Discussion Questions Lecture 1-8
What are three examples of major derivatives to which each germ layer gives rise?
Ectoderm- the surface layer of the skin, the central nervous system, and the neural crest Mesoderm- Bones, facial muscles, notochord, kidney, heart, blood Endoderm- epithelium of GI tract and respiratory tract, and thyroid cells
What is an induction?
Effect on the developmental pathway of one group of cells by a substance displayed or secreted from another group of cells There is an inducer, the tissue that produces the signal (usually through paracrine factors) and a responder that is affected by the signals
What evidence from a non-drosophilid fly suggests a role for very early specification of anterior-posterior axis, perhaps by means of gradients
Ligature of midge (smittia) embryos very early in development resulted in missing middle segments The later this occurred, the more segments developed, but still not correct UV irradiation was used, RNase treatment removed RNA, and no head was formed
Define: Isometric growth
Proportionate growth; this occurs in animals such as lizards where all parts of the body grow at approximately the same rate
What are the major physiologial changes, and their outcomes, upon egg activation?
-Activation of NAD kinase, increase NADP+ and NADPH, increase in O2 consumption - Increase in pH -sperm entry, chromatin decondensation, pronuclear migration and fusion -activation of protein synthesis -initiation of DNA synthesis -mitosis and cleavage
Diagram the anatomy of a typical eukaryotic gene
-5' -Regulatory region including positive and negative cis regulatory elements -Promoter (tata) -Begin transcription -5' UTR -Begin translation (Start codon) -Introns and exons with some enhancers in the mix -Stop translation (stop codon) -3' UTR -stop transcription (terminator sequence) -3'
What embryological, genetic, and molecular data support the idea that early cleavage pattern determines later morphology in spiral cleaving snail embryos?
-Orientation of cleavage plane to left or right is controlled by cytoplasmic factors in oocyte -Crampton analyzed embryos of mutated snails with left spiral compared to right spiraled -Mutants' early cleavage differed from norm -Orientation of cells after second cleavage was different as result of diff orientation of mitotic apparatus -Direction of snail shell coilin is controlled by one pair of genes -Rare mutatns with left spiral were mated with wt right coiled -Direction of cleavage determined by genotype of snails mother -right left axis is defined by nodal family of paracrine factors -nodal activates genes on left (dextraly) or right side (sinisterly) -changing direction of cleavage at 8 cell stage changes location of nodal gene expression
What are some examples in which a failure to maintain genomic equivalence has detrimental concequences on human health.
-Roberts syndrom: Mutations in cohesion acetyltransferase ESCO2 that is essential for mitotic chromosome pairing. Results in abnormal mitosis and severe abnormalities. -Franconi anemia- Mutations affecting 13 DNA repair genes results in growth retardation, hyperpigmentation, and an extreme increase in cancer risk among other things.
Polar lobes are striking for their role in directing putative, cytoplasmic fate determinants to specific cells in embryos of some snail species. Propose an approach to identify candidate factors responsible for polar lobe activity and two different experiments that would allow testing functional roles for such candidates
-Transient cytoplasmic protrusions in very early development of many spirally cleaving species -They are associated with CD blastomeres and eventually incorporated into just the D blastomere -Perform in situ hybridization to tell where the mRNA is located temporally and spatially -Use ChIP-chip to immunoprecipitate out candidate factor mRNAs -Use microarray to measure relative expression of each candidate and its effect on polar lobe activity -Use ChIP-chip and immunoprecipitate out protein, using SDS page to separate candidate proteins based on molecular weight and density. Using mass spectrometry, identify chemical composition and identity of that protein
What is the fast block to polyspermy?
1-3 seconds after fertilization Na+ influx to egg increases membrane potential from -70mV to +20mV preventing additional sperm-egg fusions In marine inverts and frogs but not mammals evidence- Na+ concentration up, less polyspermy. Na+ conc down, more polyspermy
What is a forward genetic screen? How were forward genetic screens employed to isolate the genes involved in Drosophila early pattern formation? How does a "forward genetic" approach differ from a "reverse genetic" approach?
1.Phenotype driven screening for discovering genes and pathways required for a particular process. When an interesting phenotype is observed: -mutagenize adults w/ chem mutagen or ionizing radiation -breed mutagenized adults and sequentially intercross resulting offspring to parents whose progeny exhibit mutant phenotypes -recover mutants, breed until one trait likely -identify affected genes by genetic mapping/molecular cloning 2.reverse genetic approach is when a gene is found that might be important, they knock out that gene to see what happens
Define: Cell lineage
A cells prior history and all of the progeny that it generates
Define: Fate map
A diagram for a particular developmental stage that shows where each part will move to at later stages and where it will be in the end
What evidence supports the idea of genomic equivalence?
A fully differentiated mammary gland epithelial cell was taken from a finn dorset ewe and cultured and starved. This cell was fused with an enucleated egg from a black faced ewe and was allowed to gestate. This resulted in the development of a Finn dorset lamb, an exact clone of the finn dorset ewe that the epithelial cells were taken from.
Defects in 'developmental' signaling pathways are often associated with human morbidity and mortality. Provide examples for Wnt
APC protein acts as a tumor suppressor. The transformation of normal adult colon epithelial cells into colon cancer is thought to occur when the APC gene is mutated and can no longer keep Beta-catenin out of the nucleus, resulting in a buildup of polyps
What are primary cilia and what evidence suggests they are involved in hedgehog signaling?
Act as an 'antenna' for hedgehog signaling When hedgehog, the ligand for this pathway, binds the receptor called patched, smoothened and a lot of other proteins can inhibit an inhibitor called suppressor fuse. In which case, Glide (protein inhibited by suppressor fuse protein) is allowed to remain attached to microtubules via KIF7 in the cilium and ultimately move to the nucleus Evidence- Mutation in KIF7 causes defects in Hh dependent phenotypes
What are the three major classes of cell fate specification?
Autonomous, Conditional, and Syncytial
Defects in 'developmental' signaling pathways are often associated with human morbidity and mortality. Provide examples for Hh
Because cholesterol is critical for the catalytic cleavage of sonic hedgehog protein, the interruption of cholesterol causes severe problems. To teratogens known to cause cyclopia in vertebrates are jervine and cycopamine which block the synthesis of cholesterol. Mutations in this pathway can cause alobar holoprosencephaly, the failure to form two lobes in the forebrain during development
What are the similarities and differences between the Hedgehog and the canonical Wnt pathways? What does this imply about the logic of a putative ancestral pathway?
Both pathways prevent phosphorylation dependent proteolysis of key effectors- Ci in hedgehog and Beta-catenin in Wnt. Both of these kinases converts a DNA-binding protein from a repressor to an activator of transcription However, they use different receptors and proteins Because both pathways display inhibitory mechs, it suggests that there might be a close evolutionary relationship
How does one analyze cell lineage?
Cell lineage involves tracking a single cell from one point in development to a later point in development and the specific pathway that a cell takes to see what they become. One way to analyze this is to directly observe the living embryo using microscopy. Many of the same techniques used to analyze fate maps can be applied for all cell lineages
What are some of they typical features of sperm? What are some examples of species differences?
Cell membrane, acrosomal vesicle, nucleus, centriole, mitochondria, and microtubule surrounded axoneme -There is varying sperm head shape, hamsters are hook-like, drosophila are needle like -Varying numbers of microtubules- 9+2 is typical and required for mobility, 9+0 in eels, 9+2 and abnormal in humans
Distinguish between cell fate and cell potential.
Cell fate is the destined phenotype of a cell, given the normal conditions, whereas cell potential describes the total of all fates of a cell or tissue that can be achieved by any environmental manipulation.
How can one assay cell fate and cell potential? Give specific examples
Cell fate- with fate mapping (a technique where certain cells are stained and tracked during differentiation) Cell potential- Dreisch's experiment where he separated the four cells of a sea urchin embryo blastomere and each developed into a smaller yet normal larva.
Explain how chromatin state is regulated
Chromatin can be in one of two states- it can be compressed and wrapped around histones, or it can be open and unwound. Histone acetylation of lysine by histone acetyltransferase neutralizes its positive charge so it cannot interact with the negatively charged DNA. Deacetylation of lysine by histone deacetylase stabilizes the positive charge so that it can interact with the DNA. Histone methylation can either cause activation or inactivation of the DNA. CpG methylation on DNA directs HDAC (hystone deacetylace) to histones so transcription start sites become inaccessible.
Distinguish between cis-regulatory elements and trans-regulatory factors. How do they work together to promote or repress gene activity?
Cis- DNA motifs that are on the same DNA strands as the gene they regulate. In general, they control the efficiency and rate of transcription by attracting enzymes that break up nucleosomes and by stabilizing Transcription Initiation Complex Trans- DNA binding factors encoded by other loci that interact with basal transcription factors Together- Trans regulatory factors bind to cis regulatory elements and can promote transcription by promoting assembly of RNA polymerase II complex. Some cells require certain TFs to bind in order for transcription to occur.
Define: Allometric growth
Disproportionate growth; different parts of the body grow at different rates. For example, in humans, the head grows at a much slower rate than the rest of the body
What are the three germ layers?
Endoderm, ectoderm, and mesoderm
How does one construct a fate map?
Fate maps are constructed by GROUPS of cells, such as specific areas in the adult organism, through the means of in situ hybridization, vital dyes, and GFP tagging. One can construct a fate map by applying vital dye to the cells of interest, allowing the embryo to develop, and observe the cells as they progress. Fluorescent dyes can also be used as their intensity can still be detected in the progeny of cells many divisions later
What is the hedgehog pathway?
Hedgehog --| Patched --| Smoothened (inhibits PKA and Slimb which cleave Ci) --> Ci protein made activator (recruits TFs) --> Transcription of hedgehog responsive genes This prevents alobar holoprosencephaly (cyclopia)
Vertebrate teeth exhibit a diverse array of forms both between species, and within the mouth of any individual organism of a given species. Tooth development requires a series of inductions between oral epithelium and underlying mesenchyme. Propose a hypothesis to explain differences in tooth shape within a species, make a prediction base on your hypothesis that can be tested experimentally, and propose an experiment with necessary controls to test this prediction.
Hypothesis- Tooth shape is determined by permissive induction and the specialization of the mesenchyme cells beneath the oral ectoderm Prediction- If mesenchyme from where canines would normally develop is transplanted to an area where molars normally develop and visa versa, then canines will develop where the molars should and molars will develop where the canines should. Experiment- Transplant mesenchyme from the canine area to the molar area and visa versa Control- remove and replace canine and molar mesenchyme where it belongs Outcomes- -The oral epithelium above the transplanted canine tissue will develop into canines and the transplanted molar tissue will develop into molars as the mesenchyme is already specialized for certain shapes of teeth and just needs oral epithelium to develop.= fail to reject the hypothesis -Teeth develop as they would without the transplant = reject hypothesis
Explain how classical ideas of cell fate specification relate to a modern understanding of transcription factors and the molecular mechanisms of transcriptional regulation
In one cell, certain activator transcription factors may be necessary in order for transcription to occur for a certain gene. However, they may not be present in that cell, or maybe just not at that time. Cells that express key transcription factors (or other genes) typical of a particular lineage are sometimes said to be "specified" for that lineage, different from experimental definition of the "specified" state.
What is syncytial cell fate specification? Provide experimental evidence
Is prominent in insects as the nuclei divide within the egg, but the cell doesn't. It is a developmental pattern determined by an mRNA and resulting protein gradient between the anterior and posterior ends of the blastoderm. Example- In drosophila, bicod mRNA from the anterior side was added to the posterior side where nanos is normally produced. This resulted in the fruit fly developing two heads.
What evidence supports roles for specific cytokeletal proteins and mRNA sequence motifs in localizing particular transcripts during early snail development?
LR-S 1. Genetic mapping and chromosomal fluorescence in situ for the chirality locus 2. Removes 5' UTR using morpholine 3. To determine how mRNA gets there, knockout diff parts of the mRNA 4. Found that a very small part of 3'UTR is vital for localization
Define: Differentiation
Occurs as a cell progresses in development. A differentiated cell produces proteins specific to that cell that are different from its previous stages and surrounding cells Results in an increasingly specialized phenotype
Clasically, what is the difference between a 'permissive' and an 'instructive' induction? provide examples
Permissive-When the responding tissue has already been specified and needs only an environment that allows the expression of these traits; eg, when newt gastrula oral ectoderm is replaced with that of a frog, the resulting newt has frog tadpole suckers. Conversly, when the frog oral ectoderm is replaced with that of a newt, a tadpole with newt balancers results Instructive- When a signal from the inducing cell is NECESSARY for initiating new gene expression in the responding cell; eg, when the inducers from the anterior neural plate of the eye induce Pax6 expression in the anterior ectoderm, it initiates observable lens formation
What are the ways used by sea urchins to ensure species-specificity of fertilization?
Sperm chemotaxis to egg mediated by small peptides (resact and speract) which is found in the jelly layer of eggs. Received by resact receptor of sperm, leading to conversion of GTP to cGMP which activates Ca++ channel opening, increasing Ca++ concentration and movement. As a result, sperm swim up the concentration gradient. Acrosomal reaction (Na_H exhanger is activated, phospholipase produced of phospholipid IP3, causing internal Ca2++ release, and the fusion of acrosome memebrane with sperm membrane, placing bindin on the outside, and creating a tip to bind with the cell) is caused by the activation of the acrosome by specific carbohydrates, which varies across species Also, the protein on the tip of the sperm, called bindin, that allows it to bind to the egg is species specific, and has a high rate of evolution as does its receptor. This prevents different species' sperm from binding to the egg (proof in bindin coated bead experiment with eggs)
Defects in 'developmental' signaling pathways are often associated with human morbidity and mortality. Provide examples for TGFbeta
TGFbeta can both suppress and metastasize tumors. Tumor cells often escape from the antiproliferative effects by mutational inactivation of the pathway-
What is the midblastula transition and what differs about early cleavage divisions before and after this event in the frog? Do all animals have a midblastula transition?
The addition of G1 and G2 phases to cell cycle shortly after the 12th cleavage Marked by exhaustion of maternal regulators of cyclin B Transition to zygotic transcription Loss of synchronized cell divisions because cells synthesize different regulators of MPF, mitosis promoting factor In most species, both the initial rate of cell dicision and the placement of the blastomeres with respect to one another are under control of the proteins and mRNAs stored in the oocyte. Only later do the rates of cell division and the placement of cell come under the control of the newly formed genome In many but not all animals, such as drosophila and xenopus
What is sorting out?
The idea that tissues can dissociate into their smaller subunits, which will each sort out in its own region.
Define: Cell fate
The normal phenotype of a cell or its progeny during a future stage
Define: Morphogenesis
The process by which biological structures are formed by changing the relationships of cells or tissues
What hypothesis was proposed to explain the morphogenic process of "sorting out" and how was this hypothesis revised in light of modern molecular data?
The spontaneous sorting out of cell types win vitro and in vivo reflects differences in adhesivity across cell types. Now it is believed that cadherin family proteins involved in adhesion often mediate sorting out behavior
What are germ layers?
The three embryological groups of cells formed and organized during gastrulation. They interact with each other to generate all the materials and organs of the body through organogenesis.
Why are fate maps important?
They map larval or adult structures onto the region of the embryo from which they originate. They constitute an important foundation for experimental embryology, providing researchers with information on which portions of the embryo normally become which larval or adult structures
What are some of they typical features of eggs? What are some examples of species differences?
Typical mature egg contains proteins, ribosomes, tRNA, mRNA, protective chemicals. There is a nucleus, and after fertilization, a protective envelope forms -At time of fertilization, human and most animals' eggs are arrested in the 2nd metaphase until sperm fusion. In many insecnts and starfish, the egg is arrested in 1st metaphase. In anemones and sea urchins meiosis is complete -Inverts have a protective vitelline envelope after fertilization, vertebrates develop a chorion
In your second high profile paper about Ntf you want to understand its function during development. Fortunately, technologies have advanced to the point that you can make a loss-of-function mutant in ntf with relatively little hassle. Propose an approach to identify direct targets of Ntf and the consequences of losing Ntf activity for downstream gene expression
Use Ch-IP- it allows one to assay if a specific TF binds to the cis-regulatory region of a specific gene 1. Treat intact cells with formaldehyde to link protein to DNA 2. Shear nucleoprotein complexes mechanically 3. Immunoprecipitate with antibody to the TF 4. Rinse out the remaining DNA and analyze with PCR amplification You should end up with antibody, Ntf protein, and the gene it regulates (if correct)
How might you identify the cis regulatory regions or specific cis-regulatory elements that control ntf expression?
Use a GFP reporter gene 1. clone each section of cis-regulatory regions I suspect react with ntf by PCR, sub clone each piece 2. add fluorescently labeled GFP gene on each piece 3. transgenically insert clones with GFP back into the 1-cell stage and let zygote develop 4. using an assay, deduce which specific 'sub piece' interacts with ntf
Imagine that you have discovered a new, candidate transcription factor, Ntf, in your favorite biomedical model organism and you find by RT-PCR that ntf mRNA is expressed differentially between stages and tissues. How could you further assess the temporal and spacial dynamics of ntf transcription during development?
Use in situ hybridyzation, gives temporal and spatial information 1. synthesize a RNA probe (riboprobe) that is antisense to the ntf transcript with fluorescein 2. fix embryo/tissue with paraformaldehyde 3. partially digest with proteinase-K 4. incubate with riboprobe 5. wash out riboprobe 6. incubate with anti-fluorescein antibody conjugated to alkaline phosphatase 7. provide a substrate will precipitate -assay it frequently at specific time intervals
What is the impact of chromatin configuration on transcription? Support with examples
When DNA is wound around the histones, it is inactive, meaning it is inaccessible to RNA polymerase, and therefore transcription cannot occur. When DNA is open, it is active, and transcription can occur. This is critical in maintaining the regulation of gene expression.
What is autonomous cell fate specification? Provide experimental evidence
When a cell inherently 'knows' what to become, as the blastomere has already inherited a set of transcription factors that regulates gene expression and direct the cell into a particular path of development. It already contains morphogenic determinants which will influence its development. Evidence- The separation of the trochoblast (ciliated) in the mollusk, Patella. Even in the isolated culture, these cells divided and still became ciliated at the correct time
What is conditional cell fate specification? Provide experimental evidence
When a cell's fate is determined by its surroundings and the signals it receives from other cells. It is largely specialized by paracrine factors secreted by its neighbors. Example- shows that when normal back cells are transplanted into the belly region of the belly region of a Leopard frog blastula, the cells form normal belly tissue
Defects in 'developmental' signaling pathways are often associated with human morbidity and mortality. Provide examples for RTK
When there is a mutation in the fibroblast growth factor receptor pathways, dwarfism results. This is because it regulates the growth of bones and epethelial cells
Give an example of "sorting out"
When skin cells are taken from E15 mouse, dissociated, and allowed to aggregate for 72 hours, skin and hair follicles reformed.
What is the slow block to polyspermy?
Within ~10 seconds Bindin meets the sperm receptor -->TK-->PLC-->PIP2-->DAG , also leadidng to IP3 binding to IP3 receptor on ER which causes Ca++ wave initiating from site of sperm entry Calcium wave initiates cortical granule-plasma membrane fusion, releasing serine proteases (cleaves protein posts tethering envelope to membrane) hyaline (sucks up H2O, forming gel and expanding) and perioxidatives (crosslinking proteins to make envelope impervious) In most species including mammals
What is the canonical Wnt pathway?
Wnt --> Frizzled (LPR co-receptor) --> Disheveled -(inactivtates)-| GSK-3 (destruction complex) -(destroys)-| Beta-catenin -->Transcription of wingless dependent genes Affects polarity of hair
