Genes & Development Exam 2 Review

Homeotic genes

specify developmental plan for each segment (usually are transcription factors).

Avian, reptilian and mammalian gastrulation

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B-catenin protein is necessary for mesoderm and endoderm formation in sea urchin development

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Distribution of signals in the Xenopus blastula

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Dominant negative knockout approach

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Fish blastula fate map

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Neurulation: formation of neural tube, brain, and spinal cord

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Regulation of b-catenin levels by the Wnt signaling pathway

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The Wnt signaling pathway in early development

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The yellow crescent of the vegetal pole (B) becomes segregated to the B4.1 blastomere pair that are fated to become muscle cells

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Xenopus fate map

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Zebrafish neurulation

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chick: neurulation begins anteriorly while gastrulation is still progressing posteriorly.

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dorsal-ventral specification of the neural tube

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human embryo and placenta after 50 days of gestation

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human neurulation: neural tube formation is similar to birds

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left-right asymmetry in the developing human

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migration of endodermal and mesodermal cells through the primitive streak

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neural closure in the mammalian embryo occurs at several different sites

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neurulation in amphibian embryo

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neurulation in amphibians

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primary and secondary neurulation and the transition zone between them

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environmental influenced on neural tube defects and the role of folic acid

An epigenetic mechanism: folic acid metabolism can influence DNA methylation or histone modification

Vegetal view of the formation of microtubules in the shear zone during the first division of Xenopus embryo.

(A) 0.40 - microtubules are short and disorganized (B) 0.50 - microtubules are strongly expressed but no directionality (polarity) (C) 0.70 - parallel array of microtubules -Cortical rotation is blocked by inhibitors of microtubule formation.

Neurulation in birds

(A) Neural plate cells elongate in the wake of the regression of Hensen's node (become highly columnar). (B) Folding begins as the medial hinge point (MHP), located where the cells are anchored to the notochord. (C) The neural folds are elevated. The presumptive epidermal cells move towards the dorsal midline. -Asymmetric constriction of actin on the apical side changes cell shapes to promote MHP bending. (D) Convergence of the neural folds occurs. Cells at the dorsolateral hinge point (DLHP) become wedge-shaped and the epidermal cells push toward the center. (E) The neural folds are brought into contact w/ one another. A new group of cells form between the closed epidermis and new neural tube: the neural crest. -The neural crest cells disperse, leaving the neural tube separate from the epidermis

chick gastrulation 24-28 hours after fertilization

(A) primitive streak at full extension (24 hours) (B) two somite stage (25hr) (C) Four somite stage (27hr) (D) At 28 hours, primitive streak has regressed to the caudal region. (E) Regression of the primitive streak leaving behind the notochord in its wake -Hours after primitive streak achieved maximum length (about 18 hrs)

Modifications/defects in the axial skeletons of transgenic mice with specific Hox genes knocked out

(B) Knockout of Hox10 paralogs converts lumbar vertebrae into ribbed thoracic vertebrae. (C) Knockout of Hox 11 paralogs transforms sacral vertebrae into limbar vertebrae.

Folate (folic acid) in neural tube closure

(see image) -Folate binding protein is expressed just prior to neural tube closure and is located at the points where the two neural folds will come and coalesce together to form a closed neural tube.

The neuroepithelium of the new neural tube gives rise to neurons and glial cells

(see image) -Neuroepithelial cells are stem cells

Epithelial Cells

*Stationary cells* -arragged in a layer or sheet: an epithelium (form barriers) -if only one layer, a simple epithelium - if multiple layers, a stratified epithelium -Held together via specialized junctions (tight junctions, adheren junctions)

Rescue Experiments

*think Spemann Organizer* Step 1: Is the process in question disrupted upon removal of the agent (gene, mRNA, protein)? -Demonstrates that a component is necessary for an event to occur. Step 2: Can the disrupted process be rescued by reintroduction of the agent (gene, mRNA, protein)? - Demonstrates that the specific component is capable of "restoring" the event disrupted by the removal step. Step 3: Is the agent on its own sufficient to direct a particular process? -This is accomplished by introducing the component at a different time or location than normal. -If sufficient, it will initiate the event in question on its own! -Common approach: heterotopic transplantation

Blastomere fate and the induction of mesoderm in Xenopus

- At 32-celled stage, isolated animal and vegetal tier cells will develop into appropriate fates of ectoderm and endoderm (no mesoderm). - At 128-celled stage, descendants of the C tier blastomeres now develop according to their fate, into mesoderm, when grown in isolation.

Current concept of mesodermal specification

- Vg1 mRNA is translated and Vg1 induces the future dorsal mesoderm to transcribe genes coding for Wnt antagonists. -The VegT turns on expression of the transcription factor "eomesodermin" (Eomes) in the presumptive ventral-biased mesoderm. -Eomesodermin and Nodal proteins activate genes encoding VegT. -VegT expression: has gone from maternal mRNAs in the presumptive endoderm to nuclear (zygotic) expression in the presumptive mesoderm. -now a mesodermal marker

tissue formation in the early mammalian embryo

-After 7 days, the bilaminar germ disc is present -After 8 days, the trophoblasts divide into the cytotrophoblasts, which then from the villi and the syncrytiotrophoblasts that ingresses into the uterine tissue. -After 9 days, the epiblast splits into the amniotic ectoderm that encircles the anionic cavity and the embryonic epiblast that will form the embryo proper. -After 10-11 days, the adult mammal forms from the embryonic epiblasts. The extraembryonic endoderm forms the yolk sac

Neuroblast development in vertebrates parallel that of Drosophila

-At the beginning of neurulation in xenopus, the group of cells that have the potential to form neuroblasts accumulate two gene products: neurogenin and Neuro D -they are members of basic helix-loop-helix protein superfamily (bHLH). -they are transcription factors -they are orthologs of the Drosophila achete and sctute proteins

double gradient model

-BMP gradient specifies dorsoventral patterning: high BMP ventralizes structures. -Wnt gradient specifies anteroposterior patterning: high Wnt posteririzes the neural tube. -we need low Wnt and low BMP to make the brain

Tissue and germ layer formation in the early human embryo

-Cytotrophoblasts: from the villi -Syncytiotrophoblast: from the chorion -Amniotic ectoderm: split from epiblasts, will form the cell layer that encircles the amniotic cavity.

laterality axis formation in mammals

-Fgf induces secretion of nodalvesicular parcels (NVPs) from cell of the right side of the node -NVPs contain sonic hedgehog and retinoic acid -Movement of the NVPs to the left side by ciliary currents -NVP contents released which stimulate a rise in Ca2+ on the left -Rise in Ca2+ leads to nodal to pitx2 expression on left only -If inhibit FGF signaling, parcels are not secreted and L/R symmetry fails to develop

Gastrulation: Early movements in Xenopus

-IMZ forms (involuting marginal zone). Fated regions have distinct transcription factors being expressed. -Vegetal rotation pushes the prospective pharyngeal endoderm to the side of the blastocoel. -Vegetal endoderm movements push the pharyngeal endoderm forward, driving the mesoderm passively into the embryo and forward to the animal pole. -The ectoderm begins epiboly.

Role of induction in Axis formation

-Neural tube: is an organ rudiment -It always forms in association w/ an underlying notochord, laterally adjacent somites, and other mesodermal structures. -This set of mostly dorsal organ rudiments is referred to as the embryonic axis in vertebrates. Axis: a set of organs arranged in the correct spatial pattern. -2 types of signals are received by tissues involuting during gastrulation; planar signals and verticals signals

Rostral-to-caudal patterning the neural tube also morphogen gradients

-Neuromesodermal progenitors (NMPs) leave the tailbud and progress into the neural mesenchyme or the paraxial mesoderm mesenchyme, where they will give rise to the neural tube or somites respectively. -Opposing antagonistic morphogens of retinoic acid (RA) and Fgf/Wnt establish inverse gradients along the rostral-caudal axis, thus setting up graded positional instructions along the axis. -High Fgf/Wnt and low RA promote early preneural progenitors to be competent to respond to TGF-b and Shh dorsoventral signals, developing into the roof plate (RP) and floor plate (FP). -As the tailbud continues to elongate, preneural progenitors will experience low Fgf/Wnt and moderate RA, which broadens their competency to initiate gene regulatory programs specific for proneural progenitor populations.

How does the Spemann organizer get its special and powerful properties?

-Nieuwkoop found that the dorsal most vegetal cells of the blastula were capable of (responsible for) inducing the organizer. - these cells are known as the Nieuwkoop center; a dorsal signaling center. - it forms on the side of the vegetal region opposite sperm entry as a result of cortical rotation and is essential for normal development.

Demonstrating that Bicoid is both necessary and sufficient to induce head structure formation

-No head structures form in bicoid -/- embryos: bicoid appears necessary for head structures to form. -Get 2 heads, the second forming at the posterior end: thus bicoid is sufficient to form head structures. Normal head structures form: Bicoid + rescued bicoid-embroys, confirms bicoid is necessary. Get head structures forming in thorax region: thus bicoid is sufficient to form head structures.

Gravity determines the anerto-posterior axis of the chick

-Rotation of egg in oviduct results in the blastoderm tilted in the direction of rotation. -The posterior marginal zone will develop at the side of the blastoderm that is the uppermost. The primitive streak will be initiated from this zone.

Simplest interpretation for the function of Shh

-Shh induces expression of regulatory genes that direct ventral patterning of the neural tube (in a concentration dependent manner). -Shh regulates suppression of regulatory genes that direct dorsal patterning of the neural tube, such as Pax3 and Pax7, in a concentration dependent manner. -The expression levels of these regulatory genes control expression of proteins that determine the type of neuron that will develop. e.g. - motor, different types of interneurons NOTE: in addition to notochord and floor plate Shh patterning the neural tube from the ventral direction, the neural tube is also patternd by dorsalizing signals coming from the epidermal ectoderm.

Dorsoventral patterning of spinal cord

-Shh is a ventralizing signal -BMPs, Wnts are dorsalizing signals *Set up opposing and competing gradients -the competing gradients of ventralizing signals (Shh) and dorsalizing signals (BMPs, Wnts) setup the dorsoventral patterning of the spinal cord. Dorsal: -roof plate -dorsal interneurons -commissural interneurons Ventral: -ventral interneurons -motor neurons -floor plate

ectodermal structures and tissues

-Surface ectoderm: primary epidermis -Neural crest: peripheral neurons, pigment, facial cartilage Neural tube: brain and spinal cord

axis and notochord formation in the mouse (day 7)

-Tbx 6 is expressed in the anterior primitive streak and epiblast surrounding the Hensen's node. -Embryos treated with retinoic acid during early gastrulation often from two axis. -Two areas of Tbx6 expression. -Sonic hedgehog is expressed in the notochord. -Two areas of sonic headgehog expression shows a bifurcating notochord.

chick development

-The chick anterior-posterior axis is specified by gravity. -The developing blastoderm cell mass is lighter than the yolk mass, and hence "floats on top". -As the egg rotates in the oviduct during shell deposition, the blastoderm shifts to the side. -The more elevated region will become the posterior of the embryo.

The avian blastula consists of two layer cells:

-The upper "epiblast" layer is an epithelial sheet that will form 3 germ layers. -The lower "hypoblast" layer will only form the extraembryonic membranes! -The hypoblast forms by delamination of cells from the epiblast layer.

Cytoplasmic rearragement are induced by sperm entry in the fertilized egg of the tunicate Styela partita

-Unfertilized: a central grey cytoplasm enveloped by a cortical layer containing yellow lipid inclusions. -Within 5 mins of sperm entry in the vegetal hemisphere, the yellow cortical and clear cytoplasm migrates to vegetal pole. -As sperm nucleus migrates animally, moves yellow and clear cytoplasm with it. -Final position of yellow crescent is where the tail muscle will form.

Dorsal patterning of neural tube

-after neural tube closure, several BMPs are synthesized by the epidermal domains covering the dorsal midline, in the roof plate and in the adjacent portions of the neural tube. -BMPs induce formation of different types of dorsal neurons in a concentration dependent manner. -Will also, at the same time inhibit the differentiation of ventral types of neurons, in a concentration dependent manner. -Just as Shh antagonizes genes necessary for dorsal neurons, dorsal signals antagonize Shh and other ventralizing signals.

Cell movements during frog gastrulation

-bottle cells move inward to form the dorsal lip and mesodermal precursors involute. -Midgastrulation -- note the moving of the animal pole, formation of the archenteron and formation of dorsal mesoderm that will form notochord. -End of gastrulation: blatsocoel is gone, embryo is surrounded by ectoderm, all the ectoderm is internalized and mesodermal cells now exit between endoderm and ectoderm.

Avian gastrulation

-cell from the entire epiblast converge to the posterior midline through cell intercalation and convergent extension. -subsequently the cells move anteriorly to form a ridge: *the primitive streak** -Hensen's node forms at the anterior tip of the primitive streak and cells involute and ingress through the primitive groove into the blastocoel to form mesoderm. completion of gastrulation occurs first in the anterior portion and continues to progress in a posterior movement as Hensen's node regresses.

Extra-embryonic membranes

-chorion: closely associated with the shell, mediates gas exchange. -amnion: a fluid filled sac that surrounds the embryo, provides protection. -yolk sac: membrane next the yolk, develops an extensive blood vessel system to supply nutrients to the embryo. -allantois: store nitrogenous wastes, such as uric acid, last to form

neurogenesis in vertebrates and Drosophila both use lateral inhibition

-clusters of potential neuroblasts use lateral inhibition to select which cells are to eventually become a neuroblast. -key protein in vertebrates: delta (Xenopus: Xdelta) -homolog of the Drosophila delta protein -it is a membrane bound protein -it is the ligand for the notch receptor, which is the homolog of the Drosophila notch receptor

Noggin proteins induce dorsal structures

-embryos at top right were UV-radiated as oocytes: results in a ventralized embryo -Rescues: As increase noggin mRNA injection, see dorsal structure appear, then normal embryo and if too much, produce an embryo that is little more than head (bottom)

What molecules and pathways are involved in neural tube formation?

-expression of N- and E-cadherin proteins during neurulation in Xenopus (see image) (B) Inject N-cadherin into the bordering epidermal cells. -Result: No separation occurs between the neural tube and epidermis. *NOTE: neural crest cells down-regulate N-cadherin expression. -Result: NC cells disperse and migrate throughout the body to form a variety of structures (express neither N- or E-cadherin).

What causes Xnr-1 to be expressed only on the left side?

-if block cortical rotation at fertilization, no left-right axis. -Vg1, while found throughout the vegetal hemisphere, appears to get processed into its active form predominantly on the left side of the embryo. -Injection of Vg1 protein into a right side blastomere causes inversion of left-right asymmetry. -In mouse, nodal is initially expressed symmetrically, but subsequently, expression becomes limited to the left side.

Comparison of dorsalizing cytoplasmic determinant movements in Xenopus and Zebrafish

-in zebrafish, the organizer is called the embryonic shield.

Types of epithelial cell movements

-invagination: the action or process of being turned inside out or folded back on itself to form a cavity or pouch. -involution: a type of cell movement during gastrulation which involves the inturning or inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells. -convergent extension: a cellular process conserved across different species, as well as in different tissues and stages of development. -epiboly: a morphogenetic process that is employed in the surface ectoderm of anamniotes during gastrulation to cover the entire embryo. -delamination: formation and separation of laminae or layers; one of the methods by which the various blastodermic layers of the ovum are differentiated. this process consists of a concentric splitting of the cells of the blastosphere into an outer layer (epiblast) and an inner layer (hypoblast).

Human (mammalian) blastula and gastrula stages

-mammalian eggs are among the smallest in the animals kingdom. -human zygote is 1/1000 the volume of the xenopus egg.

Neuroblasts move out of the neuroepithelial layer and differentiate into neurons

-migrate towardsthe external lining membrane and form a progressively thicker layer of non-dividing cells, the mantle layer. -the neuroblasts will mature and produce cytoplasmic extensions called neurites. -the longest neurite is the axon. -neurites transmit signals away from the body of the neuron to muscle cells or other neurons. -dendrites transmit signals towards the body of the neuron.

right left symmetry

-most animals are bilaterally symmetrical relative to the median plane (divides up left and right). -externally, we are bilaterally symmetrical

What is the inducer generated by the notochord that directs ventral patterning of the neural tube?

-motor neurons emerge onto the ventrolateral sides. -cells closest to the notochord become the floor plate neurons. *RESULT: Sonic hedgehog, or any Shh-secreting cell, when placed adjacent to the neural tube, induces a second floor plate as well as two other sets of motor neurons.

What are the factors that segregate to the dorsal blastomeres?

-must be present in the embryo at the required concentration -must be present in the appropriate location -must be maternally supplied in before MBT (mid-blastula transition) -must be synthesized from embryo's genome if after MBT

a localized determinant must meet 2 criteria

-must be shown to be localized -must be capable of affecting the fate of the cell

What is sonic hedgehog?

-named after Drosophila homologue hedgehog, a known patterning gene -it is secreted glycoprotein -Shh activity resides in an amino terminal peptide that is released by autoproteolytic cleavage. -The Shh peptide is diffusible and its concentration decreases w/ diffusion distance.

There is a distinct anterior to posterior gradient of developmental maturity

-occurs in avian, mammalian teleost and reptilian embryos -While posterior parts of the primitive streak are still forming mesoderm, *the anterior end is already starting to form organs*.

In both vertebrates and Drosophila

-overexpression of delta inhibits formation of neurons -overexpression of a constitutively active notch receptor inhibits formation of neurons -inhibition of delta activity leads to excessive neuron production inhibition of notch signaling leads to excessive neuron production *Thus, notch/delta lateral inhibition signaling system is an evolutionarily conserved signaling system for neurogenesis.

How does the dorsal side of the neural tube get patterned?

-pax genes are regulatory genes normally expressed in the dorsal regions of the neural tube. -if remove notochord, Pax 3, 7 are expressed dorsally (normal) and ventrally (abnormal). -this suggests there is notochord/floor plate signal that prevents expression of Pax 3, 7 in the ventral region of the neural tube. -candidate signaling molecule is sonice hedgehog (Shh).

How does cortical rotation lead to b-catenin activation in the dorsal-most blastomere?

-proteins associated w/ the microtubules move at a faster rate and travel a greater distant during cortical rotation. -in vegetal pole, disheveled is associated w/ GBP, which is bound to kinesin. Wnt is stored in vesicles in the vegetal region as well. -Fast transport during cortical rotation moves both just subequatorial to the dorsal side.

The Nieuwkoop center

-specifies the dorsal side -transplantation studies demonstrated that the vegetal cells underlying the prospective dorsal blastopore lip region are responsible for initiating gastrulation.

Formation of Hensen's node from Koller's sickle

-the anterior Koller's sickle cells will form Hensen's node (and subsequently, notochord derivatives). -the posterior Koller's sickle cells remain as the posterior region of the primitive streak.

Development and patterning the spinal cord (human)

-the developing neurons send their axons towards the outside and create the marginal layer around the mantle layer. -because of myelination, the neurons of the marginal layer have a whitish appearance: this region will become the white matter of the spinal cord.

The strength of the dorsalizing activity was demonstrated through co-culturing specific blastomeres

-the dorsal most vegetal blastomere (D1) had the greatest ability to induce the animal pole cells to form dorsal mesoderm. This cell is the Nieuwkoop center.

Neurulation

-the morphogenetic events leading to the formation of the rudiment of the nervous system. -an embryo undergoing neurulation is called a neurula. -First evident as an area of dorsal ectoderm transforming into a plate of tall cells (columnar cells): Called the neural plate. -Neural plate will subsequently form into a hollow tube, the neural tube. -Neural tube gives rise to the central nervous system (CNS): brain (anteriorly) and the spinal cord. -Neural tube will also make contributes to the peripheral nervous system (PNS): nervous tissue located outside the skull and vertebral column. From neural tube closure through post-birth, human embryo averages 250,000 new neurons per minute until the CNS contains about 100 billion neurons.

anterior-posterior patterning in the mouse embryo

-the primitive streak and other posterior tisses are the sources of Wnt and BMP proteins. -the organizer and its derivatives (notochord) produce antagonists. -fgf8 gradient in the tailbud region of a 9-day mouse embryo. The highest is found near the tip. -Posterior patterning is integrated by the Cdx family of proteins, which in tern regulates the activity of the Hox genes.

The grey matter of the spinal cord acquires a dorsoventral pattern of distinct cell types. How does this pattern arise?

-the spinal cord is patterned by signals from adjacent tissues. -start w/ induction of the neural plate tissue -key inducer: Chordomesoderm, responsible for forming the notochord.

mesenchymal cells

-typically irregular shape (stellate) -are mobile and lack polarity -remain separated from each other by ECM -The ECM they interact with is composed of -collagens -proteoglycans -glycosaminoglycans (contain chondroitin and heparin sulfate) -hyaluronic acids -fibronectin

Birds: (chickens are the model organism)

-undergo discoidal meroblastic cleavage -cleavage results in two cell layers separated above the yolk. -cells are linked by tight junctions: are epithelial cells Result: A group of cells sitting on top of the yolk, referred to as the blastoderm.

Human development: from fertilization to implantation

-zygotic transcription begins in humans in isthmus region

Generalized gastrulation

1. Convert an essentially single layered blastula composed of two basic cell types (endoderm and ectoderm) into an embryo with 3 germ layers (endoderm, ectoderm, and mesoderm). 2. Bring previously spatially separated cell types into contact with new neighbors, allowing the bells to encounter new inductive signals that will affect later cell determination and differentiation. 3. Establish the rudimentary body plan ("Form an internal tube")

factors contributing to axis formation

1. Cytoplasmic determinants - bicoid, oskar 2. External cues - point of sperm entry - gravity -light 3. Inductive interactions

common features of gastrulation

1. Involves cell movement and reorganization 2. Gut (inner tube) forms from the archenteron, replacing the blastocoel -deuterostomes - vertebrates, sea urchins -protostomes - all other inverts) 3. The three primary germ layers are formed (endoderm, ectoderm and mesoderm) 4. Form the two basic cell types -epithelia: endoderm and ectoderm -mesenchyme: mesoderm Gastrulation involves a major epithelial/mesenchymal transition -mesoderm forms from an epithelial blastula

function of gastrulation

1. Place mesodermal cell in correct location between ectoderm and endoderm. 2. Surround the embryo with all ectodermal cells. 3. Bring the endoderm inside where it will line the internal organs. **Begins on the future dorsal side.**

The organizer

1. Self differentiates into dorsal mesoderm 2. Induces surrounding mesoderm to become paraxial mesoderm (whose preferred fate was ventral mesoderm). 3. Contributes to 4 types of dorsal tissue - pharyngeal endoderm: (forebrain/midbrain) -head mesoderm: pre-chordate (forebrain/midbrain) -dorsal mesoderm: induces notochord (hind brain/trunk) 4. Dorsalizes the ectoderm and induces formation of the neural tube. 5. Initiates movements associated with gastrulation.

model for generating left-right asymmetry in the chick embryo

1. Shh activates Cerberus, which stimulates BMPs to induce Nodal, which turns on expression of pitx2---Left side. 2. Activin leads to Fgf8 expression, a protein that blocks Cerberus expression---right side ; no cerberus, no nodal and no pitx2. 3. Lefty, expressed in midline, prevents any left sided Cerberus to cros over to the right side.

Summary for mesoderm induction

1. TGF-b signals are critical for mesoderm induction -VegT (transcription factor, tethered maternal mRNA) induces activin. -activin, derriere and nodal-related proteins (TGF-b family paracrine factors) induces Xbra (brachyury), a mesodermal transcription factor, at low moderate concentrations. *high activin levels induce goosecoid* 2. Concentration gradients of inducers provide patterning cues for the type of mesoderm. -an interactive gradient between VegT and b-catenin sets up a gradient of nodal related proteins (Xnr1, Xnr2, Xnr4) along the dorsal/ventral axis. -the organizer produces inhibitors of BMP signaling setting up a ventral/dorsal gradient of BMP. 3. BMPs provide the ventral mesoderm signal and induces ectoderm to become epidermis. *However, since the organizer blocks the BMP signal from reaching the ectoderm adjacent to it, this ectoderm, that above the organizer becomes neural tissue.*

Four signals are involved in mesoderm induction

1. Ventral vegetal signal (Ex: Veg T) -specifies ventral mesoderm 2. Dorsal side (Nieuwkoop center) vegetal signal(b-catenin, TGF-b proteins) -specifies the Spemann Organizer (O) and dorsal mesoderm 3. Organizer signal -dorsalizes the adjacent mesoderm by inhibiting the ventralizing action of the fourth signal 4. Ventral mesoderm ventralizing signal (BMPs)

Sea Urchin development

1. invagination 2. convergent extension 3. archenteron extension -The stomodeum forms as a depression on the oral side (ectoderm). -Fusion of the stomodeum and the archenteron produces the mouth of the pluteus larva.

primary neurulation in amphibians: two phases

1st phase: formation of the neural plate up to the keyhole stage. -cells elongate (become highly columnar) 2nd phase: closure of the neural plate into the neural tube. -cells shape changes to a triangular formation: apical shorten, basal surface elongates

What about non-dorsal mesoderm?

Antibody staining of phosphorylates Smad 1 shows a gradient of BMP4 inactivation as you approach the dorsal blastopore lip.

The pattern of neural tube gene expression depends on the concentration of Shh

Chick neural tube: -Pax7 (dorsal-most) -Olig2 (ventromedial) -Nkx2.2 (ventral-most) Remove and grow explant of intermediate neural tube in various concentrations of Shh. Intermediate neural tube: - explants express Pax7 in the absence of Shh. - when Shh is applied in increasing doses, Pax7 expression is lost and Olig2 and Nkx2.2 are induced in a dose-dependent manner *The result indicate that Shh represses Pax7, while inducing Olig2 and Nkx2.2 in a concentration dependent manner.* *It is also known that Nkx2.2 represses Olig2 transcription.

convergence and extension during zebrafish gastrulation

Chordamesoderm: hypoblast cells that will form the future notochord.

Expression of BMP antagonists in the mammalian node

Chordin expression during mouse gastrulation: anterior primitive streak Hensen's node axial mesoderm. Are BMP antagonists important? (B) Wild type embryo (C) Choridn gene knockout defective ear, but otherwise appears normal. (D) KO chordin ad noggin. -no jaw and a single centrally located eye that protrudes a large proboscis (nose)

Human development

Cleavage: 2 weeks Gastrulation: 1 week Organogenesis: 4 weeks Specialization & Growth: 7 months

RNA transport and localization in Xenopus eggs

Early pathway: transference of mRNAs to the mitochondrial cloud. The cloud and its contents move to the vegetal pole, and the mRNA cargo gets anchored to a small vegetal cortical region. Late pathway: mRNAs are at first evenly distributed, then associate with a cone of endoplasmic reticulum to the vegetal pole where they are anchored to a broad cap of vegetal cytoplasm.

Mesodermal induction

Functions of the vegetal cells: 1. Differentiate into endoderm 2. Induce the cells above them to become mesoderm Vg1 (TGFb family)-- dorsal mesoderm VegT (TF)-- ventral mesoderm

Primitive streak formation in the chick embryo

Gastrulation: occurs as Hensen's node regresses from anterior back toward the posterior end.

How does fertilization induce cortical rotation in amphibian eggs?

In frog eggs, fertilization induces a reorganization of the cyotplasm via cortical rotation resulting in the appearance of the grey crescent. *- the first cleavage furrow will bisect this grey crescent.*

cell movements during zebrafish gastrulation

In zebrafish, epiblasts will become ectoderm. Hypoblasts will become mesoderm and endoderm

What does the notochord do?

It serves as a source of midline signals that pattern surrounding tissues and as a major skeletal element of the developing embryo.

High BMP levels prevents MHP formation by dysregulating apical-basal polarity

MHP: medial hinge point -Electroporated cells are visible through GFP expression, nuclei are visible, Par3 is expressed, marker for apical cell membranes Conclusions: -Just right BMP signaling allows apical membranes to constrict in the MHP cells, forming the MHP. -Too much BMP signaling inhibits MHP formation. Maintains a stable apical membrane. -Too little BMP signaling promotes excessing MHP folding. Allows excessive apical constriction.

epithelial-mesenchymal and mesenchymal-epithelial transitions

Many major developmental events involve their EMTs or METs Molecular basis: 1. Changes in the expression pattern of cell surface molecules. 2. Presence of different types of ECM that govern cell-substrate adhesive interactions.

localized determinants

Molecules that are localized in specific cytoplasmic regions of the unfertilized egg or zygote and affect cell fate decisions by segregating into different embryonic cells and controlling distinct gene activities in these cells - effect of cutting eggs through different axis on sea urchin development

How do localized cytoplasmic determinants work?

Most are activators: Their presence causes the development of a particular structure. -They add (direct, induce) a new capability to a cell. As Inhibitors: They prevent cells from forming a certain embryonic structure. -the inhibition restricts the cells potential without adding any new capability. **Both types of determinants can be involved in directing a particular developmental pathway.

If neurogenin expression is hig, then neurogenin activates ____________, moving the cell to a neuronal fate, i.e., they are now neuroblasts.

NeuroD

Glial cells (non-neuronal cells of the nervous system)

Neuroepithelial also give rise to glioblasts, which give rise to glial cells. -There are 10x more glial cells than neurons in the vertebrate CNS. -Glial cells have supportive roles: insulation, nutrition, etc.

Neurulation endpoint

Neurulation divides the ectodermal layer into three sets of cells. 1. The internal positioned neural tube cells that will form the brain and spinal cord (neuroepithelium). 2. The external lying epidermal ectoderm that will form epidermis (also an epithelium). 3. The neural crest cells: Cells that form between the neural tube and the overlying epidermal ectoderm. -the neural crest cells migrate throughout the body and form pigment cells (melanocytes), peripheral neurons, the adrenal medulla and other structures.

What are the inductive interactions between vegetal and animal blastomeres that result in mesoderm formation?

Nieuwkoop: isolate animal and vegetal pole cells from an early blastula and cultured them either in isolation or together with each other. -isolated animal cap cells grown in culture by themselves: ONLY ECTODERM -isolated animal cap and vegetal cells grown together: Get ECTODERM, ENDODERM, AND MESODERM -isolated vegetal cells grown in culture in culture by themselves: ONLY ENDODERM

Is your liver, stomach, or heart directly in the midline?

No, but there locations are not random. -The liver is on the right -The stomach curves to the left -The heart loops to the left

left-right asymmetry in the zebrafish embryo

Nodal-related is expressed only on the embryo's left side. -Nodal cilia in Kupffer's node create a current that releases Ca2+, which stimulates notch and BMP4 pathways, leading to the activation of TF pitx2. -On the right side FGF signaling predominates.

Morphogen regulation of hinge point formation

Noggin is expressed in the dorsal neural folds. -Surface ectoderm (green) expresses BMP. -Apical constriction occurs only in cells experiencing low BMP and low Shh. -Noggin alleviated BMP repression of the hinge points. Shh and additional signals from the notochord induce MHP morphology. Just right [BMP]. -Shh is expressed ventrally in the notochord and floor plate. NOTE: Shh is required for the specification of floor plate.

What do these organizer products do?

Noggin: a secreted protein -induces dorsal ectoderm to become neural tissue -dorsalizes mesoderm -binds BMP4 and BMP2, preventing them from binding to their receptors *It is a BMP inhibitor* Chordin: -rapidly activated by the presence of b-catenin -similar to noggin, binds BMP4 and BMP2 *It is a BMP inhibitor* Follistatin: another BMP inhibitor *They all antagonize BMP signals (which are ventralizing)

Is a Shh concentration gradient being used to determine which cell types form along the ventral to dorsal direction of the developing spinal cord?

Observations: -during neurulation, Shh is expressed in chordomesoderm and its derivative, the notochord. -Shh is both necessary and sufficient for floor plate induction. Hence, the notochord induces the most ventral neural tube cells to become floor plate. -Mice lacking Shh do not form floor plate. - Ability of notochord to induce floor plate is inhibited by antibodies to Shh-after neural tube closure, the notochord no longer associates w/ the floor plate, however, the floor plate cells become a key source of Shh expression. *CONCLUSION: a concentration gradient of Shh is responsible for directing the ventral patterning of the neural plate (tube) cells.

Schematic representation of the chick and mouse vertebral pattern along the anterior-posterior axis

Orthologs: same gene, same function different species Paralogs: gene duplication within a species and then function diverged. *Note: all paralogs and orthologs are subsets of homologous genes.*

What is the "posteriorizing" localized cytoplasmic determinant?

Oskar -an mRNA transcribed from a maternal gene - In situ hybridization shows that oskar mRNA is only found in the extreme posterior pole of the egg -Oskar -/- mutants have no polar granules, no pole cells, and produce no gametes. -Injection of wildtype (WT) oskar (oskar +/+) into okscar -/- eggs result in pole cell formtion and gamete production -Thus, oskar is necessary and sufficient for pole cell formation

Where do oskar and bicoid come from?`

Oskar and bicoid mRNAs are synthesized in the maternal nurse cells and then transferred through cytoplasmic bridges to the appropriate regions of the developing oocyte. -Gurken protein is made by oocyte. -Torpedo (its receptor) on posterior follicle cells. -Binding stimulates PKA, which orientates microtubles such that growing end is at posterior. -Bicoid mRNA binds to dynein and moves to (-) anterior. -Oskar mRNA binds to kinesin I, moves to (+) posterior and the protein Oskar is anchored there by nanos mRNA.

What is the function of the polar granules?

Polar granules are large ribonucleoprotein complexes found at the posterior end of the embryo. The granules undergo characteristic changes in morphology during development, including the assembly of multiple spherical bodies from smaller precursors.

Two major ways of forming a neural tube

Primary neurulation: -cells surrounding the neural plate direct the neural place cells to proliferate, invaginate, fold, and punch off from the surface to form a hollow tube. Secondary neurulation: -the neural tube arises from a solid cord of cells that sink into the embryo and subsequently hollows out (cavaties) to form a hollow tube.

only pole cytoplasm can

RESCUE the infertile phenotype

Why do we need these wnt antagonists?

To prevent the dorsal region from being ventralized by wnts. -Xwnt8 is a mesodermal ventralizing signal that is antagonized by Ceberus, Frzb, Dickkopf, Tiki and IGF

Frzb

Wnt inhibitor -in situ localization of Frzb and chordin mRNA

______________, _______________, and ________ are released from microtubules in the future dorsal 1/3 of the 1-celled embryo.

Wnt11 vesicles, Dsh, and GBP

Are there other dorsalizing signals?

Wnts can promote formation of the most dorsal neuron types and are also important in inducing neural crest cell formation.

Heat and gut coiling in xenopus

Xnr1-pitx2 pathway is conserved across vertebrates.

Neurogenin

a TF required for neuronal differentiation. -neurogenin stimulates Delta expression -notch signaling inhibits neurogenin expression Result: some cells express high levels of neurogenin and delta, while other cells express low neurogenin.

Technique: using dominant negative mutants to suppress pathway activation

a dominant negative mutant activin receptor (TGF b family) blocks mesoderm induction. Result: no mesoderm or axial structures *- demonstrates that TGFb family signaling molecules are involved in mesodermal induction

nodal is

a gene expressed in the lateral plane mesoderm example: Xnr-1 (Xenopus nodal related-1) -normally, only expressed in the left side -maybe the earliest signla to be expressed only on the left side - if Xnr-1 is expressed on the right-hand side, the position of the heart and coiling of the gut are randomized.

localized cytosolic determinant

a molecule that affects the specification and eventual fate of a blastomere during normal development

b-catenin binding to Tcf3 turns Tcf3 from a repressor (when by itself) into an ____________ _____________.

activator complex

Embryonic induction

an interaction between non-equivalent cells that produce a new type of cell.

Xenopus

animal-vegetal pole develops without cues from the ovarian anatomy. -It has been observed that there is an uneven distribution of pigment and yolk, and it has been shown that there is an asymmetrical distribution of several cytosolic factors.

Birds

anterior portion, primary neurulation -caudual to 27th somite pair (everything posterior to the hind limbs) is by secondary neurulation

mice (probably humans)

anterior primary -secondary neurulation begins at or around the level of the 35th somite.

Head induction

anterior regions of head and brain are induced by endomesoderm. -endomesoderm consists of pharyngeal endoderm and head mesoderm. -endomesoderm is derived from the leading edge of the dorsal blastopore lip (the most anterior portion of the organizer). -head induction involved inhibition of both BMP4 and Wnt signaling: *the head inducers are Wnt and BMP4 antagonists*

What is meant by dorsalizing activity?

any material that promotes the formation of dorsal structures *- The dorsal side of the Xenopus embryo is determined by events associated with cytoplasmic rearrangements following egg activation.

Astrocytes

are associated with endothelial cell of blood capillaries and help form the blood-brain barrier.

Hox genes

are homeotic genes that specify structures along the antero-posterior axis. -activation begins when the mesodermal precursor cells are still in the epiblast. -are activated in and anterior to posterior fashion -The anterior border of Hoxb9 is more posterior than the anterior border of Hoxb4.

Differential expression of transcription factors define progenitor domains and derived cell types along the dorsoventral axis

as the neural tube develops, these progenitor zones expand and continue to diversify with their maturing gene regulatory networks until the full differentiative program is adopted and derived cell types emerge.

Developing an body plan

axis formation

polarized epithelial cells

basal surface adheres to the basal lamina of the basement membrane -apical surface often specialized for secretion or absorption -apical surfaces often have microvilli. **major point: Differences between the apical and basal surfaces provides for unidirectional transport and directional processes.*

End of gatrulation

blastocoel is gone, embryo is surrounded by ectoderm, all the endoderm is internalized and mesodermal cells now exit between endoderm and ectoderm.

primary mechanism of cleavage was

cell division

primary mechanism of gastrulation involves

cell movements

Microglial cells (CNS)

contribute to the brain's immune system

In amphibians, bilateral symmetry results from

cortical roation. -The Nieuwkoop center forms opposite the site of sperm entry.

Fertilization causes deep

cytoplasmic movements relative to the point of sperm entry that will move dorsalizing cytoplasmic determinants to the future blastopore regions.

Should be able to rescue embryos

depleted of the activity by adding the activity back

The role of VegT mRNA on germ layer formation in Xenopus

depletion of VegT mRNA results in the vegetal pole containing mesoderm, epidermis, and CNS. There is no endodermal region. Also, there is no CNS at the animal pole. -result is dorsalized embryo

Vg1 is involved in

determining dorsal mesoderm

VegT is involved in

determining mesoderm and endoderm

chordin mRNA is first expressed in region to become the

dorsal blastopore lip, then strongly in the dorsal blastopore lip, and then in tissues

noggin mRNA expression begins in the

dorsal marginal zone, to the dorsal blastopore lip, along the pre-notochord and then under the ectoderm

The organizer induces formation of

dorsal mesoderm by inhibiting the BMP ventralizing signal. -the organizer patterns the involuting cells during gastrulation such that the first involuting cells will form head structures and the last involuting cells will form trunk and tail structures.

What happens if you block cortical rotation?

dorsal structures like the notochord or CNS do not form.

How are the axes formed?

dorsal/ventral (anterior/posterior) superior/inferior (cranial/caudal) left/right

Nuclei stain strongly for b-catenin in cells of the __________ side, but stain very weakly in blastomere nuclei of the ___________ side.

dorsal; ventral

key feature of gastrulation

epithelial and mesenchymal transitions

Ceberus mRNA is sufficient to induce

ectopic head, heart, and liver structures. *ceberus is a wnt and BMP antagonist* -it is produced by the organizer and is a secreted protein that binds wnt, thereby inhibiting wnt signaling.

Posterior pole cytoplasm is capable of forming

ectopic pole cells -growing in a place it shouldn't be

concept of local determinants

eggs have an uneven distribution of cytoplasmic components, which will differentially segregate into daughter cells as the embryo progresses through cleavage division following fertilization.

Mesodermal development: only two in states 32-cell embryo

either dorsal or other marginal cells

Effect of retinoic acid (RA) on mouse embryos

exogenous RA alters Hox gene expression and vertebrae patterning.

Organogenesis

formation of the organ rudiments -Establishes the basic body plan, however, the organ rudiments do not yet function. -Eyes do not see and brain does not coordinate control of body functions. -To perform these tasks, the cells must acquire functional specialization.

dorsoventral

forms right after fertilization and is fixed before the first cleavage division (trigger is the point of sperm entry).

Formation of the animal-vegetal axis is related to the proximity of the

germinal vesicle to the animal pole and the accumulation of the mitochondrial cloud between the germinal vesicle and the vegetal pole. It will become the anterior-posterior axis.

organizer tissue is characterized by the expression of the DNA binding protein _____________.

goosecoid

1 in 23 flies had genes that

had to come from ectopic pole cell gametes

injection of pitx2 into the right side randomizes

heart placement and coiling of the gut

Evolutionary conservation of homeotic gene organization transcriptional expression in fruit flies and mice

homeotic genes: regulate the development of anatomical structures. hox genes: are evolutionarily conserved homeotic genes that specify structures along the antero-posterior axis. -Orthologs: -ONE hom-C cluster in Drosophila genome (chr3) -Paralogs: -FOUR hox gene clusters in mammals. -Genes w/ similar structures occupy the same relative positions on each of the four chromosomes. -The higher-numbered groups expressed later in development and more posteriorly.

Formation of the dorsoventral axis in amphibians

in amphibians, fertilization can occur anywhere in the animal hemisphere. The blastopore develops opposite the site of sperm entry. Hence, the sperm entry point determines the dorsal ventral axis.

animal-vegetal axis

originates during oogenesis. The animal-vegetal poles transform into the anterior posterior axis during gastrulation.

B-catenin is sufficient to

induce a new dorsal side

2nd transplantation

inject ectopic pole cells into posterior region of a secondary host with different genetic background and let embryos develop into adult flies.

Dorsal fate can be suppressed by

injection of wild-type GSK3 and a second dorsal axis can be induced by injection of a dominant negative GSK3.

Dickkopf (DKK1)

is a protein produced by the pharyngeal endomesoderm. -inhibition of Dickkopf protein results in a small deformed head with no forebrain. -german for "thick head" -it is a Wnt antagonist

The Spemann organizer

is capable of inducing the formation of a new embryonic axis. -The Spemann organizer is the dorsal mesoderm that occupies the dorsal lip of the blastopore of the early gastrula. - it is a unique area: it has its own fate (formation of the notochord). - it will induce lateral mesoderm to give rise to its structures. - it will induce the overlying ectoderm to form brain and spinal cord.

Beta-catenin

leading candidate for the signal of the Nieuwkoop center. -is cytoplasmic protein involved in the Wnt signaling pathway.

pitx2 is expressed only on the

left side of the embryo

Nodal is involved in

left-right asymmetry

internal organs show which kind to symmetry?

left-right asymmetry

ependymal cells

line the brain ventricle and contribute to spinal fluid formation.

How universal are these mechanisms in early vertebrate development?

localization of disheveled in vegetal cells of the sea urchin embryo

b-catenin is _______________ in the dorsal blastomeres of the Xenopus embryos.

localized

In situ hybridization of Macho-1 mRNA shows that it is

localized to muscle forming tunicate cytoplasm

human (mammalian) gastrulation

mammalian (and avian) development is similar to that of reptiles.

grey matter is derived from the ____________ ______________.

mantle layer. -it contains the cell bodies of the neurons, the dendrites and some unmyelinated axons.

Most localized cytoplasmic determinants are

maternally derived mRNAs

Cortical rotation moves

membrane bound organelles and dorsalizing activity from the vegetal pole to the dorsal side. -cortex containing dorsalizing activity moves 30 degrees. -organelles like mitochondria and ER move 60 degrees.

amphibians (xenopus)

mostly primary neurulation, but tail region is via secondary.

All neural plate cells express ____________________.

neurogenin

If cell surface expression of delta is high, neighboring cells, via their notch receptors, down regulate _______________ ________________, do not become neurons, move to a glial cell fate (become glioblasts).

neurogenin expression

Injected b-catenin induced a

new dorsal side

VegT is a required protein for

normal formation of mesoderm and endoderm (becomes more important at the midblastula transition).

left-right symmetry

not recognizable until early organogenesis, but forms early. It becomes observable as internal structures develop.

Two sets of transcription factors (from Wnt and TGFb signaling) activate

organizer gene expression

VgT expression prevents

organizer genes from being expressed elsewhere

Nurse cells in drosophila generate both the

oskar and the bicoid mRNA and then transfer them to the developing oocyte.

induction of a new embryo by transplantation of Hensen's node

oxt2- head region Hoxb1- trunk neural tube

IGF is a

paracrine factor that activates pathways that antagonist Wnt and BMP signaling cascades. That is, if some Wnt and BMP ligand binds to dorsal cells, IGF signaling prevents their ability to affect signaling at the cytosolic level.

The vegetal pole has

polar granules that stain intensely for RNA (observable).

Posterior pole cytoplasm is sufficient to cause

pole cell formation

Bicoid mRNA is necessary and sufficient for

pole cell formation -Bicoid -/- embryos have an abnormal body plan -Missing their head and thorax and the abdomen is abnormally long. However, bicoid -/- embryos still form a posterior end -Thus, in Drosphila, both the posterior and anterior ends carry posterior elements, i.e, posterior formation in the default.

Injection of the posterior cytoplasm polar granules into the anterior of a pre-blastoderm embryo resulted in

pole cell formation in the anterior region

Amphibian gastrulation

problem: the large vegetal, yolk laden mass is an impediment to invagination.

Gastrulation in fish

problem: very large yolks (birds will have same problem) **Cleavage is dicoidal, resulting in a disc of blastoderm at the animal pole

Histogenesis

process of functional maturation of embryonic organs -Refers to tissues -Tissues are cells and their extracellular matrix -A tissue usually performs a particular set of functions. -Tissues typically consist of more than one cell type. Example: nervous tissue consists of neurons, glial cells and extracellular matrix.

Danilchik and Denegre 1991

pulse-labeled vitelline proteins with a fluorescent marker and then activated the eggs either with sperm or electrical current. - they observed that there was swirl in the cytoplasmic layers on the future dorsal side of the embryo during the first cell cycle.

Gastrulation

reorganizes the blastula into a three layered embryo, from which a recognizable body plan will emerge.

epidermis forms in a

rich BMP and Wnt signaling environment

Fish have mostly

secondary neurulation

axis formation in the zebrafish embryo:Induction of the embryonic sheild

similar to Xenopus, b-catenin activates organizer (embryonic shield) genes

Errors observed in left-right symmetry

sitis inversus: complete reversal of regular asymmetry -occurs about 1 in 10,000, often asymptomatic Heterotaxis: only some organs reversed Isomerisms: normally asymmetrical organs are duplicated or missing.

Wnts are

soluble ligands (paracrine factors) that bind to receptors of the frizzled family.

Heterotopic transplantation can be used to test for

sufficiency

Cleavage in Drosophila

syncytial nuclear divisions followed by peripheral cellularization. (Laser confocal micrographs of stained chromatin). -polar granules detectable -cycle 13: Cellular blastoderm formation begins by ingression of cell membranes between nuclei. Completed at cycle 14. -pole cells (germ cell precursors) form in the posterior

Gastrulation will begin in

the grey crescent, the region opposite the point of sperm entry where the greatest displacement of cytoplasm occurs.

heterotopic transplantation experiments demonstrated that

the notochord has a key role in establishing of the dorsoventral pattern of the spinal cord. *The notochord and floor plate establish the ventral side of the spinal cord and direct motor neuron differentiation.*

Putting it all together: Who is responsible for establishing the anterior/posterior axis of the drosophila embryo?

the nurse cells

Conclusion of the inductive interactions between vegetal and animal blastomeres that result in mesoderm formation.

the vegetal cells must be inducing their neighboring animal pole cells to form mesoderm. This is embryonic induction.

If their action is blocked in vivo, then

there should be no dorsoventral axis induction. (necessary)

Heterotransplantation

transfer of tissue to a place that is not normally located.

The posterior marginal zone of the chick specifies the posterior end of the antero-posterior axis

we call this kind of experiment heterotopic transplantation.

The animal pole has

unique RNAs and proteins (no visible cues)

head and brain form in a

wnt and bmp free environment

trunk and spinal cord occur in the presence of

wnts and the absence of BMP signals

Oligodendrocytes (CNS)

wrap an insulating myelin sheath around neurons.