of the somites. Cells that ingress through the middle of the streak give rise to the lateral part of the somites and to the heart and kidneys. Cells in the pos- terior portion of the streak make the lateral plate and extra- embryonic mesoderm (Psychoyos and Stern 1996). After the ingression of the mesoderm cells, epiblast cells remaining outside of but close to the streak will form medial (dorsal) structures such as the neural plate, while those epiblast cells farther from the streak will become epidermis Anterior extension, followed by posterior regression of the Primitive Streak As soon as the primitive streak has formed, epiblast cells begin to migrate through it and into the blastocoel. The streak thus has a continually changing cell population. Cells migrating through the anterior end pass down into the blastocoel and migrate anteriorly, form- ing the endoderm, head mesoderm, and noto- chord; cells passing through the more posterior portions of the primitive streak give rise to the majority of mesodermal tissues Migration of endodermal and mesoder- mal cells through the primitive streak. (A) Stereogram of a gastrulating chick embryo, showing the relationship of the primitive streak, the migrating cells, and the hypoblast and epiblast of the blastoderm. The lower layer becomes a mosaic of hypoblast and endodermal cells; the hypoblast cells eventually sort out to form a layer beneath the endoderm and contribute to the yolk sac. Above each region of the stereogram are micrographs showing the tracks of GFP-labeled cells at that position in the primitive streak. Cells migrating through Hensen's node travel anteriorly to form the prechordal plate and notochord; those migrating through the next anterior region of the streak travel laterally but converge near the midline to make notochord and somites; those from the middle of the streak form intermediate mesoderm and lateral plate mesoderm (see the fate maps in Figure 12.4). Farther posterior, the cells migrating through the primitive streak make the extraembryonic mesoderm (not shown). (B) This scanning electron micrograph shows epiblast cells passing into the blastocoel and extending their apical ends to become bottle cells.

Gene Targetting in mouse

BMP7 gene: cut gene w restriction endonuclease and insert a neo-r gene mutation (mutates and halts function of BMP). use ligase to repair gene now w the neo mutation. insert into stem cell removed from inner mass of blastocyst of mouse. The gene will undergo homologous recombination and thus some cells will be heterozygous w the neo-r mutation. select for thse cells by killing other cells w neomycin) neo-r gene is neomycin resistant). Next, inject heterozygous ES cells back into the inner cell mass cells of a mouse blastocyst, and inject into a uterus of mouse. The offspring will be chimeric (have different genotype than the mother). Breed these mice with normal (wild type) mice and get both wild type and heterozygous offspring. If you breed two heterozygous offspring tpgether, you will get some homozygous for the BMP knockout. These mice have no eyes!

The acrosome reaction and recognition at the zona pellucida

Before the mammalian sperm can bind to the oocyte, it must first bind to and penetrate the egg's zona pellucida. The zona pellucida in mammals plays a role analogous to that of the vitelline envelope in invertebrates; the zona, however, is a far thicker and denser structure than the vitelline envelope. The mouse zona pellucida is made of three major glycoproteins—ZP1, ZP2, and ZP3 (zona proteins 1, 2, and 3)—along with accessory proteins that bind to the zona's integral structure. The human zona pellucida has four major glycoproteins—ZP1, ZP2, ZP3, and ZP4. The binding of sperm to the zona is relatively, but not absolutely, species-specific, and a species may use multiple mechanisms to achieve this binding. Early evidence from rabbits and hamsters (Huang et al. 1981; Yanagimachi and Phillips 1984) sug- gested that the sperm arriving at the egg had already undergone the acrosome reaction. More recently, Jin and colleagues (2011) showed that the mouse acrosome reaction occurs prior to the sperm binding to the zona (FIGuRE 7.28A). They found that "suc- cessful" sperm—i.e., those that actually fertilized an egg—had already undergone the acrosome reaction by the time they were first seen in the cumulus. Sperm that under- went the acrosome reaction on the zona were almost always unsuccessful. Thus it appears that most sperm undergo the acrosome reaction in or around the cumulus. Moreover they probably bind to the egg through ZP2 on the zona pellucida. In a gain-of-function experiment, ZP2 was shown to be critical for human sperm-egg binding. Human sperm does not bind to the zona of mouse eggs, so Baibakov and colleagues (2012) added the different human zona pro- teins separately to the zona of mouse eggs. Only those mouse eggs with human ZP2 bound human sperm (FIGuRE 7.28b). Using mutant forms of ZP2, Avella and colleagues (2014) demonstrated that there is a particular region of the mouse ZP2 protein (between amino acids 51 and 149) that bound the sperm. This region is seen in human ZP2 and may be responsible for sperm-zona binding in humans as well. ZP3 was the other candidate for binding sperm; however, Gahlay and col- leagues (2010) provided evidence that mouse eggs with mutations in ZP3 were still fertilized. In mice, there is also evidence that acrosome-intact sperm can bind to ZP3, and that ZP3 can cause the acrosome reaction directly on the zona (Bleil and Wassarman 1980, 1983). And in humans, there is evi- dence that the reaction can also be induced by the zona proteins, per- haps by all of them acting in concert (Gupta 2015). Indeed, there may be several means to initiate the acrosome reaction and to bind to and penetrate the zona pellucida. These mechanisms may act simultane- ously, or perhaps one mechanism is used for acrosome-intact sperm and another for acrosome-reacted sperm. Given that the zona's bio- chemical composition differs in different species, the mechanisms that predominate in one species need not be the same in another. The sperm receptor that binds to the zona proteins has not yet been identi- fied. It is probably a complex containing several proteins that bind to both the protein and carbohydrate portions of the zona glycoproteins (Chiu et al. 2014).

Extracellular matrix

Cell-to-cell interactions do not happen in the absence of an environment; rather, they occur in coordination with and often due to the environmental conditions surrounding the cells. This environment is called the extracellular matrix, which is an insoluble network consisting of macromolecules secreted by cells. These macromolecules form a region of noncellular material in the interstices between the cells. Cell adhesion, cell migration, and the formation of epithelial sheets and tubes all depend on the ability of cells to form attach- ments to extracellular matrices 3 components: 1. structural proteins (collagens + elastins) 2. protein-polysaccharide complexes to embed the structural proteins (proteoglycans) 3. adhesive glycoproteins to attach cells to matrix (fibronectins and laminins) Fibronectin: The large glycoproteins are responsible for organizing the matrix and the cells into an ordered structure. fibronectin is a very large (460-kDa) glycoprotein dimer synthesized by numerous cell types. One function of fibronectin is to serve as a general adhesive molecule, linking cells to one another and to other substrates such as collagen and proteoglycans. Fibronectin has several distinct binding sites, and their interaction with the appropriate molecules results in the proper alignment of cells with their extracellular matrix (fiGure 4.11a). Fibronectin also has an important role in cell migration because the "roads" over which certain migrating cells travel are paved with this protein. Fibronectin paths lead germ cells to the gonads and heart cells to the midline of the embryo. If chick embryos are injected with antibodies to fibronectin, the heart- forming cells fail to reach the midline, and two separate hearts develop

Regulative Model

Cells are influenced by neighboring cells and regions to determine function -transplant tagged back tissue forming cells in frog blastula to belly forming region--the cells now form belly tissue in tadpole -use glass needle to remove cells from posterior region of blastula of frog, the remaining cells regulate and compensate for this loss and the embryo undergoes normal development -Driesch exp) in four cell stage, remove fertilization envelope and remove + isolate each cell from blastomere, and each cell will individually still make an embryo. Suggests no information gets partitioned in an irreversible way

Mosaic Model

Cells have predetermined fate experiments: 1. Acetylcholinesterase in the Progeny of the Muscle Lineage Blastomeres -In 8 cell stage, remove muscle forming cytoplasm cell from posterior vegetal pole of blastomere. The embryo doesn't develop correctly, lacks muscle -if remove cell from balastula, embryo loses that function

Waddington Landscape

Cells have predetermined fates that cannot be altered

CHIP-Seq

ChIP-Seq is based on two highly specific interactions. One is the binding of a transcription factor or a modified nucleo- some to very particular sequences of DNA (such as enhancer elements), and the other is the binding of antibody molecules specifically to the transcription factor or modified histone being studied Chromatin immunoprecipitation-sequencing (ChIP- Seq). Chromatin is isolated from the cell nuclei. The chromatin proteins are crosslinked to their DNA-binding sites, and the DNA is fragmented into small pieces. Antibodies bind to specific chromatin proteins, and the antibodies—with whatever is bound to them—are precipitated out of solution. The DNA fragments associated with the precipitated complexes are purified from the proteins and sequenced. These sequences can be compared with the genome maps to give a precise localization of what genes these proteins may be regulating ex) Antoni Hurtado, et al. performed knock-down of the FoxA1 "pioneer factor", resulting in reduced binding by the estrogen receptor (ER) at over 50% of known ER binding sites. They showed that FoxA1 is an important regulator of ER-mediated transcription, suggesting it may be a new and important therapeutic target in breast cancer (Hurtado 2011).

Holoblastic radial cleavage in a frog

Cleavage of a Xenopus egg. (A) The first three cleavage furrows, numbered in order of appearance. Because the vegetal yolk impedes cleavage, the second division begins in the animal region of the egg before the first division has divided the vegetal cytoplasm completely. The third division is displaced toward the animal pole. (B) As cleavage progresses, the vegetal hemisphere ultimately contains larger and fewer blastomeres than the animal hemisphere. The final draw- ing shows a cross section through a mid-blastula stage embryo. (C) Fate map of the Xenopus embryo superimposed on the mid-blastula stage. (D) SEMs of the first, second, and fourth cleavages. Note the size discrepancies of the animal and vegetal cells after third cleavage.

Cleaved ZP2 necessary for block of polyspermy

Cleaved ZP2 is necessary for ZP2Mut egg Normal 2-cell ZP2Mut 2-cell the block to polyspermy in mammals. Eggs and embryos were visualized by fluorescence micros- copy (to see sperm nuclei; top row) and brightfield microscopy (differential interference contrast, to see sperm tails; bottom row). Sperm bound nor- mally to eggs containing a mutant ZP2 that could not be cleaved. However, the egg with normal (i.e., cleavable) ZP2 got rid of sperm by the 2-cell stage, whereas the egg with the mutant (uncleav- able) ZP2 retained sperm. (From Gahlay et al. 2010, photograph courtesy of J. Dean.) Human sperm does not bind to the zona of mouse eggs, so Baibakov and colleagues (2012) added the different human zona pro- teins separately to the zona of mouse eggs. Only those mouse eggs with human ZP2 bound human sperm

AVE:Schematic Model of Interactions Between the Visceral Endoderm and Epiblast in Mice: helps set up anterior posterior axis

Creates anti-TGF−β (Nodal) domain on anterior region that blocks primitive streak formation, causes it to form on posterior region

Nucleosomes

DNA coiled around histone octamers condensed chromatin nucleosomes = methylated tails of histones = heterochromatin = decreases transcription uncondensed chromatin = acetylated tail of histones = euchromatin = increases transcription

Microarray

DNA microarrays can be used to detect DNA (as in comparative genomic hybridization), or detect RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins. The process of measuring gene expression via cDNA is called expression analysis or expression profiling. Summary of DNA Microarrays. Within the organisms, genes are transcribed and spliced to produce mature mRNA transcripts (red). The mRNA is extracted from the organism and reverse transcriptase is used to copy the mRNA into stable ds-cDNA (blue). In microarrays, the ds-cDNA is fragmented and fluorescently labelled (orange). The labelled fragments bind to an ordered array of complementary oligonucleotides, and measurement of fluorescent intensity across the array indicates the abundance of a predetermined set of sequences. These sequences are typically specifically chosen to report on genes of interest within the organism's genome.[1] A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.

RNA Splicing

Differential splicing can process the same nuclear RNA into different mRNAs by selectively using different exons. introns can be spliced out completely, or cut at 5' or 3- sites to be partially included for example, type II collagen types can be determined using this. If intron cut out completely, makes mature Chondrocytes. If intron 5' splice site, creates the large and small isoforms of the protein Bcl-X ex) A "cassette" (yellow) that can be used as an exon or removed as an intron distinguishes the type II collagen types of chondrocyte precursors and mature chondrocytes (cartilage cells). (B) Mutually exclusive exons distinguish fibroblast growth factor receptors found in the limb ectoderm from those found in the limb mesoderm. (C) Alternative 5′ splice site selection, such as that used to create the large and small isoforms of the protein Bcl-X. (D) Alternative 3′ splice sites are used to form the normal and truncated forms of Chordin. (After

Polar Lobe Formation in Certain Mollusc Embryos

During cleavage, extrusion, and reincorporation of the polar lobe (clear vegetal cytoplasm) occur twice. The CD blastomere absorbs the polar lobe material but extrudes it again prior to second cleavage. After this division, the polar lobe is attached only to the D blastomere, which absorbs its material. From this point on, no polar lobe is formed. Crampton (1896) showed that if one removes the polar lobe at the trefoil stage, the remaining cells divide normally. However, the resulting larva is incomplete (figuRe 8.10), wholly lacking its intestinal endoderm and mesodermal kidney and heart), as well as some ectodermal organs (such as eyes). Moreover, Crampton demonstrated that the same type of abnormal larva can be produced by removing the D blastomere from the 4-cell embryo. Crampton thus concluded that the polar lobe cytoplasm contains the heart and intestinal-forming determinants and that these determinants (as well as its inducing ability) is transferred to the D blastomere. Crampton also showed that the localization of these endomesodermal determinants is established shortly after fertilization.

Injection of dsRNA for E-Cadherin into the Mouse Zygote Blocks E-Cadherin Expression

E cadherin important in compaction of mouse, ehen block it you lose compaction in mouse embryo

Epithelial-mesenchymal transition (EMT)

Epithelial-mesenchymal transition, or EMT. (A) Normal epithelial cells are attached to one another through adherens junctions containing cadherin, catenins, and actin rings. They are attached to the basal lamina through integrins. Paracrine factors can repress the expression of genes that encode these cellular components, causing the cell to lose polarity, lose attachment to the basal lamina, and lose cohesion with other epithelial cells. Cytoskeletal remodeling occurs, as well as the secretion of proteases that degrade the basal lamina and extracellular matrix molecules, enabling the migration of the newly formed mesenchymal cell. (B,C) EMT is seen in vertebrate embryos during the normal formation of neural crest from the dorsal region of the neural tube (B) and during the formation of the mesoderm by mesenchymal cells delaminating from the epiblast (C).

Organizer and B catenin--a wnt pathway

Experi- mental depletion of this molecule results in the lack of dorsal structures (Heasman et al. 1994a), while injection of exogenous β-catenin into the ventral side of an embryo produces a secondary axis (Funayama et al. 1995; Guger and Gumbiner 1995)--must be present in dorsal side to make blastopore lip accumulation of Bcat is seen in the nuclei of the dorsal cells and appears to cover both the Nieuwkoop center and organizer regions The proteins Wnt11, GSK3- binding protein (GBP), and Disheveled (Dsh) all are translocated from the vegetal pole of the egg to the future dorsal side of the embryo during fertilization. From research on the Wnt pathway, we have learned that β-catenin is targeted for destruction by glycogen synthase kinase 3 (GSK3; see Chapter 4). Indeed, activated GSK3 stimulates degrada- tion of β-catenin and blocks axis formation when added to the egg, and if endogenous GSK3 is knocked out by a dominant-negative form of GSK3 in the ventral cells of the early embryo, a second axis forms GSK3 can be inactivated by GBP and Disheveled. These two proteins release GSK3 from the degradation complex and prevent it from binding β-catenin and targeting it for destruction. During the first cell cycle, when the microtubules form parallel tracts in the vegetal portion of the egg, GBP travels along the microtubules by binding to kinesin, an ATPase motor protein that travels on microtubules. Kinesin always migrates toward the growing end of the microtubules, and in this case, that means moving to the point opposite sperm entry, i.e., the future dorsal side (Figure 11.17A-c). Disheveled, which is originally found in the vegetal pole cortex, grabs onto the GPB, and it too becomes Gilbert translocated along the microtubular monorail (Miller et al. 1999; Weaver et al. 2003). Developmental Biology 10e, Sinauer Associates TDheveBcio1r1tiec_a1l1r.1o6tation iDsaptero0b1-a2b5l-y16important in orienting and straightening the microtu- bular array and in maintaining the direction of transport when the kinesin complexes occasionally jump the track (Weaver and Kimelman 2004). Once at the site opposite the point of sperm entry, GBP and Dsh are released from the microtubles. Here, on the future dorsal side of the embryo, they inactivate GSK3, allowing β-catenin to accumu- late on the dorsal side while ventral β-catenin is degraded But the mere translocation of these proteins to the dorsal side of the embryo does not seem to be sufficient for protecting β-catenin. It appears that a Wnt paracrine factor has to be secreted there to activate the β-catenin protection pathway; this is accomplished by Wnt11. If Wnt11 synthesis is suppressed (by the injection of antisense Wnt11 oligonucleotides into the oocytes), the organizer fails to form. Furthermore, Wnt11 mRNA is localized to the vegetal cortex during oogenesis and is translocated to the future dorsal portion of the embryo by the cortical rotation of the egg cytoplasm (Tao et al. 2005; Cuykendall and Houston 2009). Here it is translated into a protein that becomes con- centrated in and secreted on the dorsal side of the embryo (Ku and Melton 1993; Schroeder et al. 1999; White and Heasman 2008). BIG PICTURE: Thus, during first cleavage, GBP, Dsh, and Wnt11 are brought into the future dorsal section of the embryo where GBP and Dsh can initiate the inactivation of GSK3 and the consequent protection of β-catenin. The signal from Wnt11 amplifies the signal and stabilizes GBP and Dsh and organizes them to protect β-catenin; β-catenin can associate with other transcrip- tion factors, giving these factors new properties Model of the mechanism by which the Disheveled protein stabi- lizes β-catenin in the dorsal portion of the amphibian egg. (A) Disheveled (Dsh) and GBP associate with kinesin at the vegetal pole of the unfertilized egg. Wnt11 is also in vesicles at the vegetal portion of the egg. (B) After fertilization, these vege- tal vesicles are translocated dorsally along subcortical microtubule tracks. Cortical rotation adds a "slow" form of transportation to the fast-track microtubule ride. (C) Wnt11, Dsh, and GBP are then released from the microtubules and are distributed in the future dorsal third of the 1-cell embryo. (D) Dsh and GBP bind to and block the action of GSK3, thereby preventing the degradation of β-catenin on the dorsal side of the embryo. Wnt11 probably is needed to stabilize this reaction, keeping an active source of Dsh. (E) The nuclei of the blastomeres in the dorsal region of the embryo receive β-catenin, whereas the nuclei of those in the ventral region do not. (F) Formation of a second dorsal axis caused by the injection of both blastomeres of a 2-cell Xenopus embryo with dominant-inactive GSK3. Dorsal fate is actively suppressed by wild-type GSK3. (A-E after Weaver and Kimelman 2004; F from Pierce and Kimelman 1995, courtesy of D. Kimelman.) essentially-- wnt, dsh and GPB travel via microtubules (fast) and cortical rotation (slow) to the organizer region, where it activates wnt pathway activating frizzled then dsh, which inhibits GSK which thus cannot degrade B-cat in the lip region

Wnt/Hedgehog shared themes

G-protein-like receptor Signal lipid modified Phosphorylation role Degradation of transcription factor

Germ plasm in amphibians

Germ cells arise in endoderm, migrate into mesoderm (gonad) for anurans (frogs and toads) Not urodeles (salamanders)

Mesoderm Induction and Organizer Formation by the Interaction of β-catenin And TGF-β Proteins

High β-catenin levels activate Nodal-related gene expression. So beta catenin expressed on dorsal region, and Veg 1 expressed vegetal pole. they act through the same pathway so whgere they overlap is the most expression and makes the organizer. The remaining functions are resulted from a gradient of activation: -In the region that will become the most anterior portion of the organizer—the pharyngeal endoderm—higher levels of Nodal-related proteins produce higher concentrations of activated Smad2. Smad2 can bind to the promoter of the hhex gene, and in concert with Twin and Siamois (induced by β-catenin), Hhex activates genes that specify pharyngeal endoderm cells to become foregut endoderm and to induce anterior brain development (Smithers and Jones 2002; Rankin et al. 2011). Slightly lower levels of Smad2 are believed to activate goosecoid expression in the cells that will become the prechordal mesoderm and notochord. Even lower amounts of Smad2 result in the formation of lateral and ventral mesoderm. Model for mesoderm induction and organizer formation by the interaction of β-catenin and TGF-β proteins. (B) At late blastula stages, Vg1 and VegT are found in the vegetal hemi- sphere; β-catenin is located in the dorsal region. (C) β-Catenin acts synergistically with Vg1 and VegT to activate the Xeno- pus nodal-related (Xnr) genes. This cre- ates a gradient of Xnr proteins across the endoderm, highest in the dorsal region. (D) The mesoderm is specified by the Xnr gradient. Mesodermal regions with little or no Xnr have high levels of BMP4 and Xwnt8; they become ventral mesoderm. Those having intermediate concentra- tions of Xnr become lateral mesoderm. Where there is a high concentration of Xnr, goosecoid and other dorsal meso- dermal genes are activated and the mesodermal tissue becomes the orga- nizer

Role for non-coding RNAs: Current model for the formation and use of microRNAs

If proteins can bind to specific nucleic acid sequences to block transcription or transla- tion, you would think that RNA would do the job even better. After all, RNA can be made specifically to complement and bind a particular sequence. Indeed, one of the most efficient means of regulating the translation of a specific message is to make a small antisense RNA complementary to a portion of a particular transcript. Such a naturally occurring antisense RNA was first seen in C. elegans (Lee et al. 1993; Wight- man et al. 1993). Here, the lin-4 gene was found to encode a 21-nucleotide RNA that bound to multiple sites in the 3′ UTR of the lin-14 mRNA (FiGUrE 3.31). The lin-14 gene encodes the LIN-14 transcription factor that is important during the first larval phase of C. elegans development. It is not needed afterward, and C. elegans is able to inhibit synthesis of LIN-14 from these messages by the small lin-4 antisense RNA. The binding of these lin-4 transcripts to the lin-14 mRNA 3′ UTR causes degradation of the lin-14 message (Bagga et al. 2005). The lin-4 RNA is now thought to be the "founding member" of a very large group of micrornas (mirnas). Computer analysis of the human genome predicts that we have more than 1000 miRNA loci and that these miRNAs probably modulate 50% of the protein-encoding genes in our bodies (Berezikov and Plasterk 2005; Friedman et al. 2009). These miRNAs usually contain only 22 nucleotides and are made from longer precursors. These precursors can be in independent transcription units (the lin-4 gene is far apart from the lin-14 gene), or they can reside in the introns of other genes et al. 2003; Lagos-Quintana et al. 2003). The initial RNA tran- script (which may contain several repeats of the miRNA sequence) forms hairpin loops wherein the RNA finds complementary structures within its strand. Because short double-stranded RNA molecules can resemble pathogenic viral genomes, the cell has a mechanism to both recognize these structures and use them as guides for their eradication (Wilson and Doudna 2013). Interest- ingly, this protective mechanism has been co-opted to be used as yet another way that the cell can differentially regulate the expression of endogenous genes. The process by which miRNAs inhibit expression of specific genes by degrading their mRNAs is called rna interference (Guo and Kemphues 1995; Sen and Blau 2006; Wilson and Doudna 2013), the characterization of which garnered Andrew Fire and Craig Mello the Nobel Prize in Physi- ology or Medicine in 2006 (Fire et al. 1998). The miRNA double-stranded stem-loop structures are pro- cessed by a set of RNases (Drosha and Dicer) to make single- stranded microRNA (FiGUrE 3.32). The microRNA is then pack- aged with a series of proteins to make an rna-induced silencing complex (risc). Proteins of the Argonaute family are particularly important members of this complex. Such small regulatory RNAs can bind to the 3′ UTR of messages and inhibit their translation. In some cases (especially when the binding of the miRNA to the 3′ UTR is perfect), the RNA is cleaved. More often, however, sev- eral RISCs attach to sites on the 3′ UTR and physically block the message from being translated (see Bartel 2004; He and Hannon 2004). The binding of microRNAs and their associated RISCs to the 3′ UTR can regulate translation in two ways (Filipowicz et al. 2008). First, this binding can block initiation of translation, pre- venting the binding of initiation factors or ribosomes. The Argo- naute proteins, for instance, have been found to bind directly to the methylated guanosine cap at the 5′ end of the mRNA message (Djuranovic et al. 2010, 2011). Second, this binding can recruit endonucleases that digest the mRNA, usually starting with the polyA tail (Guo et al. 2010). The latter seems to be commonly used in mammalian cells. MicroRNAs can be used to "clean up" and fine-tune the level of gene products. We mentioned those maternal RNAs in the oocyte that allow early develoGpilmbernt t to occur. How does the embryo get rid of maternal RNAs once they have been used and the Date 03-31-16 embryonic cells are making their own mRNAs? In zebrafish, this cleanup operation is assigned to microRNAs such as miR430. That is one of the first genes transcribed by the fish embryonic cells, and there are about 90 copies of this gene in the zebrafish genome. So, the level of miR430 goes up very rapidly. This microRNA has hundreds of targets (about 40% of the maternal RNA types), and when it binds to the 3′ UTR of these target mRNAs, these mRNAs lose their polyA tails and are degraded (FiGUrE 3.33; Giraldez et al. 2006; Giraldez 2010). In addition, miR430 repress- es initiation of translation prior to promoting mRNA decay (Bazzini et al. 2012). Although the microRNA is usually 22 bases long, it recog- nizes its target primarily through a "seed" region of about 5 bases in the 5′ end of the microRNA (usually at positions 2-7). This seed region recognizes targets in the 3′ UTR of the mes- sage. What happens, then, if an mRNA has a mutated 3′ UTR? Such a mutation appears to have given rise to the Texel sheep, a breed with a large and well-defined musculature that is the dominant meat-producing sheep in Europe. Genetic techniques mapped the basis of the sheep's meaty phenotype to the myo- statin gene. We have already seen that a mutation in the myo- statin gene that prevents the proper splicing of the nRNA can produce a large-muscled phenotype (see Figure 3.26). Another way of reducing the levels of myostatin involves a mutation in its 3′ UTR sequence. In the Texel breed, there has been a G-to- A transition in the 3′ UTR of the gene for myostatin, creating a target for the miR1 and miR206 microRNAs that are abundant in skeletal muscle (Clop et al. 2006). This mutation causes the depletion of myostatin messages and the increase in muscle mass characteristic of these sheep.

Issues with mammalian development

Implantation Extraembryonic membranes, tissues Human/mouse

Types of Cadherins

Importance of the types of cadherin for correct morphogenesis. (A) The type of cadherin expressed can result in different sorting behaviors, as seen when cells expressing R-cadherin (red stain) are mixed together with an equal number of cells expressing B-cadherin (green stain). The cells form two distinct mounds with one common boundary of contact. (B) Cross section of a mouse embryo showing the domains of E-cadherin expression (left) and N-cadherin expression (right). N-cadherin is critical for separation of presumptive epidermal and neural tissues during organogenesis. (C) The neural tube separates cleanly from surface epidermis in wild-type zebrafish embryos but not in mutant embryos where N-cadherin fails to be made. in these images, the cell outlines are stained green with antibodies to β-catenin, while the cell interiors are stained blue. n vertebrate embryos, several major cadherin types have been identified. For example, e-cadherin is expressed on all early mam- malian embryonic cells, even at the zygote stage. In the zebrafish embryo, E-cadherin is needed for the formation and migration of the epiblast as a sheet of cells during gastrulation. Loss of E-cad- herin in the "half-baked" zebrafish mutant results in a failure of deep epiblast cells to move radially into the more superficial epi- blast layer, an in vivo cell sorting process known as radial interca- lation that helps power epiboly during gastrulation (fiGure 4.7; see also Chapter 11 and Kane et al. 2005). Later in development, this E-cadherin is restricted to epithelial tissues of embryos and adults. In mammals, P-cadherin is found predominantly on the pla- centa, where it helps the placenta stick to the uterus (Nose and Takeichi 1986; Kadokawa et al. 1989). n-cadherin becomes highly expressed on the cells of the developing central nervous system (Hatta and Takeichi 1986), and it may play a role in mediating neu- ral signals. r-cadherin is critical in retina formation (Babb et al. 2005). A class of cadherins called protocadherins (Sano et al. 1993) lacks the attachment to the actin cytoskeleton through catenins. Expressing similar protocadherins is an important means of keep- ing migrating epithelial cells together, and expressing dissimilar protocadherins is an important way of separating tissues (as when he mesoderm forming the notochord separates from the surrounding mesoderm that will form somites). Differences in cell surface tension and the tendency of cells to bind together depend on the strength of cadherin interactions (Duguay et al. 2003). This strength can be achieved quantitatively (the more cadherins on the apposing cell surfaces, the tighter the adhesion) or qualitatively (some cadherins will bind to different cadherin types, whereas other cadherins will not bind to different types.--found in tight junctions + ECM (need calcium)

Cell Communication

In an embryo, communication between cells can occur across short distances, such as between two neighboring cells in direct contact, called juxtacrine signaling, or across long distances through the secretion of proteins into the extracellular matrix, called paracrine signaling (fiGure 4.1). Proteins that are secreted from a cell and designed to communicate a response in another cell are generally referred to as sig- naling proteins (generally called ligands), while the proteins within a membrane that function to bind either other membrane- associated proteins or signaling proteins are called receptors. A receptor in the mem- brane of one cell that binds the same type of receptor in another cell represents a homo- philic binding. In contrast, heterophilic binding occurs between different receptor

4d blastomere

In fact, all the essential determinants for heart and intestine forma- tion are now in the 4d blastomere (also called the mesentoblast, as mentioned earlier), and removal of that cell results in a heartless and gutless larva (Clement 1986). The 4d blastomere is responsible for forming (at its next division) the two bilaterally paired blastomeres that give rise to both the mesodermal (heart) and endodermal (intestine) organs (Lyons et al. 2012; Lambert and Chan, 2014). The mesodermal and endodermal determinants of the 3D macromere, therefore, are transferred to the 4d blastomere. At least two morphogenetic determinants are involved in regulating the development of 4d. First, the cell appears to be specified by the presence of transcription factor β-catenin, which enters into the nucleus of the 4d mesentoblast and its immediate progeny (figuRe 8.11a; Henry et al. 2008; Rabinow- itz et al. 2008). When translational inhibitors suppressed β-catenin protein synthesis, the 4d cell underwent a normal pattern of early cell divisions but failed to differentiate into heart, muscles, or hindgut; and gastrulation also failed to occur in those embryos Indeed, β-catenin may have an evolutionarily conserved role in mediating autonomous specification and specifying endomesodermal fates throughout the animal kingdom; in subsequent chapters we will see a similar role for this protein in both sea urchin and frog embryos. B catenin specifies infor for heart and intestine The 4d blastomere also contains the protein and mRNA for the translation suppres- sor Nanos (figuRe 8.11B). As with β-catenin, blocking translation of Nanos mRNA prevents formation of the larval muscles, heart, and intestine from the 4d blastomere (Rabinowitz et al. 2008). In addition, the germline cells (sperm and egg progenitors) do not form. As we will see throughout the book, the Nanos protein is often involved in specification of germ cell progenitors. But the 4d blastomere not only develops autonomously, it also induces other cell lineages. The Notch signaling pathways may be critical for these inductive events of the 4d blastomere. Blocking Notch signaling after the 4d blastomere has formed causes the larva to resemble those formed when the 4d cell is removed; while the autonomous fates of the 4d cell (such as larval kidneys) are not disturbed (Gharbiah et al. 2014). The D set of blastomeres is thus the "organizer" of snail embryos. Experiments have demonstrated that the nondiffusible polar lobe [cortical] cytoplasm that is localized to the D blastomere is extremely important in normal molluscan development for several reasons: • It contains the determinants for the proper cleavage rhythm and the cleavage orientation of the D blastomere. • It contains certain determinants (those entering the 4d blastomere and hence leading to the mesentoblasts) for autonomous mesodermal and intestinal Gilbert differentiation. • It is responsible for permitting the inductive interactions (through the mate- Developmental Biology 11e, Sinauer Associates Gil_DreiavBl eion1t1eer_in08g.1th1e 3D bDlatseto0m1-2e1r-e1)5leading to the formation of the shell gland and eye.

Mouse fertilization

In mammals, it is not the tip of the sperm head that makes contact with the egg (as happens in the perpendicular entry of sea urchin sperm) but the side of the sperm head (FIGuRE 7.29). The acrosome reaction, in addi- tion to expelling the enzymatic contents of the acrosome, also exposes the inner acrosomal membrane to the outside. The junction between this inner acrosomal membrane and the sperm cell membrane is called the equato- rial region, and this is where membrane fusion between sperm and egg begins (FIGuRE 7.30A). As in sea urchin gamete fusion, the sperm is bound to regions of the egg where actin polymerizes to extend microvilli to the sperm (Yanagimachi and Noda 1970). Izumo protein and membrane fusion in mouse fertilization. (A) Localization of Izumo to the inner and outer acrosomal membrane. Izumo is stained red, acrosomal proteins green. (B) Diagram of sperm-egg cell membrane fusion. During the acrosome reaction, Izumo localized on the acrosomal becomes translo- cated to the sperm cell membrane. There it meets the complex of Juno and CD9 proteins on the egg microvil- li, initiating membrane fusion and the entry of the sperm into the egg. (After Satouh et al. 2012; photographs courtesy of M. Okabe.)

PCR

In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus.

Ovulation

Increase in FSH/LH Oocyte and follicle grow Production of E without P Leads to further growth, LH surge/ovulation/completion of 1st meiotic division Follicle becomes corpus luteum Makes E plus P Build-up of endometrial layer of the uterus 2 endings possible- 1.fertilization--implantation--trophoblast makes HCG--steady production of P from ovaries 2.no fertilization-uterine wall degrades without P

Pseudohermaphrodite

Individual with external genitalia of one sex and the internal organs of another sex Gonad differentiation-secondary sexual characteristics disagree with sex chromosomes

Embryotic induction

Induction: one region of the embryo interacts with another to alter the fate of the second region Regulative Development if transplant dorsal blastopore lip into another embryo but on the opposite side (cells determined to be epidermis) the area invaginated and forms mesodermal regions, essentially forming a whole new embryo. dorsal lip = organizer key induction—in which the progeny of dorsal lip cells induce the dorsal axis and the neural tube—is traditionally called the primary embryonic induction

Sea Urchant Gastrulation

Ingression-primary mesenchyme-adhesion changes Invagination Cell shape Proteoglycan secretion Convergent-extension of archenteron Secondary Mesenchyme Ingression of Primary Mesenchyme Cells (primary mesenchyme) Invagination of the Vegetal Plate using CSPG, a proteoglycan slide 14 in gastrulation convergent extension of archenteron makes secondary mesenchyme vegetal plate--ingression of skeletal primary mesenchyme cellls--invagination of vegetal pole

Brainbow

Is the designation of a "cell type" the most precise way of identifying a cell? To answer this question, we would have to be able to watch and analyze individual cells in an embryo over time A genetic approach to fate mapping has been developed to label cells with a seeming rainbow of possible colors, which can be used to identify each individual cell in a tissue or even a whole embryo The Brainbow system triggers the expression of different combinations and amounts of distinct fluorescent proteins (green, red, blue, etc). The resulting stochastic distribution of fluorescent protein combinations gives each cell a distinct color that is stably inherited by all its progeny. How is that achieved? The answer is that genes for each fluorescent protein are engineered into the genome of the organism being studied in such a way that they are initially inactive; upon exposure to Cre-recombinase (an enzyme that catalyzes recombination events at specific sites in the DNA), however, a random combination of fluorescent genes can become active. Different cells are then distinguishable based on the hue of fluorescence created by the different combinations of fluorescent proteins active in each cell. The Brainbow genetic system is used to randomly fix cells with a distinct fluorescent color or hue and is accomplished by inserting multiple copies of different fluorescent genes into the organism's genome. Through Cre- recombinase activity, different combinations of these fluorescent genes can be activated to produce an array of different colors. In the example here, each cell will, by default, express red fluorescent protein; upon Cre-mediated recombination, however, cyan, yellow, or green fluorescent proteins begin to be expressed in a stochastic manner. You can trace these colors through lineage for fate mapping analysis For instance, Kevin Eggan's research team has used the "Rainbow" system to label cells of the early cleavage stages of the mouse embryo to address the following question: Is the first lineage choice of becoming an embryonic cell or an extraembryonic cell a random or a regulated process? They discovered that it is nonrandom The Rainbow mouse system is a ver- sion of Brainbow and works similarly. In this experiment, recombination was initiated during early mouse blastocyst development to permanently mark differ- ent cells within the trophectoderm (TE) and inner cell mass (ICM) with unique colors. Those colors were then followed over time and the populations quantified (pie charts), which revealed a statistically significant distribution demonstrating clonal origins from the earlier labeled cells.

Neurelation in Frog

Neural plate rises above neural fold and connects, top layer becomes epidermis, the middle becomes neural crest cells and the pinched tube becomes the neural tube neural tube makes brain and spinal cord crest cells make PNS Autonomic NS

In Amphibian Oocytes, Lampbrush Chromosomes are Active in the Diplotene Germinal Vesicle During First Meiotic Prophase

Lampbrush chromosomes: Chromo- somes in an amphibian primary oocyte during the diplotene stage of the first meiotic prophase that stretch out large loops of DNA, representing sites of upregulated RNA synthesis.

Neurelation

Makes ecdoderm

Enhancer region modularity

Mediator complex brings enhancer and promoter together (folds DNA to bring enhancer region closer to/on top of promotor w help from TF). Also helps bring RNA polymerase II to the promotor for transcription **Enhancer region modularity. (A) Model for gene regulation by enhancers. (i) The top diagram shows the exons, introns, promoter, and enhancers of a hypothetical gene A. In situ hybridization (left) shows that gene A is expressed in limb and brain cells. (ii) In develop- ing brain cells, brain-specific transcription factors bind to the brain enhancer, causing it to bind to the Mediator, stabilize RNA polymerase II at the promoter, and modify the nucleosomes in the region of the promoter. The gene is transcribed in the brain cells only; the limb enhancer does not function. (iii) An analogous process allows for transcription of the same gene in the cells of the limbs. The gene is not transcribed in any cell type whose transcription factors the enhancers cannot bind. (B) The Pax6 protein is critical in the development of several widely different tissues. Enhancers direct Pax6 gene expression (yellow exons 1-7) differentially in the pancreas, the lens and cornea of the eye, the retina, and the neural tube. (C) A portion of the DNA sequence of the pancreas-specific enhancer element. This sequence has binding sites foGr tihlbeerPtbx1 and Meis transcription factors; both must be present to activate Pax6 in the pan- crDeaevse.lo(Dp)mWenhteanl Bthioeloβg-yg1a1lea,cStionsaiudearsAe srseopcoiartesr gene is fused to the Pax6 enhancers for expres- DevBio11e_03.10 Date 05-24-16 sion in the pancreas and lens/cornea, the enzyme is seen in those tissues Transcription factors bind to the enhancer and bind to nucleosome-modifying enzymes that remove nucleosomes from the area, including the enhancer and promoter. (C) The transcription factors also bind a large protein complex called the Media- tor. (D) The Mediator is able to recruit and stabilize RNA polymerase II (RNA PII) and its cofactors (TAFs IIA, IIB, etc.) at the promoter site. These factors bound with RNA polymerase II is called the pre- initiation complex. The chromatin looping is further stabilized by cohesin. (E) After RNA polymerase II leaves the promoter, there are generally two outcomes. One outcome is that it can associate with the transcription elongation complex (TEC) to elongate the nRNA while the Mediator continues to recruit new RNA polymerase II proteins to the complex. Alternatively RNA polymerase II can be instructed to stop elongation by a repressive transcription factor (NELF) that prevents the assembly of the TEC. When given a second developmental signal, NELF can be removed and transcription elongation continued. Enhancers generally activate only cis-linked promoters (i.e., promoters on the same chromosome); therefore, they are sometimes called cis-regulatory elements. Because of DNA folding, however, enhancers can regulate genes at great distances For instance, the E. coli gene for β-galactosidase (the lacZ gene) can be used as a reporter gene and fused to (1) a promoter that can be activated in any cell and (2) an enhancer that directs expres- sion of a particular gene (Myf5) only in mouse muscles. When the resulting transgene is injected into a newly fertilized mouse egg and becomes incorporated into its DNA, β-galactosidase protein reveals the expression pattern of that muscle-specific gene

Amnion structure and cell movements during human gastrulation

Movements of the epiblast cells through the primitive streak and the node and under- neath the epiblast are superimposed on the dorsal surface view. (C) At days 14 and 15 the ingressing epiblast cells are thought to replace the hypoblast cells (which contrib- ute to the yolk sac lining), and at day 16 the ingressing cells fan out to form the meso- dermal layer. Like the chick epiblast cells, mammalian mesoderm and endoderm cells originate in the epiblast, undergo epithelial- mesenchymal transition, lose E-cadherin, and migrate through a primitive streak as individual mesenchymal cells (Burdsal et al. 1993). Those cells arising from the node give rise to the notochord. However, in contrast to notochord formation in the chick, the cells that form the mouse notochord are thought to become integrated into the endo- derm of the primitive gut (Jurand 1974; Sulik et al. 1994). These cells can be seen as a band of small, ciliated cells extending rostrally from the node. They form the notochord by converging medially and "budding" off in a dorsal direction from the roof of the gut. The timing of these developmental events varies enormously in mammals. In humans, the migration of cells forming the mesoderm doesn't start until day 16—around the time that a mouse embryo is almost ready to be born The ectodermal precursors are located anterior and lateral to the fully extended primitive streak, as in the chick epiblast and (as in the chick embryo), a single cell can give rise to descendants in more than one germ layer. Thus, at the epiblast stage these lineages have not yet become fully separate from one another. Indeed, in mice, some of the visceral endoderm, which had been extraembryonic, is able to intercalate with the definitive endoderm and become part of the gut

Hedgehog Pathway

No Hedgehog: Patched inhibits Smoothened action, microtubules bind Ci-P, degrades Ci to repressor form drosophila: With Hedgehog-Patched, relieves suppression of Smoothened, now gets phosphorylated and goes to membrane, inhibits Ci degradation and its in tact form acts to increase transcription vertebrates: same pathway, but microtubules located in primary cilium and smoothened must first gain access to primary cilium via hedgehog ligand binding to patched. Also, The transcript regulator is Gli not Ci Hedgehog signal transduction pathway. Patched protein in the cell membrane is an inhibi- tor of the Smoothened protein. (A) In the absence of Hedgehog binding to Patched, Patched inhibits Smooth- ened and in Drosophila melanogaster the Ci protein remains tethered to the microtubules by the Cos2 and Fused proteins. This tether allows the proteins PKA and Slimb to cleave Ci into a transcriptional repressor that blocks the transcription of particular genes. (B) When Hedgehog binds to Patched, its conformational changes release the inhibition of the Smooth- ened protein. Smoothened then releases Ci from the microtubules, inactivating the cleavage proteins PKA and Slimb. The Ci protein enters the nucleus, and acts as a transcrip- tional activator of particular genes. In vertebrates (lower panels), the homologues of Ci are the Gli genes, which function similarly as transcrip- tional activators or repressors when a hedgehog ligand is bound to Patched or absent, respectively. Additionally in vertebrates, for Smoothened to positively regulate Gli processing into an activator form, it needs to gain access into the primary cilium— hedgehog ligand binding to patched enables the transport of Smoothened into the primary cilium. Lastly, sev- eral co-receptors such as Gas1 and Boc function to enhance hedgehog signaling ex) The Hedgehog pathway is extremely important in vertebrate limb patterning, neural differentiation and pathfinding, retinal and pancreas development, and craniofacial morphogenesis, among many other processes (fiGure 4.31a; McMahon et al. 2003). When mice were made homozygous for a mutant allele of Sonic hedgehog, they had major limb and facial abnormalities. The midline of the face was severely reduced, and a single eye formed in the center of the forehead, a condition known as cyclopia after the one-eyed Cyclops of Homer's Odyssey (fiGure 4.31B; Chiang et al. 1996). Some human cyclopia syndromes are caused by mutations in genes that encode either Sonic hedgehog or the enzymes that synthesize cholesterol (Kelley et al. 1996; Roessler et al. 1996; Opitz and Furtado 2013). Moreover, certain chemicals that induce cyclopia do so by interfering with the Hedgehog pathway (Beachy et al. 1997; Cooper et al. 1998). Two teratogens7 known to cause cyclopia in vertebrates are jervine and cyclopamine. Both are alkaloids found in the plant Veratrum californicum (corn lily), and both directly bind to and inhibit Smoothened function (see Figure 4.31B; Keeler and Binns 1968). In later development, Sonic hedgehog is critical for feather formation in the chick embryo, for hair formation in mammals, and, when misregulated, for the formation of skin cancer in humans (Harris et al. 2002; Michino et al. 2003). Although mutations that inactivate the Hedgehog pathway can cause malformations, mutations that activate the pathway ectopically can have mitogenic effects and cause cancers. If the Patched protein is mutated in somatic tissues such that it can no longer inhibit Smoothened, it can cause tumors of the basal cell layer of the epidermis (basal cell carcinomas). Heritable muta- tions of the patched gene cause basal cell nevus syndrome, a rare autosomal dominant condition characterized by both developmental anomalies (fused fingers; rib and facial abnormalities) and multiple malignant tumors (Hahn et al. 1996; Johnson et al. 1996). Interestingly, vismodegib, a compound that inhibits Smoothened function similar to cyclopamine, is currently in clinical trials as a therapy to combat basal cell carcinomas (Dreno et al. 2014; Erdem et al. 2015). (What do you think the warnings for pregnancy should be on this drug?)

Anti-BMPs (TGF β)

Noggin Chordin Follistatin Cerberus (also anti-Wnt)

nRNA Localization

Not only is the timing of mRNA translation regulated, but so is the place of RNA expression. A majority of mRNAs (about 70% in Drosophila embryos) are localized to specific places in the cell (Lécuyer et al. 2007). Just like the selective repression of mRNA translation, the selective localization of messages is often accomplished through their 3′ UTRs. There are three major mechanisms for the localization of an mRNA (see Palacios 2007): 1. Diffusion and local anchoring. Messenger RNAs such as nanos diffuse freely in the cytoplasm. When they diffuse to the posterior pole of the Drosophila oocyte, how- ever, they are trapped there by proteins that reside particularly in these regions. These proteins also activate the mRNA, allowing it to be translated (FiGUrE 3.34a). 2. Localized protection. Messenger RNAs such as those encoding the Drosophila heat shock protein hsp83 (which helps protect the embryos from thermal extremes) also float freely in the cytoplasm. Like nanos mRNA, hsp83 accumulates at the posterior pole, but its mechanism for getting there is different. Throughout the embryo, the mRNA is degraded. Proteins at the posterior pole, however, protect the hsp83 mRNA from being destroyed (FiGUrE 3.34B). 3. Active transport along the cytoskeleton. Active transport is probably the most widely used mechanism for mRNA localization. Here, the 3′ UTR of the mRNA is rec- ognized by proteins that can bind these messages to "motor proteins" that travel along the cytoskeleton to their final destination (FiGUrE 3.34c). These motor proteins are usually ATPases such as dynein or kinesin that split ATP for their motive force. We will see in Chapter 9 that this mechanism is very important for localizing transcription factor mRNAs into different regions of the Drosophila oocyte. Localization of mRNAs. (A) Diffusion and local anchoring. nanos mRNA diffuses through the Drosophila egg and is bound (in part by the Oskar protein) at the posterior end of the oocyte. This anchoring allows the nanos mRNA to be translated. (B) Localized protection. The mRNA for Drosophila heat shock protein (hsp83) will be degraded unless it binds to a protector protein (in this case, also at the posterior end of the oocyte). (C) Active transport on the cyto- skeleton, causing the accumulation of mRNA at a particular site. Here, bicoid mRNA is transported to the anterior of the oocyte by dynein and kinesin motor proteins. Meanwhile, oskar mRNA is brought to the posterior pole by transport along microtubules by kinesin ATPases Basically mRNA sometimes has to bind to specific sites or move to locations in the cell to be express or prevent degredation

Nuclear Events in the Fertilization of the Sea Urchin

Nuclear events in the fertilization of the sea urchin. (A) Sequen- (A) tial photographs showing the migration of the egg pronucleus and the sperm pronucleus toward each other in an egg of Clypeaster japonicus. The sperm pronucleus is surrounded by its aster of microtubules. (B) The two pronuclei migrate toward each other on these microtubular processes. (The pronuclear DNA is stained blue by Hoechst dye.) The microtubules (stained green with fluo- rescent antibodies to tubulin) radiate from the centrosome associated with the (smaller) male pronucleus and reach toward the female pronucleus

Research Article 1:

Often, eggs incubated at low temperatures produce one sex, whereas eggs incubated at higher temperatures produce the other. shows the abrupt temperature-induced change in sex ratios for the red-eared slider turtle. If a brood of eggs is incubated at a temperature below 28°C, all the turtles hatching from the eggs will be male. Above 31°C, every egg gives rise to a female. At temperatures in between, the brood gives rise to indi- viduals of both sexes At MPT, Kdm6b expression is up-regulated directly or by an upstream temperature-sensitive regulator such as Cirbp. KDM6B then demethylates the Dmrt1 promoter, leading to up-regulation of its expression and male development. Additionally, at MPT, transcription of Kdm6b and Jarid2 with a retained intron (IR) is up-regulated; their function is unknown. At FPT, Kdm6b and Jarid2 expression is down-regulated and they are transcribed without the retained intron. Presumably, Jarid2 is sufficiently expressed to enable PRC2 to trimethylate H3K27 on the Dmrt1 promoter and suppress its expression, leading to female development. Questions: 1. Where and when is Kdm6b expressed? 2. How does temperature effect its expression? 3. How do they test for function? 4. What is the target of Kdm6b action? 1) expressed in GMC at stage 13 2)low temps = high expression of kdm6b 3. use RNi to knockout for DMRt1 gene. Knockdown of Kdm6b abolishes the expression of Dmrt1 = female, but the male pathway can be rescued by overexpression of Dmrt1 loss of Dmrt1 redirected gonads incubating at 26°C toward female fate, whereas the gain of Dmrt1 redirected gonads incubating at 32°C toward male fate 4. Histone 3 (H3K27me-3) and acts through demethylation at the Dmrt1 locus, ehich increases expression of dmrt1

Activin

One of the most important mechanisms governing cell fate specification involves gra- dients of paracrine factors that regulate gene expression; such signaling molecules are called morphogens. A morphogen (Greek, "form-giver") is a diffusable biochemical molecule that can determine the fate of a cell by its concentration.4 That is, cells exposed to high levels of a morphogen activate different genes than those cells exposed to lower levels. Morphogens can be transcription factors produced within a syncytium of nuclei as in the Drosophila blastoderm (see Chapter 2). They can also be paracrine factors that are produced in one group of cells and then travel to another population of cells, specifying the target cells to have similar or different fates according to the concentration of the morphogen. Uncommitted cells exposed to high concentrations of the morphogen (nearest its source of production) are specified as one cell type. When the morphogen's concentration drops below a certain threshold, a dif- ferent cell fate is specified. When the concentration falls even lower, a cell that initially was of the same uncommitted type is specified in yet a third distinct manner paracrine factor of the TGF-B family Activin-secreting beads were placed on unspecified cells from an early Xenopus embryo. The activin then diffused from the beads. At high concen- trations (about 300 molecules/cell), activin induced expression of the goosecoid gene, whose product is a transcription factor that specifies the frog's dorsal-most structures. At slightly lower concentrations of activin (about 100 molecules per cell), the same tis- sue activated the Xbra gene and was specified to become muscle. At still lower concen- trations, these genes were not activated, and the "default" gene expression instructed the cells to become blood vessels and heart Activin (Or a Closely Related Protein) Is Thought to Be Responsible for Converting Animal Hemisphere Cells into Mesoderm: If isolate cells to become ectoderm of a blastula and subject them to activin, the cells change fate and become mesoderm. Different concentrations levels effected what the cells became, low levels changed cells to blood cells while high conc. changed to heart cells.

B-catenin and micromere in sea urchant

Otx and B catenin concentrated at vegetal pole of embryo. these proteins are taken up by micromeres and activate Pmar-1, which represses the repressor gene HesC. HesC usually represses transcription factors activating skeletal forming genes, so B catenin results in HesC not being able t repress these in the micromeres thus prompting their skeletal development. Also represses signaling proteins in the delta notch pathway. in veg 2 cells (located just above micromere) pmar-1 not activated so skeletal genes repressed. But, cells containing notch can be actiovated by the delta expressed from the micromere cells which form non-skeletogenic mesenchyme genes if inhibit B-catenin you get all ectoderm development of embryo and no mesoderm or endoderm

Anterior/posterior axis formation

PAR proteins and the establishment of polarity. (A) When sperm enters the egg, the egg nucleus is undergoing meiosis (left). The cortical cytoplasm (orange) contains PAR-3, PAR-6, and PKC-3, and the internal cytoplasm contains PAR-2 and PAR-1 (purple dots). (B,C) Microtubules of the sperm centrosome initiate contraction of the actin-based cytoskeleton toward the future anterior side of the embryo. These sperm microtubules also protect PAR-2 protein from phosphorylation, allowing it to enter the cortex along with its bind- ing partner, PAR-1. PAR-1 phosphorylates PAR-3, causing PAR-3 and its binding partners PAR-6 and PKC-3 to leave the cortex. (D) The posterior of the cell becomes defined by PAR-2 and PAR-1, while the anterior of the cell becomes defined by PAR-6 and PAR-3. The meta- phase plate is asymmetric, as the microtubules are closer to the posterior pole. (E) The meta- phase plate separates the zygote into two cells, one having the anterior PARs and one the posterior PARs. (F) In this dividing C. elegans zygote, PAR-2 protein is stained green; DNA is stained blue. (G) In second division, the AB cell and the P1 cell divide perpendicularly (90° dif- ferently from each other) PAR-3 and PAR-6, interact- ing with the protein kinase PKC-3 (mutations of which cause defective partitioning), are uniformly distributed in the cortical cytoplasm. PKC-3 restricts PAR-1 and PAR-2 to the internal cytoplasm by phosphorylating them. The sperm centrosome (microtubule-organizing center) contacts the cortical cytoplasm through its micro- tubules and initiates cytoplasmic movements that push the male pronucleus to the nearest end of the oblong oocyte. That end becomes the posterior pole (Goldstein and Hird 1996). Moreover, these microtubules locally protect PAR-2 from phosphorylation, thereby allowing PAR-2 (and its binding partner, PAR-1) into the cortex nearest the centrosome. Once PAR-1 is in the cortical cytoplasm, it phosphorylates PAR-3, caus- ing PAR-3 (and its binding partner, PKC-3) to leave the cortex. At the same time, the sperm microtubules induce the contraction of the actin-myosin cytoskeleton toward the anterior, thereby clearing PAR-3, PAR-6, and PKC-3 from the posterior of the 1-cell embryo. During first cleavage, the metaphase plate is closer to the posterior, and the fertilized egg is divided into two cells, one having the anterior PARs (PAR-6 and PAR-3) and one having the posterior PARs (PAR-2 and PAR-1)

Types of cell movement during gastrulation

Page 41 invagination--sea urchant endoderm Involution-- amphibian mesoderm ingression--Sea urchant mesoderm, fly neuroblasts Epiboly--Ectoderm formation in sea urchins, tunicates, and amphibians

The Roles of Inositol Phosphates in Releasing Calcium from the ER and the Initiation of Development

Phospholipase C splits PIP2 into IP3 and DAG. IP3 releases calcium from the endoplasmic reticulum (initiates cortical granule rxn), and DAG, with assistance from the released Ca2+, activates the sodium-hydrogen exchange pump in the membrane--increases intracellular PH and Stimulation of protein synthesis, DNA replication, and cytoplasmic movements of morphogenetic material so how does phospholipase C get activated?: The bindin receptor (perhaps acting through a G protein) acti- vates tyrosine kinase (TK, an Src kinase), which activates PLC. This is probably the mechanism used by sea urchins.

Testosterone dependent development:

Seminal vessicle, epididymis, Vas Deferans

Progesterone

Progesterone is produced by the ovaries, placenta, and adrenal glands. Supports endometrium buildup inhibits contractility of uterine muscle firms cervix ad inhibits dilation

Transgenic Mammals to Produce Protein Pharmaceuticals

Protein drugs such as human insulin, protease inhibitors, and clotting factors are difficult to manufacture. Because of immunological rejection problems, human proteins are usually much better tolerated by patients than proteins extracted from other species. Similarly, our bodies often reject proteins that have been synthesized by genetically engineered bacteria. The problem thus becomes how to obtain large amounts of human proteins. One of the most efficient ways to produce these proteins is to insert the human genes encoding them into the oocyte DNA of sheep, goats, or cows. Animals containing a gene from another individual (often of a different species)—a transgene—are called transgenic animals. A female sheep or cow made transgenic for the human protein gene might express this gene in her mammary tissue and secrete the protein in her milk. Obtain gene (such as AAT gene in humans) and add B-Lactoglobulin promotor to make recombinant DNA. Inject this DNA into pronucleus of a sheep ovum and implant into foster mother. Identify transgenic progeny via PCR which will have expression of AAT secreted into the sheep milk. You obtain the milk and fractionate the milk proteins to obtain the pure human AAT protein Thus, cloning these sheep will produce the protein to use in human drugs (producing transgenic sheep is innefective, while cloning the "elite transgenics" is very effective because they all produce high amounts of protein

RNA-seq

RNA is isolated from samples and converted to complementary DNA (cDNA) with standard procedures using reverse transcriptase. This cDNA is broken up into smaller fragments, and known adaptor sequences are added to the ends. These adaptors allow immobilization and PCR-based amplification of these tran- scripts. Next-generation sequencing can analyze these tran- scripts for both nucleotide sequence and quantity (Goldman and Domschke 2014). RNA-seq has been particularly power- ful for comparing transcriptomes between identical samples differing only in select experimental parameters. Deep sequencing: RNA-Seq. (Top) Research- ers begin with specific sorts of tissues, often comparing different conditions, such as embryos of different ages (chick embryos, as shown here), isolated tissues (such as the eye; boxed regions) or even single cells, samples from different genotypes, or experimen- tal paradigms. (1) RNA is isolated to obtain only those genes that are actively expressed; (2) these transcripts are then fragmented into smaller stretches and used to create cDNA with reverse tran- scriptase. (3) Specialized adaptors are ligated to the cDNA ends to enable PCR amplification and immobilization for (4) subsequent sequencin can be graphed to only show expressed axons Steps: 1. Bind poly-AAA tail to RNA to purify 2. Fragment mRNA 3. convert to cDNA by reverse transcriptase and add random primers 4. Ligate adaptors and amplify using PCR One can ask, how does the array of transcripts differ between tissues located in different regions of the embryo, or the same tissue at different times of development, or the same tissue treated or untreated with a specific compound? These comparisons only scratch the surface of what is possible and what we can learn from differences in transcriptomes. The advent of fluorescence activated cell sorting (typically spoken as FACS for short) and microdissection has allowed for the precise isolation of tissues and individual cells, and recent advances in RNA-seq sensitiv- ity has permitted transcriptomics of single cells. A common experimental approach has been to design a targeted deep-sequencing experiment to arrive at a list of genes associated with a given condition. Researchers then use bioinformatics and an understanding of developmental biology to select gene candidates from the list to test the func- tion of these genes in their system. used to identify differential gene expression between samples--shows all genes actually being expresses (transcriptome)

BMP Ligand

Receptor is a Dimer, when phosphorylated recruits SMAD 1,5 which recruits binding of SMAD4 which allows it to enter the nucleus and promote or repress transcription

Activin of TGF-B ligand

Receptor is a Dimer, when phosphorylated recruits SMAD 2,3 which recruits binding of SMAD4 which allows it to enter the nucleus and promote or repress transcription

Hormones in spermatogenesis

Release of LH from pituitary to the testes causes lyedig cells to release testosterone, which diffuses into the sertoli cells to be converted to estradiol by aromatase. The testosterone also binds to the germ cell region which binds to ABP (androgen binding protein). SOme testosterone also remains free to stimulate steps of spermatogenesis: FSH ot testosterone stimulates mitosis I for germ cells to make primary spermatocyte, and testosterone is then needed to make the secondary spermatocyte and eventually the spermatid. FSH is needed to turn the spermatid to spermatozoa, and finally LH is needed to make spermiation. LH and FSH also aid in sperm production. FSH binds to sertoli cells and stimulates production of ABP, aromatase, inhibin, and STP. STP leaves tubule and helps LH stimulate testosterone production. LAstly, estrogen inhibin and testosterone enter blood to control target organs and negative feedback regulate GnRH in hypothalamus

Capacitation (sperm maturation) in mammals

Remove cholesterol, proteins from sperm PM, pH change, cAMP, influx of Ca++ 1. Lipidchanges. The sperm cell membrane is altered by the removal of cholesterol by albumin proteins in the female reproductive tract (Cross 1998). The cholesterol efflux from the sperm cell membrane is thought to change the location of its "lipid rafts," isolated regions that often contain receptor pro- teins that can bind the zona pellucida and participate in the acrosome reac- tion (Bou Khalil et al. 2006; Gadella et al. 2008). Originally located throughout the sperm cell membrane, after cho- lesterol efflux lipid rafts are clustered over the anterior sperm head. The outer acrosomal membrane changes and comes into contact with the sperm cell membrane in a way that prepares it for the acrosome reaction 2. Protein changes. Particular proteins or carbohydrates on the sperm surface are lost during capacitation (Lopez et al. 1985; Wilson and Oliphant 1987). It is possible that these compounds block the recognition sites for the sperm proteins that bind to the zona pellucida. It has been suggested that the unmasking of these sites might be one of the effects of cholesterol depletion (Benoff 1993). The membrane poten- tial of the sperm cell becomes more negative as potassium ions leave the sperm. This change in membrane potential may allow calcium channels to be opened and permit calcium to enter the sperm. Cal- cium and bicarbonate ions are critical in activating cAMP production and in facilitating the membrane fusion events of the acrosome reac- tion (Visconti et al. 1995; Arnoult et al. 1999). The influx of bicar- bonate ions (and possibly other ions) alkalinizes the sperm, raising its pH. This will be critical in the subsequent activation of calcium channels (Navarro et al. 2007). As a result of cAMP formation, pro- tein phosphorylation occurs (Galantino-Homer et al. 1997; Arcelay et al. 2008). Once they are phosphorylated, some proteins migrate to the surface of the sperm head. One of these proteins is Izumo, which is critical in sperm-egg fusion (see Figure 7.30; Baker et al. 2010).

The Binding of Cytoskeleton to the Extracellular Matrix Through the Integrin Molecule

Simplified diagram of the fibronectin receptor complex. The integrins of the complex are membrane-spanning receptor proteins that bind fibronectin on the outside of the cell while binding cytoskeletal proteins on the inside of the cell. This family of receptor proteins are called integrins because they integrate the extra- cellular and intracellular scaffolds, allowing them to work together (Horwitz et al. 1986; Tamkun et al. 1986). On the extracellular side, integrins bind to the amino acid sequence arginine-glycine-aspartate (RGD), found in several extracellular matrix adhe- sive proteins, including fibronectin, vitronectin (found in the basal lamina of the eye), and laminin (Ruoslahti and Pierschbacher 1987). On the cytoplasmic side, integrins bind to talin and α-actinin, two proteins that connect to actin microfilaments. This dual binding enables the cell to move by contracting the actin microfilaments against the fixed extracellular matrix. Integrins can also signal from the outside of the cell to the inside of the cell, altering gene expression (Walker et al. 2002). Bissell and colleagues (Bissell et al. 1982; Martins- Green and Bissell 1995) have shown that integrin is critical for inducing specific gene expression in developing tissues, especially those of the liver, testis, and mammary gland. In the mammary gland, extracellular laminin is able to signal the expression of estro- gen receptor and casein protein genes through the integrin proteins The presence of bound integrin prevents the activation of genes that promote apoptosis, or programmed cell death (Montgomery et al. 1994; Frisch and Ruoslahti 1997). For instance, the chon- drocytes that produce the cartilage of our vertebrae and limbs can survive and differentiate only if they are surrounded by an extracellular matrix and are joined to that matrix through their integrins (Hirsch et al. 1997). If chondrocytes from the developing chick sternum are incubated with antibodies that block the bind- ing of integrins to the extracellular matrix, they shrivel up and die. Indeed, when focal adhesions linking an epithelial cell to its extra- cellular matrix are broken, the caspase-dependent apoptosis path- way is activated, and the cell dies. Such "death-on-detachment" is a special type of apoptosis called anoikis, and it appears to be a major weapon against cancer (Frisch and Francis 1994; Chiarugi and Giannoni 2008). Although the mechanisms by which bound integrins inhibit apoptosis remain controversial, the extracellular matrix is obvi- ously an important source of signals that can be transduced into the nucleus to produce specific gene expression. Some of the genes induced by matrix attachment are being identified. When plated onto tissue culture plastic, mouse mammary gland cells will divide (fiGure 4.13). Indeed, genes for cell division (c-myc, cyclinD1) are expressed, whereas genes for differentiated products of the mammary gland (casein, lactoferrin, whey acidic protein) are not expressed. If the same cells are plated onto plastic coated with a basal lamina, the cells stop dividing, and the genes of differenti- ated mammary gland cells are expressed. That happens only after the integrins of the mammary gland cells bind to the laminin of the basal lamina. Then the gene for lactoferrin is expressed, as is the gene for p21, a cell division inhibitor. The c-myc and cyclinD1 genes become silent. Eventually, all the genes for the develop- mental products of the mammary gland are expressed, and the cell division genes remain turned off. By this time, the mam- mary gland cells have enveloped themselves in a basal lamina, forming a secretory epithelium reminiscent of the mammary gland tissue. The binding of integrins to laminin is essential for transcription of the casein gene, and the integrins act in concert with prolactin

C elegans Nematode Axis formation

Sperm are stored such that a mature egg must pass through the sperm on its way to the vulva. (B) The germ cells undergo mitosis near the distal tip of the gonad. As they move farther from the distal tip, they enter meiosis. Early meioses form sperm, which are stored in the spermatheca. Later meioses form eggs, which are fertilized as they roll through the spermatheca. (C) Early develop- ment occurs as the egg is fertilized and moves toward the vulva. The P lineage consists of stem cells that will eventually form the germ cells. Separate AB from P1 blastomere: P goes on to make all its derivatives AB not able to make all of its derivatives AB loses something and cant make all necessary cells So, some signaling back and forth--AB senses its missing something. SO in mosaic embryo, there is still a need for communication-- thus it mosaic development still has regulative properties (C elegans undergo mosaic development) regulative- mosaic-

Chick Egg

Stage X embryo, where islands of hypoblast cells can be seen, as well as a congregation of hypoblast cells around Koller's sickle. (B) By stage XII, a sheet of cells that grows anteriorly from Koller's sickle combines with the hypoblast islands to form the complete hypoblast layer. (C) By stage XIII, just prior to primitive streak formation, the formation of the hypoblast just been completed. (D) By stage 2 (12-14 hours after the egg is laid), the primi- tive streak cells form a third layer that lies between the hypoblast and epiblast cells. (E) By stage 3 (15-17 hours post laying), the primitive streak has become a definitive region of the epiblast, with cells migrating through it to become the mesoderm and endoderm **primitive streak ingresses, then elongates through intercalation 1.Thickening>Koller's sickle 2.Posterior MZ Sickle-derived cells 3/In blue, migrate anteriorly 4. Delamination-hypoblast islands (green)> primary hypoblast 5. Meanwhile, blue cells link up> secondary hypoblast, like blastula stage 6. Primitive streak (like blastopore), red, from epiblast, ingress 7. Cells replace hypoblast, become endoderm 8. Second wave, become mesoderm

Mechanism for primary sex determination of internal genetalia

Starts w genital ridge. If Sry is not present (pink region), the interactions between paracrine and transcription factors in the develop- ing genital ridge activate Wnt4 and Rspo1. Wnt4 activates the canonical Wnt pathway, which is made more efficient by Rspo1. The Wnt pathway causes the accumulation of b-catenin, and large accumulation of b-catenin stimulates further Wnt4 activity. This continual production of b-catenin both induces the transcription of ovary- producing genes and blocks the testis- determining pathway by interfering with Sox9 activity. If Sry is present (blue region), it may block b-catenin signaling (thus halt- ing ovary generation) and, along with Sf1, activate the Sox9 gene. Sox9 activates Fgf9 synthesis, which stimulates testis development and promotes further Sox9 synthesis. Sox9 also prevents b-catenin's activation of ovary-producing genes. Sry may also activate other genes (such as TCF21 and NT3) that help generate Sertoli cells. In summary, then, a Wnt4/b-catenin loop specifies the ovaries, whereas a Sox9/Fgf9 loop specifies the testes. One of the targets of the Wnt pathway is the fol- listatin gene, whose product organizes the granulosa cells of the ovary. Transcription factor Foxl2, which is activated (in a still unknown way) in the ovary, is also involved in inducing follistatin synthesis. The XY pathway appears to have an earlier initia- tion; if it does not function, the XX pathway takes over.

Receptor serine kinases: the TGFBeta family

Steps: 1. Ligand binds to receptor II--TGF-B like ligands 2. Receptor II phosphorylates I 3. Smad activated ( by phosphorylation) 4. Smad associates with co-Smad 5. Complex enters nucleus, represses or activates transcription SMAD pathway: Members of the TGF-β superfamily activate members of the smad family of transcription factors (Heldin et al. 1997; Shi and Massagué 2003). The TGF-β ligand binds to a type II TGF-β receptor, which allows that receptor to bind to a type I TGF-β receptor. Once the two receptors are in close contact, the type II receptor phosphorylates a serine or threonine on the type I receptor, thereby activating it. The activated type I receptor can now phosphorylate the Smad12 proteins (fiGure 4.36a). Smads 1 and 5 are activated by the BMP family of TGF-β factors, whereas the receptors binding activin, Nodal, and the TGF-β family phosphorylate Smads 2 and 3. These phosphorylated Smads bind to Smad4 and form the transcription factor complexes that will enter the nucleus The Smad pathway is activated by TGF-β superfamily ligands (A) An activation complex is formed by the binding of the ligand by the type I and type II receptors, which allows the type II receptor to phosphorylate the type I receptor on particular serine or threonine residues. The phosphorylated type I receptor protein can now phosphorylate the Smad proteins. (B) Those receptors that bind TGF-β family proteins or mem- bers of the activin family phosphorylate Smads 2 and 3. Those receptors that bind to BMP family proteins phosphory- late Smads 1 and 5. These Smads can complex with Smad4 to form active tran- scription factors. A simplified version of the pathway is shown on the left. TGF-B family: -Among members of the TGf-β family, TGF-β1, 2, 3, and 5 are important in regulating the formation of the extracellular matrix between cells and for regulating cell division (both positively and negatively). TGF-β1 increases the amount of extracellular matrix that epithelial cells make (both by stimulating collagen and fibronectin synthesis and by inhibiting matrix degradation). TGF-β proteins may be critical in controlling where and when epithelia branch to form the ducts of kidneys, lungs, and salivary glands (Daniel 1989; Hardman et al. 1994; Ritvos et al. 1995). The effects of the individual TGF-β family members are difficult to sort out because members of the TGF-β family appear to function similarly and can compensate for losses of the others when expressed together. • The members of the BmP family can be distinguished from other members of the TGF-β superfamily by having seven (rather than nine) conserved cysteines in the mature polypeptide. Because they were originally discovered by their ability to induce bone formation, they were given the name bone morphogenetic proteins. It turns out, though, that bone formation is only one of their many functions; the BMPs are extremely multifunctional. They have been found to regulate cell division, apoptosis (programmed cell death), cell migration, and differentiation (Hogan 1996). They include proteins such as BMP4 (which in some tissues causes bone formation, in other tissues specifies epidermis, and in other instances causes cell proliferation or cell death) and BMP7 (which is important in neural tube polarity, kidney development, and sperm formation). The BMP4 homologue in Drosophila is critically involved in forming appendages, including the limbs, wings, genitalia, and antennae. Indeed, the malformations of 15 such structures have given this homologue the name Decapentaplegic (DPP). As it (rather oddly) turns out, BMP1 is not a member of the BMP family at all; rather, it is a protease. BMPs are thought to work by diffusion from the cells producing them (Ohkawara et al. 2002). Inhibitors such as Noggin and Chordin that bind directly to BMP reduce BMP-receptor inter- actions. We will cover this morphogenetic mechanism more directly when we discuss dorsoventral axis specification in the gastrula. • The nodal and activin proteins are extremely important in specifying the different regions of the mesoderm and for distinguishing the left and right sides of the vertebrate body axis. The left-right asymmetry of bilateral organisms is strongly influenced by a gradient of Nodal from right to left across the embryo. In vertebrates, this Nodal gradient appears to be created by the beating of motile cilia that promotes the graded flow of Nodal across the midline (Babu and Roy 2013; Molina et al. 2013; Blum et al. 2014; Su 2014). ex) Regulation by gradients of paracrine factor concentration was elegantly demonstrated by the specification of different mesodermal cell types in the frog Xenopus laevis by activin, a paracrine factor of the TGF-β family (fiGure 4.23; Green and Smith 1990; Gurdon et al. 1994). Activin-secreting beads were placed on unspecified cells from an early Xenopus embryo. The activin then diffused from the beads. At high concen- trations (about 300 molecules/cell), activin induced expression of the goosecoid gene, whose product is a transcription factor that specifies the frog's dorsal-most structures. At slightly lower concentrations of activin (about 100 molecules per cell), the same tis- sue activated the Xbra gene and was specified to become muscle. At still lower concen- trations, these genes were not activated, and the "default" gene expression instructed the cells to become blood vessels and heart Negative regulators: Extracellular anti-TGF-betas bind ligand-preventing productive interaction with receptor. examples include: Noggin Chordin Follistatin Cerberus

Receptor Tyrosine Kinase Cascade

Steps: 1. Ligand dimerizes (ligand usually a paracrine factor) 2. Receptor dimerize 3. Activate Ras 4. Activate MEKinase 5. Phosphorylate and activate transcription factor Ligand RTK GEF RAS RAF MEK ERK Transcription factor Transcription ex) FGF-2 important in angiogenesis and neural stem cell proliferation; FGF-4/8 limb morphogenesis Structure and function of a receptor tyrosine kinase. The binding of a paracrine factor (such as Fgf8) by the extracellular portion of the receptor protein activates the dormant tyrosine kinase, whose enzyme activity phosphorylates its recipro- cal receptor partner followed by specific tyrosine residues of certain intracellular proteins. One member of this family, Fgf8, is especially important during segmentation, limb development, and lens induction. Fgf8 is usually made by the optic vesicle that contacts the outer ectoderm of the head (fiGure 4.25a; Vogel-Höpker et al. 2000). After contact with the outer ectoderm occurs, Fgf8 gene expression becomes concentrated in the region of the presumptive neural retina (the tissue directly apposed to the presumptive lens) (fiGure 4.25B). Moreover, if Fgf8-contain- ing beads5 are placed adjacent to head ectoderm, this ectopic Fgf8 will induce this ectoderm to produce ectopic lenses and express the lens-associated transcription factor l-Maf (fiGure 4.25C). FGFs often work by activating a set of receptor tyrosine kinases called the fibroblast growth factor receptors (fGfrs). For instance, the Branchless protein is an FGFR in Drosophila ex) Fgf8 in the developing chick. (A) Fgf8 gene expres- sion pattern in the 3-day chick embryo, shown by in situ hybridiza- tion. Fgf8 protein (dark areas) is seen in the distalmost limb bud ectoderm (1), in the somitic mesoderm (the segmented blocks of cells along the anterior-posterior axis (2), in the branchial arches of the neck (3), at the boundary between the midbrain and hindbrain (4), in the optic vesicle of the developing eye (5), and in the tail (6). (B) In situ hybridization of Fgf8 in the optic vesicle. The Fgf8 mRNA (purple) is localized to the presumptive neural retina of the optic cup and is in direct contact with the outer ectoderm cells that will become the lens. (C) Ectopic expression of l-Maf in competent ecto- derm can be induced by the optic vesicle (above) and by an Fgf8- containing bead (below) The widely used RTK signal transduction pathway can be activated by fibroblast growth factor. The receptor tyrosine kinase is dimerized by the ligand (a paracrine factor, such as FGF) along with heparan sulfate proteoglycans (HSPG), which together cause the dimerization and autophosphoryla- tion of the RTKs. The adaptor protein recog- nizes the phosphorylated tyrosines on the RTK and activates an intermediate protein, GEF, which activates the Ras G-protein by allowing phosphorylation of the GDP-bound Ras. At the same time, the GAP protein stimulates hydrolysis of this phosphate bond, returning Ras to its inactive state. The active Ras activates the Raf protein kinase C, which in turn phosphorylates a series of kinases (such as MEK). Eventually, the activated kinase ERK alters gene expression in the nucleus of the responding cell by phosphory- lating certain transcription factors (which can then enter the nucleus to change the types of genes transcribed) and certain translation factors (which alter the level of protein synthe- sis). In many cases, this pathway is reinforced by the release of calcium ions. EGF signaling also uses RTK, as example of conservation of molecular mechanism use of this cascade during development used in cell cycle

Sea urchant acrosomal reaction

Summary of events leading to the fusion of egg and sperm cell membranes in sea urchin fertilization, which is external. (1) The sperm is chemotactically attracted to and activated by the egg. (2, 3) Contact with the egg jelly triggers the acrosome reac- tion, allowing the acrosomal process to form and release proteo- lytic enzymes to digest jelly layer. (4) The sperm adheres to the vitelline envelope and lyses a hole in it. (5) The sperm adheres to the egg cell membrane and fuses with it. The sperm pronucleus/Actin can now enter the egg cytoplasm. the acrosome membrane is lined with the Bindin adhesion molecule, that moves from inside sperm head to protruding out with the acrosomal process once acrosomal vessicle diffuses out. This binds to EBR1 (bindin receptor in vitelline layer of egg) to anchor sperm to egg ex) iSoploegcyie1s1-es, pSiencaiufiecrbAinssdoincgiatoefsthe acrosomal process to the egg sDuervfaBcioe1i1nes_e07a.1u0rchins. D(Aa)tAec0t2u-a2l2c-1o6ntact of a sperm acrosomal process with an egg microvillus. (B) In vitro model of species-specific binding. The agglu- tination of dejellied eggs by bindin was measured by adding bindin particles to a plastic well containing a suspension of eggs. After 2-5 minutes of gentle shaking, the wells were photographed. Each bindin bound to and agglutinat- ed only eggs from its own species bindin receptors are species specific Biochemical studies have confirmed that the bindins of closely related sea urchin spe- cies have different protein sequences. This finding implies the existence of species-spe- cific bindin receptors on the egg vitelline envelope

II. MEROBLASTIC (INCOMPLETE) CLEAVAGE

Telolecithal (Dense yolk throughout most of cell) The eggs of most bony fish are telolecithal, meaning that most of the cytoplasm is occupied by yolk. Cleavage can take place only in the blastodisc, a thin region of yolk- free cytoplasm at the animal pole. The cell divisions do not completely divide the egg, so this type of cleavage is called meroblastic (Greek meros, "part"). Since only the blas- todisc becomes the embryo, this type of meroblastic cleavage is referred to as discoidal. Discoidal meroblastic cleavage in a zebrafish egg. (A) 1-Cell embryo. The mound atop the cytoplasm is the blastodisc. (B) 2-Cell embryo. (C) 4-Cell embryo. (D) 8-Cell embryo, wherein two rows of four cells are formed. (E) 32-Cell embryo. (F) 64-Cell embryo, wherein the blastodisc can be seen atop the yolk cell.

DHT dependent

Testes, penis, urethra, pubis, prostate Group in Dominican Republic without the DHT converting enzyme XY individuals appear female till puberty when excess testosterone is made

Notch Pathway

The Ligand also membrane bound to another cell (ligand called delta). Delta binds to Notch receptor, Receptor cytoplasmic domain cleaved by protease , cleaved receptor enters nucleus and acts as co-factor for transcription by binding to CSL, relieving its bound repressor and recruiting p300. Mechanism of Notch activity. (A) Prior to Notch signaling, a CSL transcription factor (such as Suppressor of hairless or CBF1) is on the enhancer of Notch-regulated genes. The CSL binds repressors of transcription. (B) Model for the activation of Notch. A ligand (Delta, Jagged, or Serrate protein) on one cell binds to the extracellular domain of the Notch protein on an adjacent cell. This binding causes a shape change in the intracellular domain of Notch, which acti- vates a protease. The protease cleaves Notch and allows the intracellular region of the Notch protein to enter the nucleus and bind the CSL transcription factor. This intracellular region of Notch displaces the repressor proteins and binds activators of transcription, including the histone acetyl- transferase p300. The activated CSL can then transcribe its target genes. (After K. Koziol-Dube, personal communication.) Notch proteins are involved in the formation of numerous vertebrate organs—kid- ney, pancreas, and heart—and they are extremely important receptors in the nervous system. In both the vertebrate and Drosophila nervous systems, the binding of Delta to Notch tells the receiving cell not to become neural (Chitnis et al. 1995; Wang et al. 1998). In the vertebrate eye, the interactions between Notch and its ligands regulate which cells become optic neurons and which become glial cells (Dorsky et al. 1997; Wang et al. 1998). WEB ToPiC 4.3 noTCh muTaTions Humans have genes for more than one Notch protein and more than one ligand. Their interactions are critical in neural development, and mutations in Notch genes can cause nervous system abnormalities. ex) Formation of anchor cell in c elegans vulva Induction does indeed occur on the cell-to-cell level, and one of the best examples is the formation of the vulva in the nematode worm C. elegans. Remarkably, the signal transduction pathways involved turn out to be the same as those used in the formation of retinal receptors in Drosophila; only the targeted transcription factors are different. In both cases, an epidermal growth-factor-like inducer activates the RTK pathway, leading to the differential regulation of Notch-Delta signaling. noTCh-DelTa anD laTeral inhiBiTion: We have discussed the reception of the EGF- like LIN-3 signal by the cells of the equivalence group that forms the vulva. Before this induction occurs, however, an earlier interaction has formed the anchor cell. The forma- tion of the anchor cell is mediated by lin-12, the C. elegans homologue of the Notch gene. In wild-type C. elegans hermaphrodites, two adjacent cells, Z1.ppp and Z4.aaa, have the potential to become the anchor cell. They interact in a manner that causes one of them to become the anchor cell while the other one becomes the precursor of the uterine tissue. In loss-of-function lin-12 mutants, both cells become anchor cells, whereas in gain-of-function mutations, both cells become uterine precur- sors (Greenwald et al. 1983). Studies using genetic mosaics and cell ablations have shown that this decision is made in the second larval stage, and that the lin-12 gene needs to function only in that cell destined to become the uterine precursor cell. The presumptive anchor cell does not need it. Seydoux and Greenwald (1989) speculate that these two cells originally synthesize both the signal for uterine differentiation (the LAG-2 protein, homologous to Delta) and the receptor for this molecule (the LIN-12 protein, homologous to Notch; Wilkinson et al. 1994). During a particular time in larval development, the cell that, by chance, is secreting more LAG-2 causes its neighbor to cease its production of this differen- tiation signal and to increase its production of LIN-12. The cell secreting LAG-2 becomes the gonadal anchor cell, while the cell receiving the signal through its LIN-12 protein becomes the ventral uterine precursor cell (fiGure 4.45). Thus, the two cells are thought to determine each other prior to their respective differentiation events. When LIN-12 is used again during vulva formation, it is activated by the primary vulval lineage to stop the lateral vulval cells from form- ing the central vulval phenotype (see Figure 4.44). Thus, the anchor cell/ventral uterine precursor decision illustrates two important aspects of determination in two originally equivalent cells. First, the initial difference between the two cells is created by chance. Second, this initial difference is reinforced by feedback. This Notch-Delta mediated mechanism of restricting adjacent cell fates is called lateral inhibition--increases in Lin-12 initially causes cels to differentiate from anchor cell to uterus cell. LAter in vulval development, increases in Lin-12 stop the lateral cells (forming from the primary lineage cell) from becoming the central lineage cell, therefote showing a negative feedback effect called lateral inhibition C. elegans vulval precursor cells (VPCs) and their descendants. (A) Location of the gonad, anchor cell, and VPCs in the second instar larva. (B,C) Relationship of the anchor cell to the six VPCs and their subsequent lineages. Primary (1°) lineages result in the central vulval cells, secondary (2°) lineages constitute the lat- eral vulval cells, and tertiary (3°) lineages gener- ate hypodermal cells. (C) Outline of the vulva in the fourth instar larva. The circles represent the positions of the nuclei. (D) Model for the deter- mination of vulval cell lineages in C. elegans. The LIN-3 signal from the anchor cell causes the determination of the P6.p cell to generate the central vulval lineage (dark purple). Lower concentrations of LIN-3 cause the P5.p and P7.p cells to form the lateral vulval lineages. The P6.p (central lineage) cell also secretes a short-range juxtacrine signal that induces the neighboring cells to activate the LIN-12 (Notch) protein. This signal prevents the P5.p and P7.p cells from generating the primary central vulval cell lineage. (After Katz and Sternberg 1996.) Model for the generation of two cell types (anchor cell and ventral uterine pre- cursor cell) from two equivalent cells (Z1.ppp and Z4.aaa) in C. elegans. (A) The cells start off as equivalent, producing fluctuating amounts of signal and receptor. The lag-2 gene is thought to encode the signal, and the lin-12 gene is thought to encode the receptor. Reception of the signal turns down LAG-2 (Delta) production and upregulates LIN-12 (Notch). (B) A stochastic (chance) event causes one cell to produce more LAG-2 than the other cell at some particular critical time, which stimulates more LIN-12 production in the neighboring cell. (C) This differ- ence is amplified because the cell producing more LIN-12 produces less LAG-2. Eventually, just one cell is delivering the LAG-2 signal, and the other cell is receiving it. (D) The signaling cell becomes the anchor cell, and the receiving cell becomes the ventral uterine precursor cell. (After Greenwald and Rubin 1992.)

Anterior-posterior polarity in the oocyte

The anterior-posterior polarity of the embryo is established while the oocyte is still in the egg chamber, and it involves interactions between the developing egg cell and the Gilbert Developmental Biology 11/e follicular cells that enclose it. The follicular epithelium surrounding the developing oocyte is initially uniform with respect to cell fate, but this uniformity is broken by two signals organized by the oocyte nucleus. Interestingly, both of these signals involve the same gene, gurken. The gurken message appears to be synthesized in the nurse cells, but it is transported into the oocyte. Here it becomes localized between the oocyte nucleus and the cell membrane, and it is translated into Gurken protein (Cáceres and Nilson 2005). At this time the oocyte nucleus is very near what will become the poste- rior tip of the egg chamber, and the Gurken signal is received by the follicle cells at that position through a receptor protein encoded by the torpedo gene2 (FIGURE 9.8A). This signal results in the "posteriorization" of these follicle cells (FIGURE 9.8B). The poste- rior follicle cells send a signal back into the oocyte. This signal, a lipid kinase, recruits the Par-1 protein to the posterior edge of the oocyte cytoplasm (FIGURE 9.8c; Doer- flinger et al. 2006; Gervais et al. 2008). Par-1 protein organizes microtubules specifically with their minus (cap) and plus (growing) ends at the anterior and posterior ends of the oocyte, respectively. The orientation of the microtubules is critical, because different microtubule motor proteins will transport their mRNA or protein cargoes in different directions. The motor protein kinesin, for instance, is an ATPase that will use the energy of ATP to trans- port material to the plus end of the microtubule. Dynein, however, is a "minus-directed" motor protein that transports its cargo in the opposite direction. One of the messages transported by kinesin along the microtubules to the posterior end of the oocyte is oskar mRNA (Zimyanin et al. 2008). The oskar mRNA is not able to be translated until it reaches the posterior cortex, at which time it generates the Oskar protein. Oskar recruits more Par-1 protein, thereby stabilizing the microtubule orientation and allowing more material to be recruited to the posterior pole of the oocyte (Doerflinger et al. 2006; Zimyanin et al. 2007). The posterior pole will thereby have its own distinctive cytoplasm, called pole plasm, which contains the determinants for producing the abdomen and the germ cells. This cytoskeletal rearrangement in the oocyte is accompanied by an increase in oocyte volume, owing to transfer of cytoplasmic components from the nurse cells. These components include maternal messengers such as the bicoid and nanos mRNAs. These mRNAs are carried by motor proteins along the microtubules to the anterior and posterior ends of the oocyte, respectively (FIGURE 9.8D-F). As we will soon see, the protein products encoded by bicoid and nanos are critical for establishing the anterior- posterior polarity of the embryo oskar, nanos travel to posterior pole and anchor and orient microtubules bicoid goes anterior

Postulated cascades leading to male and female phenotypes in mammals

The conversion of the genital ridge into the bipotential gonad requires, among others, the Sf1, Wt1, and Lhx9 genes; mice lacking any of these genes lack gonads. The bipo- tential gonad appears to be moved into the female pathway (ovary development) by the Foxl2, Wnt4, and Rspo1 genes and into the male pathway (testis development) by the Sry gene (on the Y chromosome), which triggers the activity of Sox9. (Lower levels of Wnt4 are also present in the male gonad.) The ovary makes thecal cells and granu- losa cells, which together are capable of synthesizing estrogen. Under the influence of estrogen (first from the mother, then from the fetal gonads), the Müllerian duct differen- tiates into the female reproductive tract, the internal and external genitalia develop, and the offspring develops the secondary sex characteristics of a female. The testis makes two major hormones involved in sex determination. The first, anti-Müllerian hormone (AMH), causes the Müllerian duct to regress. The second, testosterone, causes dif- ferentiation of the Wolffian duct into the male internal genitalia. In the urogenital region, testosterone is converted into dihydrotestosterone (DHT), which causes the morpho- genesis of the penis and prostate gland The reason the Y chromosome is able to direct testis formation even when more than one X chromosome is present may be a matter of timing. It appears there is a crucial window of opportunity during gonad development during which the testis- determining factor (now known to be the product of the Sry gene) can function. If the Sry gene is present, it usually acts during this duration to promote testis formation and to inhibit ovary formation. If the Sry gene is not present (or if it fails to act at the appro- priate time), the ovary-forming genes are the ones that will function does SRY and SOX9 inhibit Respondin and Wnt? In the absence of gonads, it appears the female phenotype is generated. When Jost (1947, 1953) removed fetal rabbit gonads before they had differentiated, the resulting rabbits had a female phenotype, regardless of whether their genotype was XX or XY. The general scheme of primary sex determination is shown in Figure 6.2B. If the embryonic cells have two X chromosomes and no Y chromosome, the gonadal pri- mordia develop into ovaries. The ovaries produce estrogen, a hormone that enables the development of the müllerian duct into the uterus, oviducts, cervix, and upper portion of the vagina (Fisher et al. 1998; Couse et al. 1999; Couse and Korach 2001). If embryonic cells contain both an X and a Y chromosome, testes form and secrete two major factors. The first is a TGF-b family paracrine factor called anti-müllerian hor- mone (amh; sometimes called müllerian-inhibiting factor, mif). AMH destroys the Müllerian duct, thus preventing formation of the uterus and oviducts. The second factor is the steroid hormone testosterone. Testosterone masculinizes the fetus, stimulating formation of the penis, male duct system, scrotum, and other portions of the male anatomy, as well as inhibiting development of the breast primordia. wolffian duct makes internal male genetalia (epididymis, seminiferous tunules) testosterone converted to DHT makes external genetalia such as penis scrotum and prostate indifferent gonads differentiate at week 4 of development, at 6 weeks both mulerian and wolffian ducts are seen. Week 8 testes and ovaries differentiate, still have both wolf and mullerian ducts but genetal ridge has begun to differentiate and testes cords form. Week 16 males mullerian duct starts degenerating and testes cord connects to wolf, females this happens at week 20 w the wolffian duct degeneration and formation of follicles of ovarian cortex

ectoderm to neural tissue

The ectoderm is induced to become epidermal tissue by binding bone morphogenetic proteins (BmPs), whereas the nervous system forms from that region of the ectoderm that is protected from epidermal induction by BMP-inhibiting molecules (Hemmati- Brivanlou and Melton 1994, 1997). In other words, (1) the "default fate" of the ectoderm is to become neural tissue; (2) certain parts of the embryo induce the ectoderm to become epidermal tissue by secreting BMPs; and (3) the organizer tissue acts by secreting molecules that block BMPs, thereby allowing the ectoderm "protected" by these BMP inhibitors to become neural tissue. one such mlecule secreted is chordin. chordin mRNA expression starts in blastopore lip and is high in organizer region as gastrulation progresses. Others include noggin--expose embryo to uv light to kill dorsal regions, if you then inject noggin you form dorsal structures The major epidermal inducers are BMP4 and its close relatives BMP2, BMP7, and ADMP (promote BMP production) BMP4 (along with certain other molecules) is a powerful ventralizing factor. Organizer proteins such as Chordin, Noggin, and Follistatin block the action of BMP4; their inhibitory effects can be seen in all three germ layers--organizer proteins block BMP4 and make mesoderm, dorsal endoderm and neural ectoderm

Inner cell mass

The first segregation of cells within the inner cell mass forms two layers. The lower layer, in contact with the blastocoel, is called the primitive endoderm, and it is homologous to the hypoblast of the chick embryo. The remaining inner cell mass tissue above it is the epiblast. The primitive endoderm will form the yolk sac of the embryo, and like the chick hypoblast, will be used for positioning the site of gastrulation, regulating cell move- ments in the epiblast, and promoting the maturation of blood cells. Moreover, the primitive endoderm, like the chick hypoblast, is an extraembryonic layer and does not provide many (if any) cells to the actual embryo (see Stern and Downs 2012).

Chromosome diminution

The fragmen- tation of chromosomes just prior to cell division, resulting in cells in which only a portion of the original chromosome survives. Chromosome diminution occurs during cleavage in Parascaris aequorum in the cells that will generate the somatic cells while the future germ cells are protected from this phenom- enon and maintain an intact genome. In Nematode Parascaris-chromosome diminution, this occurs due to a change in the cleavage plane.

embryo development order

The germinal stage refers to the time from fertilization through the development of the early embryo until implantation is completed in the uterus. The germinal stage takes around 10 days.[2] During this stage, the zygote begins to divide, in a process called cleavage. A blastocyst is then formed and implanted in the uterus. Embryogenesis continues with the next stage of gastrulation, when the three germ layers of the embryo form in a process called histogenesis, and the processes of neurulation and organogenesis follow. i.e.) clevage--gastrulation--neurelation feRtilizatioN giveS the oRgaNiSm a new genome and rearranges its cytoplasm. Once this is accomplished, the resulting zygote begins the production of a multicellular organism. During cleavage, rapid cell divisions divide the zygote cytoplasm into numer- ous cells. These cells undergo dramatic displacements during gastrulation, a process whereby the cells move to different parts of the embryo and acquire new neighbors. The different patterns of cleavage and gastrulation were described in Chapter

-cortical granule reaction

The mechanism of cortical granule exocytosis is similar to that of the exocytosis of the acrosome, and it may involve many of the same molecules. Upon fertilization, the concentration of free Ca2+ in the egg cytoplasm increases greatly. In this high-calcium environment, the cortical granule membranes fuse with the egg cell membrane, releas- ing their contents (see Figure 7.17A). Once the fusion of the cortical granules begins near the point of sperm entry, a wave of cortical granule exocytosis propagates around the cortex to the opposite side of the egg. In sea urchins and mammals, the rise in Ca2+ concentration responsible for the cortical granule reaction is not due to an influx of calcium into the egg, but comes from within the egg itself. The release of calcium from intracellular storage can be monitored visually using calcium-activated luminescent dyes such as aequorin (a protein that, like GFP, is isolated from luminescent jellyfish) or fluorescent dyes such as fura-2. These dyes emit light when they bind free Ca2+. When a sea urchin egg is injected with dye and then fertilized, a striking wave of calcium release propagates across the egg and is visualized as a band of light that starts at the point of sperm entry and proceeds actively to the other end of the cell (FIGuRE 7.18; Steinhardt et al. 1977; Hafner et al. 1988). The entire release of Ca2+ is complete within roughly 30 seconds, and free Ca2+ is re-sequestered shortly after being released ex) several experiments have demonstrated that Ca2+ is directly responsible for propa- gating the cortical granule reaction, and that these ions are stored within the egg itself. The drug A23187 is a calcium ionophore—a compound that allows the diffusion of ions such as Ca2+ across lipid membranes, permitting them to travel across otherwise imper- meable barriers. Placing unfertilized sea urchin eggs into seawater containing A23187 initiates the cortical granule reaction and the elevation of the fertilization envelope. Moreover, this reaction occurs in the absence of any Ca2+ in the surrounding water; thus the A23187 must be stimulating the release of Ca2+ that is already sequestered in organelles within the egg In sea urchins and vertebrates (but not snails and worms), the Ca2+ responsible is stored in the endoplasmic reticulum of the egg. this reticulum is pronounced in the cortex and surrounds the cortical granules (FIGuRE 7.19; Gardiner and Grey 1983; Luttmer and Longo 1985). The cortical granules are themselves tethered to the cell membrane by a series of integral membrane proteins that facilitate calcium- mediated exocytosis (Conner et al. 1997; Conner and Wessel 1998). Thus, as soon as Ca2+ is released from the endoplasmic reticulum, the cortical granules fuse with the cell membrane above them. Once initiated, the release of calcium is self-propagating. Free calcium is able to release sequestered calcium from its storage sites, thus causing a wave of Ca2+ release and cortical granule exocytosis.

random egg facts

The membrane enclosing the egg cytoplasm regulates the flow of specific ions during fertilization and must be capable of fusing with the sperm cell membrane. Outside this egg cell membrane is an extracel- lular matrix that forms a fibrous mat around the egg and is often involved in sperm-egg recognition (Wasserman and Litscher 2016). In invertebrates, this structure is usually called the vitelline envelope (FIGuRE 7.4A). The vitelline envelope contains several different glycoproteins. It is supplemented by extensions of membrane glycoproteins from the cell membrane and by proteinaceous "posts" that adhere the vitelline envelope to the cell membrane (Mozingo and Chandler 1991). The vitelline envelope is essential for the species-specific binding of sperm. Many types of eggs also have a layer of egg jelly outside the vitelline envelope. This glycoprotein meshwork can have numerous functions, but most commonly it is used either to attract or to activate sperm. The egg, then, is a cell specialized for receiving sperm and initiating development. Lying immediately beneath the cell membrane of most eggs is a thin layer (about 5 μm) of gel-like cytoplasm called the cortex. The cytoplasm in this region is stiffer than the internal cytoplasm and contains high concentrations of globular actin mol- ecules. During fertilization, these actin molecules polymerize to form long cables of actin microfilaments. Microfilaments are necessary for cell division. They are also used to extend the egg surface into small projections called microvilli, which may aid sperm entry into the cell (FIGuRE 7.4b). Also within the cortex are the cortical granules (see Figures 7.4B). These membrane-bound, Golgi-derived structures contain proteolytic enzymes and are thus homologous to the acrosomal vesicle of the sperm. However, whereas a sea urchin sperm contains just one acrosomal vesicle, each sea urchin egg contains approximately 15,000 cortical granules. In addition to digestive enzymes, the cortical granules contain mucopolysaccharides, adhesive glycoproteins, and hyalin pro- tein. As we will soon describe, the enzymes and mucopolysaccharides help prevent polyspermy—that is, they prevent additional sperm from entering the egg after the first sperm has entered—while hyalin and the adhesive glycoproteins surround the early embryo, providing support for cleavage-stage blastomeres. In mammalian eggs, the extracellular envelope is a separate, thick matrix called the zona pellucida. The mammalian egg is also surrounded by a layer of cells called the cumulus (FIGuRE 7.5), which is made up of the ovarian follicular cells that were nur- turing the egg at the time of its release from the ovary. Mammalian sperm have to get past these cells to fertilize the egg. The innermost layer of cumulus cells, immediately adjacent to the zona pellucida, is called the corona radiata

Spiral Cleavage in snails

The mitotic spindles, sketched in the early stages, divide the cells unequally and at an angle to the vertical and horizontal axes. Each successive quartet of micro- meres (lowercase letters) is displaced clockwise or counterclockwise relative its sister macromere (uppercase letters), creating the characteristic spiral pattern. this happens at the third cleavage Nodal is activated on the left ventral side of sinestral (left hand spiral) embryos and on the right ventral side of dextral (righ spiral) embryos. (C) The Pitx1 transcription factor, seen expressed in the embryo (above) is responsible for organ formation, as seen in the ventral view of the adults. Pitx1 active on same side as nodal, but also in middle too

Ectodermal Competence and the Ability to Respond to the Optic Vesicle Inducer in Xenopus

The optic vesicle is able to induce lens formation in the anterior por- tion of the ectoderm (1) but not in the pre- sumptive trunk and abdomen (2). If the optic vesicle is removed (3), the surface ectoderm forms either an abnormal lens or no lens at all. Most other tissues are not able to substi- tute for the optic vesicle (4). The head ecto- derm is competent to respond to the paracrine factors made by these brain bulges (the optic vesicles), and the head ectoderm receiving these paracrine factors is induced to form the lens of the eye. The genes for lens proteins become induced in the head ecto- derm cells and are expressed in these cells. The Rho-family GTPases are activated to control the elongation and curvature of the lens fibers (see Chapter 16; Maddala et al. 2008). Moreover, the prospective lens cells secrete paracrine factors that instruct the optic vesicle to form the retina. Thus, the two major parts of the eye co-construct each other, and the eye forms from reciprocal paracrine interactions. The head ectoderm is the only region capable of responding to the optic vesicle

diSHeveled and β-caTenin: SPeciFying THe MicroMereS

The specification of the micromere lineage (and hence the rest of the embryo) begins inside the undivided egg. The initial regulatory inputs are two transcription regulators, Disheveled and β-catenin, both of which are found in the cytoplasm and are inherited by the micromeres as soon as they are formed (i.e., at the fourth cleavage). During oogenesis, Disheveled becomes located in the vegetal cortex of the egg here it prevents the degradation of β-catenin in the micromere and veg2-tier macromere cells. The β-catenin then enters the nucleus, where it combines with the TCF transcription factor to activate gene expression from specific promoters. Several pieces of evidence suggest that β-catenin specifies the micromeres. First, during normal sea urchin development, β-catenin accumulates in the nuclei of those cells fated to become endoderm and mesoderm (FigUre 10.7b). This accumulation is autonomous and can occur even if the micromere precursors are separated from the rest of the embryo. Second, this nuclear accumulation appears to be responsible for specifying the vegetal half of the embryo. It is possible that levels of nuclear β-catenin accumulation help determine the mesodermal and endodermal fates of the vegetal cells (Kenny et al. 2003). Treating sea urchin embryos with lithium chloride allows β-catenin to accumulate in every cell and transforms presumptive ectoderm into endoderm (Fig- Ure 10.7c). Conversely, experimental procedures that inhibit β-catenin accumulation in the vegetal cell nuclei prevent the formation of endoderm and mesoderm Role of the Disheveled and β-catenin proteins in specifying the vegetal cells of the sea urchin embryo. (A) Localization of Disheveled (arrows) in the vegetal cortex of the sea urchin oocyte before fertilization (left) and in the region of a 16-cell embryo about to become the micromeres (right). (B) Dur- ing normal development, β-catenin accu- mulates predominantly in the micromeres and somewhat less in the veg2 tier cells. (C) In embryos treated with lithium chlo- ride, β-catenin accumulates in the nuclei of all blastula cells (probably by LiCl's blocking the GSK3 enzyme of the Wnt pathway), and the animal cells become specified as endoderm and mesoderm. (D) When β-catenin is prevented from entering the nuclei (i.e., it remains in the cytoplasm), the vegetal cell fates are not specified, and the entire embryo develops as a ciliated ectodermal ball.

Cortical rotation reveals grey crescent future site of dorsal lip of the blastopore in frog

The sperm nucleus has entered at one side and is migrating inward. At right, 80% into first cleavage, the cortical cytoplasm has rotated 30o relative to the internal cyto- plasm. Gastrulation will begin in the gray crescent—the region opposite the point of sperm entry, where the greatest dis- placement of cytoplasm occurs Fertilization can occur anywhere in the animal hemisphere of the amphibian egg. The point of sperm entry is important because it determines dorsal-ventral polarity. The point of sperm entry marks the ventral (belly) side of the embryo, while the site 180o opposite the point of sperm entry will mark the dorsal (spinal) side. The sperm cen- triole, which enters the egg with the sperm nucleus, organizes the microtubules of the egg into parallel tracks in the vegetal cytoplasm, separating the outer cortical cytoplasm from the yolky internal cytoplasm (Figure 11.2A,B). These microtubular tracks allow the cortical cytoplasm to rotate with respect to the inner cytoplasm. Indeed, these par- allel arrays are first seen immediately before rotation starts, and they disappear when rotation ceases. In the zygote, the cortical cytoplasm rotates about 30o with respect to the internal cytoplasm. In some cases, this exposes a region of gray-colored inner cytoplasm directly opposite the sperm entry point. This region, the gray crescent, is where gastrulation will begin.

Bar Coding

The use of a well-characterized gene sequence to identify distinct species, using the phylogenetic species concept. Use RNAseq combined with barcoding to follow how transcriptome changes over time ????????

C. elegans vulval precursor cells (VPCs) and their descendants

This pathway has both RTK and Notch signaling The formation of the vulva occurs during the larval stage from six cells called the vulval precursor cells (vPCs). The cell connecting the overlying gonad to the vulval precursor cells is called the anchor cell (fiGure 4.44). The anchor cell secretes LIN-3 protein, a paracrine factor (similar to mammalian epidermal growth factor, or EGF) that activates the RTK pathway (Hill and Sternberg 1992). If the anchor cell is destroyed (or if the lin-3 gene is mutated), the VPCs will not form a vulva and instead become part of the hypodermis or skin (Kimble 1981) But, the anchor cell itself forms from a notch pathway in which one of 2 cells randomly forms anchor cell, the other forms uterus cell. Depends on Lag2 (delta) or lin-12 (notch) presence. random increase in lag2 in one cell causes it to form anchor cell and the other to increase lin 12 and form the uterus cell. anchor cell then secretes LIN 3 in a gradient of concentrations, acing on the RTK pathway and making cells recieving the highest conc of LIn3 (the cell directly underneath) to form the central lineage, and cells further out to form lateral vulval lineages, and the farthest making hypodermis. Furthermore, Lin-12 secretion from the central cell uses notch pathway again to cause the lateral cells to be unable to turn int central cells, this being called lateral inhibition The six VPCs influenced by the anchor cell form an equivalence group. Each mem- ber of this group is competent to become induced by the anchor cell and can assume any of three fates, depending on its proximity to the anchor cell. The cell directly beneath the anchor cell divides to form the central vulval cells. The two cells flanking that central cell divide to become the lateral vulval cells, whereas the three cells farther away from the anchor cell generate hypodermal cells. If the anchor cell is destroyed, all six cells of the equivalence group divide once and contribute to the hypodermal tis- sue. If the three central VPCs are destroyed, the three outer cells, which normally form hypodermis, generate vulval cells instead. LIN-3 secreted from the anchor cell forms a concentration gradient, in which the VPC closest to the anchor cell (i.e., the P6.p cell) receives the highest concentration of LIN-3 and generates the central vulval cells. The two adjacent VPCs (P5.p and P7.p) receive lower amounts of LIN-3 and become the lateral vulval cells. VPCs farther away from the anchor cell do not receive enough LIN-3 to have an effect, so they become hypodermis Isnt this a notch pathway? C. elegans vulval precursor cells (VPCs) and their descendants. (A) Location of the gonad, anchor cell, and VPCs in the second instar larva. (B,C) Relationship of the anchor cell to the six VPCs and their subsequent lineages. Primary (1°) lineages result in the central vulval cells, secondary (2°) lineages constitute the lateral vulval cells, and tertiary (3°) lineages generate hypodermal cells. (C) Outline of the vulva in the fourth instar larva. the circles represent the positions of the nuclei. (D) Model for the determination of vulval cell lineages in C. elegans. the LIN-3 signal from the anchor cell causes the determination of the P6.p cell to generate the central vulval lineage (dark purple). Lower concentrations of LIN-3 cause the P5.p and P7.p cells to form the lateral vulval lineages. The P6.p (central lineage) cell also secretes a short-range juxtacrine signal that induces the neighboring cells to activate the LIN-12 (Notch) protein. This signal prevents the P5.p and P7.p cells from generating the primary central vulval cell lineage Most C. elegans individuals are hermaphrodites. In their early development, they are male, and the gonad produces sperm, which are stored for later use. As they grow older, they develop ovaries. The eggs "roll" through the region of sperm storage, are fertilized inside the nematode, and then pass out of the body through the vulva (see Chapter 8; Barkoulas et al. 2013). The formation of the vulva occurs during the larval stage from six cells called the vulval precursor cells (vPCs). The cell connecting the overlying gonad to the vulval precursor cells is called the anchor cell (fiGure 4.44). The anchor cell secretes LIN-3 protein, a paracrine factor (similar to mammalian epidermal growth factor, or EGF) that activates the RTK pathway (Hill and Sternberg 1992). If the anchor cell is destroyed (or if the lin-3 gene is mutated), the VPCs will not form a vulva and instead become part of the hypodermis or skin (Kimble 1981). The six VPCs influenced by the anchor cell form an equivalence group. Each mem- ber of this group is competent to become induced by the anchor cell and can assume any of three fates, depending on its proximity to the anchor cell. The cell directly beneath the anchor cell divides to form the central vulval cells. The two cells flanking that central cell divide to become the lateral vulval cells, whereas the three cells farther away from the anchor cell generate hypodermal cells. If the anchor cell is destroyed, all six cells of the equivalence group divide once and contribute to the hypodermal tis- sue. If the three central VPCs are destroyed, the three outer cells, which normally form hypodermis, generate vulval cells instead. LIN-3 secreted from the anchor cell forms a concentration gradient, in which the VPC closest to the anchor cell (i.e., the P6.p cell) receives the highest concentration of LIN-3 and generates the central vulval cells. The two adjacent VPCs (P5.p and P7.p) receive lower amounts of LIN-3 and become the lateral vulval cells. VPCs farther away from the anchor cell do not receive enough LIN-3 to have an effect, so they become hypodermis (Katz et al. 1995).

Human implantation

Tissue and germ layer formation in the early human embryo. Days 5-9: Implantation of the blastocyst. The inner cell mass delaminates hypoblast cells that line the blastocoel, forming the extraembryonic endoderm of the primitive yolk sac and a bilayered (epiblast and hypoblast) blastodisc. Days 10-12: The trophoblast divides into the cytotropho- blast, which will form the villi, and the syncytiotrophoblast, which will ingress into the uterine tissue to form the chorion. Days 12-15: Gastrulation and formation of primitive streak. Mean- while, the epiblast splits into the amniotic ectoderm (which encircles the amniotic cavity) and the embryonic epiblast. The adult mammal (ectoderm, endoderm, mesoderm, and germ cells) forms from the cells of the embryonic epiblast. The extraembryonic endoderm forms the yolk sac. The actual size of the embryo at this stage is about that of the period at the end of this sentence.

Translational regulation in oocytes

Translational regulation in oocytes. (A) In Xenopus oocytes, the 3′ and 5′ ends of the mRNA are brought together by maskin, a protein that binds CPEB on the 3′ end and eukaryotic ini- tiation factor 4E (eIF4E) on the 5′ end. Maskin blocks the initiation of translation by preventing eIF4E from binding eIF4G. (B) When stimulated by progesterone during ovulation, a kinase phosphorylates CPEB, which can then bind CPSF. CPSF can bind polyA polymerase and initiate growth of the polyA tail. PolyA binding protein (PABP) can bind to this tail and then bind eIF4G in a stable manner. This initiation factor can then bind eIF4E and, through its association with eIF3, position a 40S ribosomal subunit on the mRNA

Immunocyto/histo chemistry

Use antibody to localize protein that is cell type specific can stain specific cells in regions of embryo and see how they manifest in a stem cell? Mouse-brain slice stained by Immunohistochemistry. Immunohistochemistry (IHC) involves the process of selectively imaging antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see immunoperoxidase staining).[4] Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine (see immunofluorescence).

Summary of Experiments by Nieuwkoop and by Nakamura and Takasaki, Showing Mesodermal Induction by Vegetal Endoderm

When Nieuwkoop (1973) took embryonic newt cells from the roof of the blastocoel in the animal hemisphere (a region often called the animal cap) and placed them next to the yolky vegetal cells from the base of the blastocoel, the animal cap cells differentiated into mesodermal tissue instead of ectoderm. Thus, the blastocoel prevents the premature contact of the vegetal cells with the animal cap cells, and keeps the animal cap cells undifferentiated. If in contact, cells might commmunicate and the vegetal cells would induce the animal cap to mesoderm --took animal cap and put it on top of endoderm vegetal cells and animal cap became mesoderm--suggests induction Experiments by Pieter Nieuwkoop and Osamu Nakamura showed that the organizer receives its special properties from signals com- ing from the prospective endoderm beneath it. Nakamura and Takasaki (1970) showed that the mesoderm arises from the marginal (equatorial) cells at the border between the animal and vegetal poles. The Nakamura and Nieuwkoop laboratories then dem- onstrated that the properties of this newly formed mesoderm can be induced by the vegetal (presumptive endoderm) cells underlying them Nieuwkoop (1969, 1973, 1977) removed the equatorial cells (i.e., presumptive mesoderm) from a blastula and showed that neither the animal cap (presumptive ectoderm) nor the vegetal cap (presumptive endoderm) produced any mesodermal tissue. However, when the two caps were recom- bined, the animal cap cells were induced to form mesodermal structures such as noto- chord, muscles, kidney cells, and blood cells. The polarity of this induction (i.e., whether the animal cells formed dorsal mesoderm or ventral mesoderm) depended on whether the endodermal (vegetal) fragment was taken from the dorsal or the ventral side: ventral and lateral vegetal cells (those closer to the site of sperm entry) induced ventral (mes- enchyme, blood) and intermediate (kidney) mesoderm, while the dorsalmost vegetal cells specified dorsal mesoderm components (somites, notochord)—including those having the properties of the organizer. These dorsalmost vegetal cells of the blastula, which are capable of inducing the organizer, have been called the nieuwkoop center (Gerhart et al. 1989). Formation of a new gastrulation site and body axis by the transplantation of the dorsalmost vegetal cells of a 64-cell embryo into the ventralmost vegetal region of another embryo

D blastomere mollusks

When one removes the D blastomere or its first or second macromere derivatives (i.e., 1D or 2D), one obtains an incomplete larva lacking heart, intestine, velum, shell gland, eyes, and foot. This is essentially the same pheno- type seen when one removes the polar lobe (see Figure 8.10B). Since the D blastomeres do not directly contribute cells to many of these structures, it appears that the D-quad- rant macromeres are involved in inducing other cells to have these fates.

Chromosome-based sex determination in humans

XX female XY male X0 female, sterile XXY male XYY male males need a Y chromosome! ex) The Y chromosome carries a gene that encodes a testis-determining factor that organizes the bipotential gonad into a testis. This was demonstrated in 1959 when karyotyping showed that XXY individuals (a condition known as Klinefelter syndrome) are male (despite having two X chromosomes), and that individuals having only one X chromosome (XO, sometimes called Turner syndrome) are female. XXY men have functioning testes. Women with a single X chromosome begin making ovaries, but the ovarian follicles cannot be maintained without the second X chromosome. Thus, a second X chromosome completes the ova- ries, whereas the presence of a Y chromosome (even when multiple X chromosomes are present) initiates the development of testes

Sry more important in regulating testes development than sox9

XX mice transgenic for Sry develop normal male phenotype, while XX mice transgenic for the SOX9 gene have descended testes, but the seminiferous tubules lack sperm (due to the presence of two X chromosomes in the Sertoli cells) conflicting: For all its importance in sex deter- mination, the Sry gene is probably active for only a few hours during gonadal develop- ment in mice. During this time, it synthesizes the Sry transcription factor, whose primary role appears to be to activate the sox9 gene (Sekido and Lovell-Badge 2008; for other targets of Sry, see Web Topic 6.2). Sox9 is an autosomal gene involved in several develop- mental processes, most notably bone formation. In the gonadal rudiments, however, Sox9 induces testis formation. XX humans who have an extra activated copy of SOX9 develop as males even if they have no SRY gene, and XX mice transgenic for Sox9 develop testes (fiGure 6.7a-D; Huang et al. 1999; Qin and Bishop 2005). Knocking out the Sox9 gene in the gonads of XY mice causes complete sex reversal (Barrionuevo et al. 2006). Thus, even if Sry is present, mouse gonads cannot form testes if Sox9 is absent, so it appears that Sox9 can replace Sry in testis formation. This is not altogether surprising; although the Sry gene is found specifically in mammals, Sox9 is found throughout the vertebrate phyla. Indeed, Sox9 appears to be the older and more central sex determination gene in vertebrates (Pask and Graves 1999). In mammals, it is activated by Sry protein; Thus sry may act merely as a "switch" operating during a very short time to activate Sox9, and the Sox9 protein may initiate the conserved evolutionary pathway to testis formation. So, borrowing Eric Idle's phrase, Sekido and Lovell-Badge (2009) propose that Sry initiates testis formation by "a wink and a nudge.

Androgen deficiency-DHT deficiency

XY Testosterone levels fine missing 5-alpha reductase enzyme so no DHT Comes in range of outcomes depending on whether low or no DHT Testis and male plumbing, underdeveloped penis, prostate At puberty with high testosterone, overcome DHT deficiency, testis descend-penis enlarges Sterile

Hamster acrosome reaction

acrosomal membrane fuses with outermost sperm cell membrane

RU486 (abortion pill)

blocks Progesterone from binding to receptor, no endometrium forms and egg cant implant/breaks down

DNA Methylation

can selectively inhibit promotors suppress certain genes methylated DNA is associated with stable DNA silencing either (1) by interfering with the binding of gene-activating transcription factors or (2) by recruiting repressor proteins that stabilize nucleosomes in a restrictive manner along the gene. The presence of a methyl group in the minor groove of DNA can pre- vent certain transcription factors from binding to the DNA, thereby preventing the gene from being activated The addition of methyl groups to bases of DNA after DNA synthesis; may serve as a long-term control of gene expression. imprinting: the chromosomes from the male and the female are not equivalent; only the sperm-derived or only the egg-derived allele of the gene is expressed. This depends on which allele is methylated--methylated represses and causes he other allele to be expressed The methyl groups are placed on the DNA during spermatogenesis and oogenesis by a series of enzymes that first take the existing methyl groups off the chromatin and then place new sex-specific ones on the DNA

How is early lineage established?

cell polarity determines outside/inside cell Changing cleavage plane changes polariy of cell--polar cells make outside, nonpolar inside outside makes trophectoderm (placenta/nourishment outside embryo), inside makes ICM (inner cell mass) The decision to become either trophoblast or inner cell mass is the first binary decision in mam- malian life. Possible pathway ini- tiating the distinction between inner cell mass and trophoblast. (A) The Tead4 transcription factor, when active, pro- motes transcription of the Cdx2 gene. Together, the Tead4 and Cdx2 tran- scription factors activate the genes that specify the outer cells to become the trophoblast. (B) Model for Tead4 activa- tion. In the outer cells, the lack of cells surrounding the embryo sends a signal (as yet unknown) that blocks the Hippo pathway from activating the Lats protein. In the absence of functional Lats, the Yap transcriptional co-factor can bind with Tead4 to activate the Cdx2 gene. In the inner cells, the Hippo pathway is active and the Lats kinase phosphory- lates the Yap transcriptional co-activator. The phosphorylated form of Yap does not enter the nucleus and is targeted for degradation.

Mouse axis formation

endoderm surrounds embryo The mammalian embryo appears to have two signaling centers: one in the node (equivalent to Hensen's node and the trunk portion of the amphibian organizer), and one in the anterior visceral endoderm (ave; Beddington and Robertson 1999; Foley et al. 2000). The node appears to be responsible for neural induction and for the pat- terning of most of the anterior-posterior axis, while the AVE is critical for positioning the primitive streak (see Bachiller et al. 2000). Axis and notochord formation in the mouse. (A) In the 7-day mouse embryo, the dorsal surface of the epiblast (embryonic ecto- derm) is in contact with the amniotic cavity. The ventral surface of the epiblast contacts the newly formed mesoderm. In this cuplike arrangement, the endoderm covers the surface of the embryo. The node is at the bottom of the cup, and it has generated chordamesoderm. The two signaling centers, the node and the anterior visceral endo- derm (AVE), are located on opposite sides of the cup. Eventually, the notochord will link them. The caudal side of the embryo is marked by the pres- ence of the allantois. (B) Confocal fluorescence image of Cerberus gene expression, with the Cer- berus gene fused to a gene for GFP. At this stage, the Cerberus-synthesizing cells are migrating to the most anterior region of the visceral endoderm

A Burst of Protein Synthesis at Fertilization Uses mRNAs Stored in the Oocyte Cytoplasm

experiments show seawater treated with actinomyocin inhibits new mRNA transcription. So first burst of protein synthesis on stored mRNA!

vitellogenesis

formation of yolk at vegetal pole of egg Extent depends on how long embryo must go it on its own before feeding Proteins made outside and within oocyte Hormone regulated

Germ cells in chicks

germ cells travel through blood from brain to gonads why? If mutation in germ cells, maybe they wont migrate and thus survival of the fittest for the germ cells to ensure only the healthy ones migrate Something about Journey informs cells of their fate Gonad might not be mature enough to harbor cells

Nuclear RNA

has introns

Sperm structure

head: nucleus and acrosomal vessicle midpiece: To make compact nucleus exchange histones for Protamines--Histones maintained at high CpG density (cpg are promotor regions, have high conc of C and G) and Low DNA methylation--so ready for transcription?. The ATP needed to move the flagellum and propel the sperm comes from rings of mitochondria located in the midpiece of the sperm (see Figure 7.1B). In many species (notably mammals), a layer of dense fibers has interposed itself between the mitochondrial sheath and the cell membrane. This fiber layer stiffens the sperm tail. Because the thickness of this layer decreases toward the tip, the fibers probably prevent the sperm head from being whipped around too suddenly. Thus, the sperm cell has undergone extensive modifica- tion for the transport of its nucleus to the egg plasma membrane flagellum

B-catenin

helps make organizer

Driesch regulative development

in four cell stage, isolate each cell and they can each individually make a functional pluteus larvae

GFP-reporter cell line

infect cell embryos with a virus whos genes have been altered such that they express the gene for a fluorescently active protein such as green fluorescent protein, or GfP. When the infected embryonic cells are transplanted into a wild-type host, only the donor cells will express GFP; these emit a visible green glow. Variations on transgenic labeling can give us a remarkably precise map of the developing body, and thus can see specific fate of genes cells engineered to express GFP downstream from the promoter of the gene of interest-Advantage see in living material ex) Neural differentiation of mouse embryonic stem cells Fate mapping with transgenic DNA shows that the neural crest is critical in making the gut neurons. (A) A chick embryo containing an active gene for green fluorescent protein expresses GFP in every cell. The brain is forming on the left side of the embryo, and the bulges from the forebrain (which will become the retinas) are contacting the head ectoderm to initiate eye formation. (B) The region of the neural tube and neural crest in the presumptive neck region (rectangle in A) is excised and transplanted into a similar position in an unlabeled wild- type embryo. One can see it by its green fluorescence. (C) A day later, one can see the neural crest cells migrating from the neural tube to the stomach region. (D) In 4 more days, the neural crest cells have spread in the gut from the esophagus to the anterior end of the hindgut For example, Freem and colleagues (2012) used transgenic techniques to study the migration of neural crest cells to the gut of chick embryos, where they form the neurons that coordinate peristalsis—the muscular contractions of the gut necessary to eliminate solid waste. The parents of the GFP-labeled chick embryo were infected with a replica- tion-deficient virus that carried an active gene for GFP. This virus was inherited by the chick embryo and expressed in every cell. In this way, Freem and colleagues generated embryos in which every cell glowed green when placed under ultraviolet light (fiGure 1.16a). They then transplanted the neural tube and neural crest of a GFP-transgenic embryo into a similar region of a normal chick embryo (fiGure 1.16B). A day later, they could see GFP-labeled cells migrating into the stomach region (fiGure1.16C), and by 7 days, the entire gut showed GFP staining up to the anterior region of the hindgut -Sox1-GFP reporter (neural stem cells), Sox1 gene expressed in early neural lineage, use this promoter upstream of GFP, so cells turn green as they become neural

dsRNA mediated gene silencing

inject dsRNA, dicer enzyme complex binds to dsRNA and cleaves it into many siRNA. The antisense strand of siRNA interacts w a RISC complex protein, which then recognizes the target sequence in the tissue mRNA. This target mRNA then gets cleaved and these fragments can bind to more regions of mRNA, silencing them ex) Injection of dsRNA for E-Cadherin into the Mouse Zygote Blocks E-Cadherin Expression This phenomenon was demonstrated recently in Caenorhabditis eleganswhen dsRNA was injected into the worm and the corresponding gene products disappeared from both the somatic cells of the organism as well as in its F1 progeny

Cre-Lox

make a mutation only in the tissue in question or at a specific point in time using an enhancer (Cre-recombinase) For example, the tran- scription factor Hnf4α is expressed in liver cells, but it is also expressed prior to liver formation in the visceral endoderm of the yolk sac. If this gene is deleted from mouse embryos, the embryos die before the liver can even form. So, if you wanted to study the consequence of eliminating this gene's function in the liver, you would need to create a mutation that would be conditional; that is, you would need a mutation that would appear only in the liver and nowhere else. How can that be done? Parviz and colleagues (2002) accomplished it using a site-specific recombinase technology called Cre-lox. The Cre-lox system allows for control over the spatial and temporal pattern of a gene knockout and gene misexpression. The Cre-lox technique for conditional mutagenesis, by which gene mutations can be generated in specific cells only. Mice are made wherein wild-type alleles (in this case, the genes encoding the Hnf4α tran- scription factor) have been replaced by alleles in which the second exon is flanked by loxP sequences. These mice are mated with mice having the gene for Cre-recombinase transferred onto a pro- moter that is active only in particular cells. In this case, the promoter is that of an albumin gene that functions early in liver development. In mice with both these altered alleles, Cre-recombinase is made only in the cells where that promoter was activated (i.e., in these cells synthesizing albumin). The Cre-recombinase binds to the loxP sequences flanking exon 2 and removes that exon. Thus, in the case depicted here, only the developing liver cells lack a functional Hnf4a gene.

Gametogenesis

making germ cells

Mesoderm

middle germ layer; develops into muscles, and much of the circulatory, reproductive, and excretory systems muscle, notochord, connecting tissue

Sea urchant blastula

moves from circular shape to a flattened vegetal plate and cilliary tuft

Germ cells origin in mice

no germ plasm Germ cells arise at border Embryo/extraembryonic Accumulate in extraembryonic mesoderm Migrate along endoderm into genital ridge Primordial germ cell migration in the mouse. (A) On embryonic day 8, PGCs established in the posterior epiblast migrate into the definitive endo- derm of the embryo. The photo shows four large PGCs (stained for alkaline phosphatase) in the hindgut of a mouse embryo. (B) The PGCs migrate through the gut and, dorsally, into the genital ridges. (C) Alkaline phosphatase-staining cells are seen entering the genital ridges around embryonic day 11.

induction and competence

one group of cells changing the behavior of an adjacent set of cells, thereby causing them to change their shape, mitotic rate, or cell fate. This kind of interaction at close range between two or more cells or tissues of dif- ferent histories and properties is called induction The first component is the inducer, the tissue that pro- duces a signal (or signals) that changes the cellular behavior of the other tissue. Often this signal is a secreted protein called a paracrine factor. Paracrine factors are proteins made by a cell or a group of cells that alter the behavior or differentiation of adjacent cells. Cells of the respond- ing tissue must have both a receptor protein for the inducing factor and the ability to respond to the signal. The ability to respond to a specific inductive signal is called competence

mRNA

only exons

Cleavage cycle

only two phases, mitosis w active cdc2 bound to cyclin b complex (together protein kinase M), and the synthesis phase with inactive cdc2. Mitosis has G1, M, G2, and S phases with different cyclins, and a Go phase. cleavage cycle thus happens rapidly because much longer M phase (half cycle) compared to normal mitosis, where M phase is less than a fourth.

Ectoderm

outermost germ layer; produces sense organs, nerves, and outer layer of skin skin + nervous system

Gastrulation

reorganize the embryo Create 3 primary germ layers, characterized by cells changing shape and cell movement Ectoderm-epidermis, nervous system Mesoderm-muscle, skeleton, circulatory,mes. Endoderm-gut, respiratory tracts Establish anterior/posterior axis Facilitate interactions that guide organogenesis--making organs

Silencers

repress transcription and gene activity ex) neuron specific repressor sequence (NRSE) represses lacz gene function A silencer represses gene transcription. (A) Mouse embryo containing a transgene composed of the L1 promoter, a portion of the neuron-specific L1 gene, and a lacZ gene fused to the L1 second exon, which contains the NRSE sequence. (B) Same-stage embryo with a similar transgene but lacking the NRSE sequence. Dark areas reveal the presence of β-galactosidase (the lacZ product)

seminiferous tubule and spermatogenesis

sperm made from basal lamina surrounding seminiferous tubule from out to in--with primary spermatocytes near he outer circle and fully developed sperm on the inside lumen. lyedig cells surround seminiferus tubule and produce testosterone sertoli cells surround developing sperm in seminiferus tubule and nourish development. g Sertoli cells surround the incoming germ cells and organize themselves into the testis cords. These cords form loops in the central region of the developing testis and are connected to a network of thin canals, called the rete testis, located near the developing kidney duct (fiGure 6.3c,D). Thus, when germ cells enter the male gonads, they will develop within the testis cords, inside the organ. Later in development (at puberty in humans; shortly after birth in mice, which procreate much faster), the testis cords mature to form the seminiferous tubules Spermatogonia appear to take up residence in stem cell niches at the junction of the Sertoli cells (the epithelium of the seminiferous tubules), the interstitial (testosterone- producing) Leydig cells, and the testicular blood vessels. Adhesion molecules join the spermatogonia directly to the Sertoli cells, which will nourish the developing sperm The Desert hedgehog protein is found in the Sertoli cells of the testes, and mice homozygous for a null allele of dhh exhibit defective spermatogenesis w no germ cells and a smaller testes

Cleavage

subdivision of zygote into blastomeres cleavage comes before gastrulation, has rapid cell production

Endoderm

the inner germ layer that develops into the lining of the digestive and respiratory systems gut + respiratory tract

Formation of external genetalia

the mesen- chyme in the urogenital swellings secretes inhibitors of Wnt signaling. In the absence of Wnt signaling, estrogen modifies the genital tubercle into the clitoris and the labioscrotal folds into the labia majora surrounding the vagina. In males, however, androgens (such as testosterone and dihydrotestosterone) bind to the androgen receptor in the mesenchymal cells and prevent the synthesis of the Wnt inhibitors. Wnt signaling is permitted, and it causes the genital tubercle to become the penis and the labioscrotal folds to become the scrotum

The Pattern of Sex-Specific RNA Splicing in Three Major Drosophila Sex-Determining Genes

the result of the sex determination cascade in flies comes down to the type of mRNA processed from the doublesex transcript. If there are two X chromasomes, the transcription factors activating the early promoter of Sxl reach a critical concentration, and Sxl makes a splicing factor that causes the trans- DevBio11e_06.16 Date 03-08-16 former gene transcript to be spliced in a female-specific manner. This female-specific protein interacts with the tra2 splicing factor, causing dsx pre-mRNA to be spliced in a female-specific manner. If the dsx transcript is not acted on in this way, it is processed in a "default" manner to make the male-specific message. Interestingly, the doublesex gene of flies is very similar to the Dmrt1 gene of vertebrates, and the two types of sex determination may have some common denominators.

Chick Neurelation

the specification of the ectoderm is accomplished during gastrulation, primarily by regulating the levels of BMP experienced by the ectodermal cells. High levels of BMP specify the cells to become epidermis. Very low levels specify the cells to become neural plate. Intermediate levels effect the formation of the neural crest cells. Neurulation directly follows gastrulation gastrulation and neurulation graient where neurulation starts at hensens node anteriorly, and progressively moves posterior thus also moving hensens node posterior Sox family of tran- (A) DORSAL SURFACE scription factors (Sox1, 2, and 3). These factors (1) activate the genes that specify cells to be neural plate and (2) inhibit the formation of epidermis and neural crest by blocking the transcription and signaling of BMPs Primary neurulation can be divided into four distinct but spatially and temporally overlapping stages: 1. Elongation and folding of the neural plate. Cell divisions within the neural plate are preferentially in the anterior-posterior direction (often referred to as the rostral- caudal, or beak-to-tail, direction), which fuels continued axial elongation associ- ated with gastrulation. These events occur even if the neural plate tissue is isolated from the rest of the embryo. To roll into a neural tube, however, the presumptive epidermis is also needed 2. . Bending of the neural plate. The bending of the neural plate involves the formation of hinge regions where the neural plate contacts surrounding tissues. In birds and mammals, the cells at the midline of the neural plate form the medial hinge point, or mhP (Schoenwolf 1991a,b; Catala et al. 1996). MHP cells are reported to be firmly anchored to the notochord beneath them and form a hinge, which enables the creation of a furrow, or neural groove, at the dorsal midline 3. Convergence of the neural folds. Shortly thereafter, two dorsolateral hinge points (DLhPs) are induced by and anchored to the surface (epidermal) ectoderm. After the initial furrowing of the neural plate, the plate bends around the hinge regions. Each hinge acts as a pivot that directs the rotation of the cells around it (Smith and Schoenwolf 1991). Continued convergence of the surface ectoderm pushes toward the midline of the embryo, providing another motive force for bending the neural plate, causing the neural folds to converge (FigUre 13.6D; Alvarez and Schoen- wolf 1992; Lawson et al. 2001). This movement of the presumptive epidermis and the anchoring of the neural plate to the underlying mesoderm may also be impor- tant for ensuring that the neural tube invaginates inward, or into the embryo, and not outward (Schoenwolf 1991a). 4. Closure of the neural tube. The neural tube closes as the paired neural folds are brought in contact together at the dorsal midline. The folds adhere to each other, and the neural and surface ectoderm cells from one side fuse with their respective counterparts from the other side. During this fusion event, cells at the apex of the neural folds delaminate and become neural crest cells regulation of hinge points: To fold the neural plate means to bend a sheet of epithelial cells. How can a row of attached boxes be bent? While in the shape of a rectangular box (i.e., epithelial), they cannot; however, if the surface area of one side of each box in a region were reduced relative to its apposing side (creating the shape of a truncated pyramid), each of these cells should introduce a displacing angle with its neighboring cells and cause the row of boxes to bend. The MHP and two DLHPs are three regions of the neural plate where such cell shape changes occur (see Figure 13.6B- D). The epithelial cells in these locations adopt a "wedge-shaped" morphology along the apicobasal axis, one that is wider basally than apically (Schoenwolf and Franks 1984; Schoenwolf and Smith 1990). Similar to the bottle cells that initiate invagination during gastrulation (see Figure 11.4), localized contraction of actinomyosin complexes at the apical border reduces the size of the apical half of the cell relative to the basal compartment, a process known as apical constriction. This apical constriction pairs with the basal retention of nuclei to yield the wedge-shaped hinge point cells (see Fig- ure 13.6C,D; Smith and Schoenwolf 1987, 1988). In addition, recent findings suggest that the division rates in the dorsolateral domains of the neural plate are significantly faster than in ventral regions; this increases the cell density in the neural folds and adds a force that is hypothesized to promote buckling at the DLHP (McShane et al. 2015). The physical forces exerted by different regions of the neural plate have yet to be quantified, but at the cellular level hinge points are formed by (1) apical constriction, (2) basal thickening with retention of the nucleus within the basal portion of cells, and (3) cell packing in the neural folds. What regulates these cellular changes in the correct locations of the neural plate? BMP prevents MHP formation by regulating apical-basal polarity The morphogen Sonic hedgehog (Shh) is expressed in the notochord and is required for the induction of floor plate cells in the neural plate (Chiang et al. 1996), which in turn form the MHP. In the DLHP, Noggin appears to be critical for proper hinge formation. Noggin binds to and inhibits BMP--Activated BMP signaling leads to neural tube defects. BMP prevents MHP formation by regulating apical-basal polarity. This experiment demonstrates that high levels of BMP signaling inhibit MHP formation while low levels promote excessive folding at the midline. BMP activates SMAD pathway Specifically, when BMP signals are present in higher amounts, they promote the recruitment of proteins that serve to stabilize junctional proteins and maintain size equality between the apical and basal membranes, which prevents folding. In contrast, attenuation of BMP signaling (by Noggin) leads to a relaxation in these cell-to-cell junctions, which then permits api- cally restricted actinomyosin contractions and a shortening of the apical membrane. In summary, hinge point formation appears to center around the precise control of BMP signaling. BMP inhibits MHP and DLHP formation, whereas repression of BMP by Noggin enables DLHPs to form, and Shh from the notochord and floor plate prevent precocious and ectopic hinges from forming in the neural plate BMP signaling on the apical surface leads to an apically stabilized Par complex that segregates basal defining components such as LGL, all of which promotes an equal epithelial morphology. Attenuation of BMP signaling by Noggin can disrupt the division of these the compartments, leading to an expansion of typical basal components and a loosening of junc- tional complexes, all of which enables apical constriction forming of neural tube (fusing of neural crests): Expression of N- and E-cadherin adhesion proteins during neurulation in Xen- opus. (A) Normal development. In the neural plate stage, N-cadherin is seen in the neural plate, whereas E-cadherin is seen on the presumptive epidermis. Eventually, the N-cadherin-bearing neural cells separate from the E-cadherin-containing epidermal cells. (Neural crest cells express neither N- nor E-cadherin, and they disperse.) (B) No separation of the neural tube occurs when one side of the frog embryo is injected with N-cadherin mRNA so that N-cadherin is expressed in the epidermal cells as well as in the presumptive neural tube. The neural tube eventually forms a closed cylinder that separates from the surface ectoderm. This separation appears to be mediated by the expression of different cell adhesion molecules. Although the cells that will become the neural tube originally express E-cadherin, they stop producing this protein as the The neural tube eventually forms a closed cylinder that separates from the surface ectoderm. This separation appears to be mediated by the expression of different cell adhesion molecules. Although the cells that will become the neural tube originally express E-cadherin, they stop producing this protein as the neural tube forms and instead synthesize N-cadherin (FigUre 13.13a). As a result, the surface ectoderm and neural tube tissues no longer adhere to each other.

Meroblastic cleavage in centrolecithal fly egg

upon fertalization, the nuclei migrate to the periphery formiong an outer cell boundary. The outer cells become the somatic cells, and the outermost cells become the pole cells that will make germ cells

Antisense Approach

use complimentary mRNA (antisense RNA) to block translation can inject into an embryo to block function can halt disease causing proteins

Western Blot

used to detect specific proteins n brief, the sample undergoes protein denaturation, followed by gel electrophoresis. Synthetic or animal-derived antibodies are created that recognize and bind to a specific target protein, known as the primary antibody. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein. It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane. In addition, applying a dilution series of a purified protein of known concentrations can be used to allow a more precise estimate of protein concentration. The western blot is routinely used for verification of protein production after cloning. It is also used in medical diagnostics, e. g. in the HIV test or BSE-Test. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody Western analysis looks at actual proteins made, more useful in determining function of gene because sometimes mRNA genes still dont get expressed. Shows how much and when proteins get made during development

Sea urchant egg

vitellin envelope instead of zona pellucida. Surrounded by jelly layer

spermatids connected by cytoplasmic bridge

why? The spermatids that are connected in this manner have haploid nuclei but are functionally diploid, since a gene product made in one cell can readily diffuse into the cytoplasm of its neighbors Keeps cytoplasm constatnt and allows for synchrony of communication between cells Allows for sharing of diploid genome, so if one spot on genome is damaged the next cell can share its genome to help fix this hgh transgene ex) ??

Wnt signal transduction pathway

wnt--frizled/LRP5/6--desheveled--inhibits GSK3--inhibits beta catenin--affects transcription Wnt signal transduction pathways. (A) the canonical, or β-catenin-dependent, Wnt pathway. The Wnt protein binds to its receptor, a member of the Frizzled family, but it often does so in combination with interactions with LRP5/6 and Lgr receptors. During periods of Wnt absence, β-catenin interacts with a complex of proteins, including GSK3, APC, and Axin, that target Wnt for protein degradation in the proteasome. The downstream transcriptional effector of Wnt signaling is the β-catenin transcription factor. in the presence of certain Wnt proteins, Frizzled then activates Disheveled, allowing Disheveled to become an inhibitor of glycogen synthase kinase 3 (GSK3). GSK3, if it were active, would prevent the dissociation of β-catenin from the APC protein. So, by inhibiting GSK3, the Wnt signal frees β-catenin to associate with it's co-factors (LEF or TCF) and become an active transcription factor. (B,C) Alternatively, noncanonical (β-catenin-independent) Wnt signaling pathways can regulate cell morphology, division, and movement. (B) Certain Wnt proteins can similarly signal through Frizzled to activate Disheveled but in a way that leads to the activation of Rho GTPases, like Rac and RhoA. These GTPases coordinate changes in cytoskeleton organization and also through janus kinase (JNK) regulate gene expression. (C) In a third pathway, certain Wnt proteins activate Frizzled and Ryk receptors in a way that releases calcium ions and can result in Ca2+-dependent gene expression The planar cell polarity, or PCP, pathway functions to regulate the actin and microtubule cytoskeleton, thus influenc- ing cell shape, and often results in bipolar protrusive behaviors necessary for a cell to migrate. In vertebrates, this regulation of cell division and migration is important for establishing germ layers and for anterior-posterior axis extension during gastrulation and neurulation Ryk and calcium release has been demonstrated to be cleaved and transported into the nucleus, where it plays roles in mammalian neural development and C. elegans vulval development (Lyu et al. 2008; Poh et al. 2014). ex) Mutation in APC gene resuts in loss of APC protein, thus never being able to bind B-catenin and thus allowing B-catenin to always act as a cofactor, continuously stimulating transcription and leading to colon cancer ex) formation of ovaries w lack of Sry gene

Regulation of the Mitosis-or-Meiosis Decision by the Distal Tip Cell of the C. elegans Ovotestis

works in a notch pathway, where the distal tip cell is the delta signal and the glp-1 cells act as the notch receptor, where cells in contact with the distal tip delta cap undergo mitosis, and the other regions of the gonad has the cells undergoing meiosis. glp-1 mutation has all cells undergo meiosis

Downstream of B-catenin-Events Hypothesized to Bring about the Induction of the Organizer in the Dorsal Mesoderm

β-catenin can combine with a ubiquitous transcription factor known as Tcf3, converting the Tcf3 repressor into an activator of transcription. Expres- sion of a mutant form of Tcf3 that lacks the β-catenin binding domain results in embryos without dorsal structures Bcat and Tcf3 enhancer go on to enhance siamois and twin gene, and these proteins along with the Smad2 transcription factor activated by vegetal TGF-β family members (Nodal-related proteins, Vg1, activin, etc.) activate the "organizer" genes such as chordin, noggin, and goosecoid. The presence of the VegT transcription factor in the endoderm prevents the organizer genes from being expressed outside the organizer area. The β-catenin /Tcf3 complex binds to the promoters of several genes whose activity is critical for axis formation. Two of these genes, twin and siamois, encode homeodomain transcription factors and are expressed in the organizer region immediately follow- ing the mid-blastula transition. If these genes are ectopically expressed in the ventral cells, a secondary axis emerges on the former ventral side of the embryo; and if cortical microtubular polymerization is prevented, siamois expression is eliminated (Lemaire et al. 1995; Brannon and Kimelman 1996). The Tcf3 protein is thought to inhibit sia- mois and twin transcription when it binds to those genes' promoters in the absence of β-catenin. However, when β-catenin binds to Tcf3, the repressor is converted into an activator, and twin and siamois are transcibed (Figure 11.18). The proteins Siamois and Twin bind to the enhancers of several genes involved in organizer function (Fan and Sokol 1997; Bae et al. 2011). These include genes encod- ing the transcription factors Goosecoid and Xlim1 (which are critical in specifying the dorsal mesoderm) and the paracrine factor antagonists Noggin, Chordin, Frzb, and Cer- berus (which specify the ectoderm to become neural; Laurent et al. 1997; Engleka and Kessler 2001). In the vegetal cells, Siamois and Twin appear to combine with vegetal transcription factors to help activate endodermal genes (Lemaire et al. 1998). Thus, one could expect that if the dorsal side of the embryo contained β-catenin, this β-catenin would allow the region to express Twin and Siamois, which in turn would initiate for- mation of the organizer. Gilbert

Fate Mapping

•Cells of an early embryo are marked with a dye which does not spread or fade (now done with intracellular markers; originally external). •Determine which structures have dye in later stages. •Overall Results: Provided that the amphibian embryo is not disturbed, a region of the later embryo consistently develops from a particular region of the earlier embryo. The fate of the cells can therefore be determined by this technique. Cells dyed with Agar chips w fluorescent die ex) quail vs. chick embryo take a fluorescently tagged cell from a 24 hr old quail embryo and place it into the same spot on a chick embryo. The quail cells will develop into the neural tube of the chick embryo