Vertebrate Early Development

Question 1

The first step is to generate a multicellular structure that is capable of being
patterned into multicellular tissues.

Answer 1

In Xenopus, fertilization triggers a series of rapid cleavage divisions that creates distinct tissues within the two visibly different animal and vegetal hemispheres:

Question 2

The specific problem: how does a mass of cells such as the blastula become
transformed into a free-swimming tadpole?

Answer 2

This then reduces the problem slightly to how the mass of cells within the blastula is transformed into the free-swimming tadpole.

Question 3

Step 1: Formation of the embryonic Germ Layers – ectoderm, mesoderm and endoderm

Answer 3

• > Cells arising from within the Vegetal and Animal Hemispheres are differentiated with respect to one another.
• > Cells derived from the Vegetal (lower) Hemisphere SIGNAL to cells derived from the Animal Hemisphere
• > Equatorial Animal Hemisphere cells (in the Marginal Zone) close to the Vegetal Hemisphere become mesoderm.
• > Animal Hemisphere cells further away from the Vegetal Hemisphere become ectoderm.
• > Vegetal Hemisphere cells become endoderm.

Question 4

The unfertilized egg is polarized such that the yolky Vegetal Hemisphere contains molecules missing
from the Animal Hemisphere – localized, yolk-associated molecules – “cytoplasmic determinants”

Answer 4

In Xenopus, this egg is partitioned into two types of cytoplasm by gravity. The lower, “vegetal” hemisphere is beige and contains dense yolky platelets and a large number of RNA and protein molecules – so-called cytoplasmic determinants. The upper, animal hemisphere, is opaque and contains pigment granules that give it a dark brown colour.

Question 5

A vegetally-localized determinant: mRNA encoding the T-box transcription factor VegT. VegT protein binds to the promoters of genes encoding molecules related to the secreted morphogen Nodal (Xnr genes) and activates their transcription

Answer 5

After fertilization and the initial rounds of cell division, one vegetally localised cytoplasmic determinant, VegT mRNA, is translated to produce the VegT protein, which is a transcription factor.
Within these vegetally-positioned cells, the VegT transcriptoin factor binds to the transcription regulatory elements of genes encoding Nodal-related morphogens, the Xnr genes, and promotes their transcription.

Nodal-related proteins then diffuse from the VegT-expressing cells and into the equatorial region of the embryo.

Question 6

Cells closest to the source of Nodal-related proteins become endoderm
Cells at intermediate distances from the source of Nodal-related proteins become mesoderm
Cells furthest from the source of Nodal-related proteins become ectoderm

Answer 6

In the equatorial region of the embryo, Nodal-related proteins induce mesoderm in a broad band of equatorial tissue, and closest to the VegT-expressing vegetal tissue, a narrow band of endoderm is also induced within cells experiencing the highest concentration of Nodal protein. Cells furthest away from the VegT-expressing tissue do not receive Nodal-related signals and retain their ectodermal fate.

Question 7

The presence of localized determinants such as VegT in the Vegetal Hemisphere explains
how mesoderm and endoderm are formed by the process of induction, but:

Answer 7

This observation does not explain how endoderm, mesoderm and ectoderm become
organized and patterned within the embryo along antero-posterior and dorsal-ventral axes.

Tissue organization and axial patterning are established during the process of gastrulation.

Gastrulation is prefigured by a symmetry-breaking event that occurs at the time of fertilization,
leading to the formation of the Nieuwkoop Centre in the blastula.

The Nieuwkoop Centre induces the formation of the Spemann-Mangold Organizer, which then
initiates gastrulation.

Question 8

The future dorsal side of the embryo develops from a region of the fertilized egg that is
opposite the site of sperm entry. A 30o rotation of cortical cytoplasm redistributes and
activates maternal dorsalizing factors

Answer 8

Components of the Wnt signalling pathway are activated on the side of the fertilized egg
opposite the site of sperm entry, within the Nieuwkoop Centre.
The Nieuwkoop Centre will eventually induce the Spemann-Mangold Organizer, which produces
signals that pattern the antero-posterior and dorso-ventral axes of the embryo.

Question 9

Within the Nieuwkoop Centre, Wnt11 mRNA and Dishevelled protein are transported along
microtubules, away from the vegetal pole towards the animal hemisphere of the egg.

Answer 9

After several cleavage divisions, activated b-catenin accumulates in the nuclei of cells on the
future dorsal side of the embryo, causing transcription of Wnt-pathway target genes.

Question 10

Canonical Wnt signalling causes the accumulation of beta-catenin transcription factor in the nuclei of target cells

Answer 10

Consequently, beta-catenin protein accumulates in the cell nuclei, promoting transcription of Wnt pathway target genes in the cells of the Nieuwkoop Centre.

Question 11

Wnt target genes activated by beta-catenin include the Nodal-related genes already mentioned as being activated by VegT in the vegetal hemisphere.

Answer 11

The presence of beta-catenin on the dorsal side then -increases Xnr gene expression on the future dorsal side, creating what is essentially a dorsal-vegetal gradient of Nodal-related protein underneath the developing mesoderm.
The Nieuwkoop Centre: a region of vegetal tissue within the blastula
where b-catenin and Nodal are both present

Question 12

A combination of b-catenin and VegT produces a gradient of Nodal-related gene transcription in
Xenopus vegetal tissue

Answer 12

A combination of high levels of Nodal-related and b-catenin signalling from the Nieuwkoop Centre
induce the Spemann-Mangold Organizer
This gradient functions as a morphogen gradient within the mesoderm, and the combined action of VegT and Beta-catenin within the Nieuwkoop Centre on the dorsal side of the embryo promotes high enough levels of Nodal-related protein signalling to induce the Spemann-Mangold Organiser in the dorsal mesoderm.

Question 13

Nodal-related signalling proteins signal through cell surface receptors with Serine-Threonine
kinase intracellular domains that phosphorylate the Smad2 transcription factor

Answer 13

Wnt signalling stabilizes
b-catenin and promotes
its nuclear translocation
There are, however, additional consequences of high levels of Wnt-signalling and Xnr/Nodal signalling on the dorsal side of the embryo:

Question 14

Transcription of Spemann-Mangold Organizer-specific genes (chordin, noggin, goosecoid) requires a
combination of b-catenin-induced and Xnr-induced DNA-binding transcription factors

Answer 14

Transcription of genes encoding Organizer-specific determinants (intercellular signals and transcription factors) requires a combination of beta-catenin-induced and Xnr-activated DNA binding transcription factors.

Thus, beta-catenin induces transcription or the homeodomain transcription factor Siamois, whereas Xnr / Nodal-related signalling activates the Smad2 transcription factor.

And together, Siamois and Smad2 activate Organizer-specific genes such as chordin, noggin and goosecoid.

So how does this process functionalise the Spemann-Mangold Organizer? What does this structure do within the gastrula?

Question 15

The Goosecoid, Not1, Lim1 transcription factors are specifically expressed in the Spemann-Mangold Organizer

The Brachyury transcription factor is expressed throughout the mesoderm, including in the Organizer

The targets of Organizer-specific transcription factors in combination with Brachyury targets
create dorsal axial mesoderm which initiates the gastrulation process

Answer 15

The Spemann-Mangold Organizer initiates the process of Gastrulation. It does this because it expresses a combination of transcription factors (Organizer-specific Goosecoid, Not1, Lim1 plus pan-mesodermal Brachyury) that specify dorsal axial mesoderm: prechordal plate and notochord.

Question 16

Gastrulation (and neurulation) of the Xenopus embryo

Answer 16

Shows the process of gastrulation, first initiated in the dorsal blastopore lip, which then spreads laterally and then ventrally. Neural plate formation can be seen in the video, which then undergoes major morphogenetic changes to fold and elongate, forming the neural tube.

Question 17

During gastrulation, the Spemann-Mangold Organizer creates and patterns the embryos anterior-posterior and dorsal-ventral axes, by regulating:

Answer 17

(a) initiating and co-ordinating involution, intercalation and migration of dorsal axial mesoderm cells over the inner surface of the blastocoel roof, as parts of the process of convergent extension.
(b) regulating the production of multiple distinct fates in the correct positions and proportions within the mesoderm: prechordal mesoderm, notochord, somites, paraxial mesoderm, intermediate mesoderm, lateral plate mesoderm and blood islands.

Question 18

The Anterior-Posterior Axis of the embryo is formed during gastrulation under the direction of the Spemann-Mangold Organizer
Key point 1:

Answer 18

The Organizer is a dynamic mix of axial mesendodermal progenitor cells that sequentially produces anteriorly migrating pharyngeal endoderm, prechordal mesoderm and notochord during gastrulation, and organizes the co-ordinate induction and anterior-ward migration of paraxial, intermediate, lateral plate and ventral mesoderm.

Question 19

The Anterior-Posterior Axis of the embryo is formed during gastrulation under the direction of the Spemann-Mangold Organizer
Key point 2:

Answer 19

The Organizer drives the process of Convergence and Extension of dorsal axial mesoderm in a stepwise sequence of involution at the blastopore lip, followed by cell convergence to the midline and cell intercalation at the midline, which allows the axial mesoderm to elongate and extend enteriorly, as can be seem with this in whole mount situ hybridisation for expression of Brachyury in early and late gastrulae. The finger of Brachyury-expressing tissue is the dorsal axial mesoderm, mostly notochord, after convergence of the tissue between the blue bars in the early gastrula, followed by extension in the later stage of gastrulation.

Question 20

The process of gastrulation is fundamentally similar in Xenopus, chick and human embryos,
despite the overall structural differences
(hollow multilayered sphere, versus flattened multilayered disc)

Answer 20

If we look at the process of gastrulation in other vertebrates, we see fundamentally very similar processes, as in Chick, where three germ layers are organised around a line of tissue involution on the surface of the embryo proper, the primitive streak, through which the mesoderm migrates and then self-organises, as in Xenopus.

Question 21

In the chick gastrula, as in the Xenopus gastrula, pharyngeal endoderm extends along
the dorsal midline first, followed by prechordal mesoderm, then notochord

Answer 21

Looking down on the chick embryo as it sits in the egg, you can clearly see the linear landmark of the primitive streak, which is the equivalent of the Xenopus blastopore lip, and Hensen’s Node at the anterior end of the streak, which is the equivalent of the Spemann-Mangold Organizer.

Question 22

12 day-old human embryo: cross-section

Answer 22

By 12 days, the embryo has implanted into the uterine wall. CRUCIALLY, the morula has separated into a top epiblast layer and the bottom hypoblast layer. This is very similar to the situation in an early Xenopus embryo with animal and vegetal hemispheres.

Question 23

The Anterior-Posterior Axis of the embryo is formed during Gastrulation, under the direction of the Spemann-Mangold Organizer
The Organizer comprises axial mesendodermal progenitor cells that give rise to three distinct
embryonic tissues: pharyngeal endoderm, prechordal mesoderm and notochord.

Answer 23

One of the main questions we are grappling with in this lecture, is how and when the anterior-posterior axis of a vertebrate embryo is formed. We know most about this process from studies of amphibian embryos over the last 100 years or so, which have demonstrated that the Spemann-Mangold Organizer, which, as I mentioned in the last lecture, can first be recognized as the dorsal blastopore lip at the onset of gastrulation. The Organizer is a dynamic structure comprising axial mesodermal progenitor cells that will give rise to three distinct embryonic midline tissues: pharyngeal endoderm here marked in orange), prechordal mesoderm (marked in brown) and notochord (marked in red). Gastrulation is the process by which these and other mesendodermal progenitors move from the blastopore lip to the interior of the embryo. In the case of the Organizer, the pharyngeal endoderm, prechordal plate and notochord precursors move through a fibronectin rich pathway across the roof of the blastocoel. In doing so, these progenitor cells define the embryo’s anterior-posterior axis.

Question 24

The Spemann-Mangold Organizer Graft

Answer 24

The Spemann-Mangold Organizer was identified by Hans Spemann and his PhD student Hilde Mangold in the early 1920s in their experimental embryology studies carried out in the newt, Ambystoma mexicanum. They found that when the dorsal blastopore lip was removed from one early gastrula-stage newt embryo and transplanted into the ventral side of a second newt embryo, then both the host dorsal lip and the transplanted dorsal lip contributed to a twinned embryo with two neural tubes and sets of dorsal axial mesodermal tissues – notochord and somites. Interestingly , the grafted dorsal lip only contributed to small portions of the second neural tube and axial mesodermal structures, indicating that most of the second axis had been induced by the transpslanted tissue in the host tissue - i.e. the host tissue fate had been reprogrammed.
The dorsal blastopore lip was thus called the Organizer, because of its ability to induce new patterns of development and differentiation in surrounding tissue.

Question 25

A small piece of tissue removed from the dorsal blastopore lip and transplanted to the ventral side of a host embryo can induce a secondary embryonic axis in the host embryo comprising mesodermal and neural tissues and organs However, the inductive properties of the Organizer change during gastrulation:

Answer 25

Sadly, Hilde Mangold died in a house fire shortly after being awarded her PhD, but her husband, Otto, continued her research on the Organizer, and discovered that its secondary axis-inducing properties changed during gastrulation. If the grafted Organizer was taken from the dorsal blastopore lip of an early gastrula donor and transplanted to an early gastrula host, then a complete second axis with head and trunk developed. But if the grafted Organizer was taken from the dorsal blastopore lip of a late gastrula donor embryo and transplanted to an early gastrula host, then only a partial second axis, with a portion of trunk and no head, developed. This experiment revealed that the Spemann-Mangold Organizer is a dynamic tissue, and this next slide shows what is going on inside the gastrula during gastrulation.

Question 26

As gastrulation proceeds, so the Early Organizer tissue migrates into the embryo and under the ectoderm that forms the blastocoel roof, creating the anterior-posterior axis of the embryo as it moves. The Organizer properties of the dorsal blastopore lip persist in an altered form, as the Organizer tissue that was at the blastopore lip in the early gastrula migrates anteriorly across the blastopore roof

Answer 26

In the Early gastrula, the dorsal lip tissue begins to migrate over the blastocoel roof. By the mid-gastrula stage, the early dorsal lip tissue has moved deep into the interior of the embryo – this is the tissue fated to become pharyngeal endoderm and prechordal mesoderm. Back at the dorsal blastopore lip, a new set of cells is beginning its journey into the embryo – this will become the posterior of the notochord. So the Organizer tissue that gastrulates early and becomes the pharyngeal endoderm / prechordal mesoderm, has greater secondary axis inducing potential than the Organizer tissue that gastrulates late, because the responses to their inducing activities need to be different.

Question 27

Organizer tissues from early, mid- and late gastrulae have different eventual fates in the embryo. Organizer grafts taken from early, mid- and late gastrula-stage donors have different inducing potential because the Organizer tissues express different signalling molecules.

Answer 27

and we now know that these early and late Organizers have different inducing potential because they express different combinations of signalling molecules.

Question 28

The Spemann-Mangold Organizer is a dynamic tissue with several related functions:

Answer 28

1. It creates the anterior-posterior axis of the embryo 2. It induces neural tissue from ectoderm 3. It patterns this neural tissue whilst creating the anterior-posterior axis of the embryo 4. It introduces anterior-posterior and dorso-ventral pattern into the mesoderm The primary “inducing” and “patterning” functions of the Organizer are achieved by secreting molecules that INHIBIT Wnt and BMP signalling pathway activities!

Question 29

Organizer grafts taken from early, mid- and late gastrula-stage donors have different inducing potential because the Organizer tissues express different signalling molecules

Answer 29

The pharyngeal endoderm and prechordal plate mesoderm secrete multiple different, secreted, specific Wnt inhibitors that prevent Wnt proteins from interacting with the Frizzled receptors – Dickkopf, Frzb, for example. Prechordal plate mesoderm and Notochord (but NOT pharyngeal endoderm) express specific inhibitors of BMPs: Chordin, noggin and follistatin. Interestingly Pharyngeal Endoderm and Prechordal plate mesoderm also secrete a protein called Cerberus, and Insulin-like Growth Factor (IGF), which inhibit both Wnt and BMP signalling. Therefore, an EARLY or MID-GASTRULA stage Organizer will inhibit both Wnt and BMP signalling, whereas a LATE GASTRULA stage Organizer will ONLY inhibit BMP signalling.   This has profound consequences for the CHARACTER of the second axis that an EARLY or LATE Organizer can induce.

Question 30

Early Organizer tissue expresses Wnt Antagonists and BMP Antagonists -> Head and Brain Mid- and Late Organizer tissue expresses only BMP Antagonists ->Trunk and Spinal Cord

Answer 30

So, accordingly, in the early gastrula, the Organizer secretes BMP and Wnt inhibitors, which prevent BMP4 and Wnt8 signalling on the dorsal side, and consequently head and brain tissue are induced.   In the late gastrula, by contrast, the Organizer no longer secretes Wnt inhibitors and only BMP signalling is inhibited, which leads to trunk and spinal cord induction instead of head and brain.   Don’t confuse the activities of the Organizer with the activities in the vegetal hemisphere that give rise to the Nieuwkoop Centre in the blastula. They are quite different. Remember vegetal blastula tissue produces a gradient of Xnr gene expression which hactivates SMAD2 signalling in the mesoderm, and Wnt11 mRNA is translated in the Dorsal blastopmeres that will give rise to the Nieuwkoop Centre. The Wnt and BMP inhibitors of the Organizer act much later in embryogenesis and inhibit Wnt8 and BMP4 signals.

Question 31

BMP Antagonists Noggin, Chordin and Cerberus are expressed in the Spemann-Mangold Organizer and its axial mesoderm derivatives, and they act on ectoderm to suppress epidermal fate and induce neural fate

Answer 31

On the left are some in situ hybridisation data showing the expression domains of the BMP antagonist/inhibitors Noggin and Chordin. You can see that both are expressed in the Spemann-Mangold Organizer of the Early gastrula and also in the axial mesoderm derivative of the Late Organizer, the notochord. The figure also makes the point that by inhibiting BMP siganlling in the ectoderm neural fate is realised BECAUSE epidermal fate is suppressed, i.e. in a real sense, NEURAL fate is the default fate of embryonic ectoderm, and BMP is an inducer of EPIDERMAL fate.

Question 32

Wnt Antagonists Cerberus, Frzb and Dickkopf are expressed in the Spemann-Mangold Organizer and they induce anterior character within neural tissue, promoting brain development, by antagonizing the posteriorizing and ventralizing functions of wnt8 in the mesoderm, preserving prechordal mesodermal identity

Answer 32

If we look at the Wnt Antagonists, we can see a much more anterior localisation of Wnt-antagonist frzb here to prechordal mesoderm and pharyngeal endoderm, in comparison to the domain of the BMP-antagonist Chordin, which is expressed in Prechordal Mesoderm and Pharyngeal Endoderm but also the Notochord. Remember the functions of Wnt antagonists are to block interaction of Wnt molecules with Frizzled receptors, INDUCING ANTERIOR CHARACTER in neural tissue and inducing brain development by antagonizing the POSTERIORIZING AND VENTRALIZING functions of Wnt8 in the mesoderm, preserving prechordal mesodermal identity. Figure at bottom right is an embryo that has developed a second head by injecting Cerberus mRNA into a ventrally located blastomere of the Xenopus blastula-stage embryo.

Question 33

The Double Gradient Model of embryonic axis formation Spemann-Mangold Organizer’ generates:

Answer 33

(a) An Anterior-Posterior gradient of Wnt signalling activity in the developing neural plate - lowest in anterior neural plate (where Wnt antagonist activity is highest) (b) A Dorsal-Ventral gradient of BMP signalling activity throughout the embryo - lowest in neural plate and dorsal axial mesoderm (where BMP antagonist activity is highest)

Question 34

Make sure you fully-understand what we mean by ‘induction’

Answer 34

Cell A makes a signal that acts on neighbouring Cell B Cell B is induced to become Cell C. Induction is an example of a change in fate mediated by an extrinsic / non-cell-autonomous signal

Question 35

Not to be confused with intrinsic / cell-autonomous differentiation

Answer 35

Cell A divides asymmetrically. One daughter cell realizes the same fate as mother cell (A’). Second daughter adopts a different fate X because it may lack certain components present in the mother cell and then cell-autonomously differentiates to the alternate fate X This should not be confused with intrinsic, non-autonomous differentiation, e.g. where Cell A divides asymmetrically, reproducing itself in one daughter A’ but the second daughter X may lack a component present in the mother cell, and then differentiate spontaneously as a result of this difference.

Question 36

1. Dual inhibition of Wnt and BMP signalling promotes brain formation in the anterior neural plate 2. BMP inhibition without Wnt inhibition promotes spinal cord formation

Answer 36

But the brain and spinal cord are complex, richly patterned structures How are the more detailed elements of anterior-posterior pattern in the brain and spinal cord created?

Question 37

Early, Mid and Late gastrula

Answer 37

The general mechanism is that in the Early Gastrula, prechordal mesoderm / pharyngeal endoderm first signal to the anterior neural plate, which then activates Anterior Transcription Factors that specify Brain tissue. Forming notochord then represses Anterior Transcription Factors in the Mid-Gastrula, and by the Late Gastrula stage has activated Posterior Transcription Factors in the spinal cord. For a more detailed explanation of the actual process, we need to know more about some other signals that posteriorize the neural tube.

Question 38

The Activation-Transformation Model of Neural Tube Anterior-Posterior Patterning Activation:

Answer 38

Activation The Spemann-Mangold Organizer in the early gastrula secretes Wnt Antagonists and BMP Antagonists. 2. Neurectoderm exposed to these Organizer signals adopts forebrain fate.

Question 39

The Activation-Transformation Model of Neural Tube Anterior-Posterior Patterning Transformation:

Answer 39

Transformation 3. From mid-gastrula stages onwards, secretion of Wnt Antagonists by the Spemann-Mangold Organizer is attenuated. 4. Posterior neural plate is exposed to multiple gradients of posteriorizing factors, e.g. Wnts, Retinoic Acid and FGFs. 5. Forebrain fate is suppressed and over time, increasingly posteriorized neural fates are induced by posteriorizing factors. 6. Neural plate elongates and transitions to neural tube.

Question 40

Activation – Transformation involves simultaneous integration of information from multiple signalling pathways

Answer 40

Thus, Activation-Transformation involves simultaneous integration of information from multiple signalling pathways. In addition to Wnt and BMP signalling, Retinoic Acid and FGF signalling are now involved. Retinoic Acid diffuses into cells and binds to its ligand-regulated Retinoic Acid Receptors, members of the Nuclear Hormone Receptor class of transcription factors. Fibroblast Growth Factors signal to the nucleus via a cascade of Serine-Threonine kinases, leading to activation of ETS-domain containing transcription factors.

Question 41

Anterior-Posterior regionalization of the neural tube is achieved by two opposing gradients of antagonistic signals – “anteriorizing” and “posteriorizing” factors

Answer 41

The Activation-Transformation Model and the supporting evidence indicate that Anterior-Posterior regionalization of the neural tube is achieved by two opposing gradients of antagonistic signals: Wnt Antagonists and BMP Antagonists promoting Anteriorization and Neuralization, and Wnt, FGF and Retinoic Acid promoting Posteriorization. These opposing gradients introduce complex patterns of differential gene expression across the Anterior-Posterior Axis, allowing high resolution regionalization of the CNS

Question 42

Pattern refinement: the distinct domains of the vertebrate spinal cord and vertebral column are defined through their specific combinations of Hox transcription factor expression (“Hox signatures”)

Answer 42

``` A Retinoic acid gradient induces different patterns of Hox gene transcription at different positions along the anterior-posterior axis. An example of this is that the distinct domains of the vertebrate spinal cord and vertebral column are defined by specific combinations of Hox transcription factors, which can be modulated by exposure to exogenous Posteriorizing factors such as Retinoic Acid. ```

Question 43

Pattern refinement: an interaction between forebrain and hindbrain cells induces midbrain cells at the boundary between them. Further events result in regionalisation of forebrain into diencephalon and telencephalon.

Answer 43

Similarly, in the brain, subdivision into forebrain, mid-brain and hindbrain is regulated by posteriorizing factors such as Wnt, RA and FGFs.

Question 44

Neural tube formation | ‘primary neurulation’

Answer 44

I want to switch topics slightly now and consider the process of neural tube formation. The neural plate is induced initially as a single cell thick epithelial sheet, defined at its periphery by a group of cells called the Dorso-Lateral-Hinge Point (DLHP) cells, that will eventually delaminate from the neurectoderm as Neural Crest cells, and thereby separate the closed neural tube from overlying epidermis.

Question 45

The vertebrate neural tube is polarized along its dorsal-ventral axis by two opposing signalling systems:

Answer 45

1. TGF-b ligands (mainly BMPs) secreted by the dorsal Roof Plate – induce dorsal neuronal fates 2. Sonic Hedgehog (Shh) secreted by the ventral Floor Plate (and notochord) – induces ventral neuronal fates 3. Wnt signals are also secreted by the Roof Plate. Their functions include promoting expression of BMPs in roof plate and contributing to induction of neural crest.

Question 46

In the dorsal neural tube, different members of the TGF-b family are expressed in different domains, and induce different dorsal neuronal fates.

Answer 46

In the ventral neural tube, different concentrations of Shh induce different ventral neuronal fates. In the ventral neural tube, different concentrations of Shh induce different ventral neuronal fates

Question 47

A gradient of Shh protein is established in the ventral neural tube which determines neural progenitor fate by regulating expression of Homeodomain Transcription Factors such as Pax7, Pax6 and Nkx6.1 in a Shh-concentration-dependent manner

Answer 47

In the Ventral Neural Tube, a gradient of Shh protein is established which determines neural progenitor fate by regulating expression of Homeodomain Transcription Factors such as Pax7, Pax6 and Nkx6.1 in a Shh-concentration-dependent manner The neuronal subtypes specified at different positions along the dorso-ventral axis activate specific transcription factors in response to the particular combination of TGF-b ligands or the concentration of Shh detected. In this figure, different concentrations of Shh induce different transcription factors in ventral neural progenitors of the chick neural tube: high Shh in the most ventral part of the neural tube induces Nkx6.1, slightly lower concentration induces Pax6, and a lower still concentration induces Pax7. Some neural progenitors express both Nkx6.1 and Pax6, whereas other progenitors express both Pax6 and Pax7. These different combinations result in the differentiation of different neuronal subtypes. e.g. Nkx6.1 only  V3 interneurones Nkx6.1 + Pax6  Motorneurones and V2 interneurones Pax6 only  V1 interneurones

Question 48

TGF-b and Shh induce distinct domains of transcription factor expression in neural progenitordomains along the dorsal-ventral axis of the early neural tube

Answer 48

``` These transcription factors define distinct neural identities that are realised when progenitors terminally differentiate into neurons a few days later ```