Hox genes are expressed collinearly along the antero-posterior axis in the endoderm and mesoderm. Distinct Hox expression domains oft coincide with distinct intestinal domains. This is reminiscent of Hox gene expression in morphologically distinct rhombomere and somite units. However, Hox deletions generally cause malformations (instead of homeotic transformations) in the intestine.

For example, Hoxa5^-/- mutants (expressed in mesoderm around the outgrowing tracheal bud) have small trachea and lungs. Hox13^-/- mutants (the most posteriorly restricted Hox gene) have cloaca (hindgut) and anal sphincter defects. Hox mutations impact tissues at the anterior boundary of the Hox gene’s expression — as in rhombomeres and somites, this is called posterior prevalence.

The mechanism that restricts Hox gene expression to specific antero-posterior levels of the endodermal tube is unknown. In some cases, Shh has been shown to act as an inducing signal controlling Hox gene expression. This may explain why Shh mutants exhibit transformations in identity of endodermal organs along the A/P axis. FGFs produced in the lateral plate mesoderm may also be involved.

ParaHox is a complex of three homeodomain genes also expressed sequentially along the antero-posterior axis that reflects their chromosomal order. Of these three genes (Cdx, Pdx and Nkx in vertebrates), Caudal is the most posteriorly expressed ParaHox gene. Vertebrate Cdx^-/- mutants have posterior gut defects; Drosophila, Caudal^-/- mutants flat-out lack a posterior gut. Caudal thus has a conserved role in posterior gut development.

Other ParaHox genes are also required for endoderm development. Pdx1 expression is induced in the region of the midgut endoderm that has the potential to develop into pancreas before any overt sign of pancreatic bud formation. Pdx expression persists in the pancreatic bud once it has formed. Nkx2.1 is expressed in the ventral foregut region. It is required for the lung and thyroid, both ventral invaginations of the foregut endoderm.

The pancreas and liver develop as buds (invaginations) at the same antero-posterior location. However, they are on opposite sides of the endodermal tube. Adjacent tissues secret signals to induce the endoderm into liver or pancreas.

The dorsal mesoderm is closest to the notochord. The notochord secretes TGFβ and chordin, reducing Shh activity in the dorsal mesoderm. This reduced Shh activity (via notochord signals) initiates its differentiation as pancreas.

The ventral mesoderm is furthest from the notochord. It therefore has high Shh activity. Also, adjacent cardiac mesoderm secretes BMPs. Shh and BMP induce the ventral mesoderm to differentiate as liver.

In other words, BMP and Shh induce the ventral mesoderm to differentiate as liver. The BMP is secreted by adjacent cardiac mesoderm. Low Shh activity induces the dorsal mesoderm to differentiate as pancreas. Shh activity is reduced by TGF-β and chordin secretions by the adjacent notochord.

Different tissues control pancreas development at different times. Pancreas development is initiated by chording and TGF-β secreted by the notochord. Later, the aorta forms between the notochord and prospective pancreas. The aorta secretes signals to continue pancreas development.

The mature pancreas is derived from dorsal mesoderm. It contains endocrine and exocrine cells. Endocrine cells (aka β cells) produce insulin to stimulate glucose uptake by cells. Exocrine cells produce amylase, a digestive enzyme.

Type I diabetes arises from a loss of β cells. Different transcription factors specify immature pancreatic cells as β or exocrine cells. Endoderm → Pancreatic cells (by Pdx1, Ptf1a) → Exocrine cells (Ptf1a) and β cells (Ngn3).

Intestinal vili are self-renewing epithelium. Deep Cyrpts of Lieberkühn (aka Crypts) contain, from the bottom of the crypt up to its cusp: slowly dividing stem cells; rapidly dividing stem cells; enteroendocrine cells; gobelet cells; and enterocytes. Cells migrate up out of the crypt to the very tips of the vili, where they shed away to make room for new cells.

β-catenin is required for stem cell proliferation in the intestine. In the deep crypt are proliferating undifferentiatd precursors; β-catenin/TCF is activatedhere. At the crypt’s rim are nonproliferating differentiated cells; β-catenin/TCF is turned off. BMPs and other secreted signals promote differentiation.

Wnt signaling blocks the GSK/APC complex from degrading β-catenin. Thus, Wnt signaling stabilizes β-catenin and maintains stem cells. Wnt^-/-, β-catenin^-/-, TCF^-/- and LEF^-/- mice have defective stem cell proliferation and thus few and shorter or even no intestinal vili. Wnt → Stabilized β-catenin → β-catenin + TCF/LEF in Nucleus → Wnt-Regulated Genes.

One of the leading causes of death in the US (2nd leading cause of cancer-related death). Mutations in the Wnt signaling pathway that lead to high levels of TCF/LEF activity are responsible for most cases of colorectal cancer in humans.

Colorectal patients with Wnt signaling mutations frequently have an overactive Wnt that essentially ablates APC, allowing for permanent β-catenin activation. This causes over-proliferation of stem cells. Also, mutant β-catenin may be resistant to degradation.

Ephrin signaling controls localization of differentiated cells in villi (cell sorting).

Respiratory systems form via branching morphogenesis. The laryngotracheal groove is a respiratory diverticulum (an outgrowth) that emanates via invagination from the foregut. This area of the foregut forms the pharynx, posteriorly it develops into esophagus. In vertebrates, the trachea forms two lung buds at its posterior end. Localized ECM breakdown (by hyaluronidase secretion) at tips of growing buds lets branches burst through.

In Drosophila, Branchless encoded FGF and Breathless encoded the FGF receptor. Branchless is expressed around tracheal primordium in advance of in advance of outgrowth. Cells at the tip of the tracheal bud grow filopodia. FGF expression is downregulated by Sprouty (Drosophila) and Shh (vertebrates). FGF^-/-, Sprouty⁺⁺⁺⁺⁺⁺ and Shh⁺⁺⁺⁺⁺⁺ mutants have unbranched tracheal buds.

Left/right asymmetry is controlled early in development by Hensen’s Node (or just the node). Shh is expressed only on the left side of the node. Shh induces the expression of nodal, a secreted protein, only on the left side of the embryo. If the pattern of nodal expression is made symmetric by implanting a pellet of cells expressing Shh on the right side of the node, then organ asymmetry is lost, and organs are randomly placed in the body.

Cilia located in the node may provide this left-localization of Shh, perhaps by causing a directional flow of signaling molecules. Mutations in left-right dynein (lrd) cause randominzation of organ placement; dyneins are motor proteins that move along microtubules and are involved in cilia movement. Furthermore, individuals with Kartagener’s Syndrome (where all cilia are immotile) have random organ placement.

Apical Ectodermal Ridge
The Apical Ectodermal Ridge (AER) controls identity along the proximal/distal axis. The AER is a ridge of thickened ectoderm that forms along the entire edge of the limb bud at the dorsal/ventral boundary. This thickened ridge is analogous to ectodermal placodes in that each are areas of thickened ectoderm created by cell shape changes and they each signal to the underlying mesoderm. The mesoderm underlying the AER is called the Progress Zone.
Experiment	Overview
Removal	The AER was removed from the limb at various times. This resulted in limb truncations. Thus, the AER signals the underlying mesenchyme to allow proximal/distal outgrowth. Late removes results in only the most distal elements (ie, digits) missing; early removal results in more proximal elements also missing. Thus, proximal-distal patterning occurs progressively over time with proximal elements forming first.
Hybridization	In situ hybridization was used to determine what factors are expressed by the AER. Among those factors were FGF-4 and -8. To test if these are the factors responsible for AER’s proximal-distal patterning activity, implantation of beads was performed in embryos stripped of their AER.
Implantation	The AER was removed and beads soaked in FGF-8 were implanted. The implanted beads were nearly as effective as the AER itself. Thus, the AER is clearly a signaling center that secretes FGFs to induce underlying mesenchyme.

Zone of Polarizing Activity
The Zone of Polarizing Activity (aka ZPA or Polarizing Region) controls identity along the anterior/posterior axis (and thus digit identity). It is a mesenchyme located at the posterior end of the limb bud. Digit 1 is most anterior numeration proceeds posteriorly.
Experiment	Overview
Transplantation	A ZPA was grafted to an ectopic anterior position. This caused formation of a new anterior/posterior axis. This is similar to the grafting of the frog embryo DBL to an ectopic position to induce a new dorsal/ventral axis with a new neural tube and complete set of structures. The interpretation for the ZPA results is the same as in the DBL results: the grafted tissue must be a source of a secreted signal that acts upon neighboring cell.
Hybridization	In situ hybridization identified Shh as the potential signal secreted by the ZPA.
Implantation	Implanting a bead soaked with Shh at the anterior end of the limb will cause the limb to form with two anterio-posterior axes as though it were a reflection of itself.
Formation of structures along the anterio-posterior axis is dependent on the concentration of Shh. Naturally, the [Shh] is highest posteriorly where the ZPA is located. Adding a source of Shh at the anterior end will form a concentration gradient that is high anteriorly and posteriorly and intermediate in the middle. Mutations that lead to ectopic Shh are the most frequent cause of limb/digit duplication.

Progress Zone, Part I
The Progress Zone controls identity along the proximo-distal axis. The Progress Zone is the limb mesenchyme directly underneath the AER. The proximal/distal fate of a cell is determined by the amount of time it spends in the Progress Zone. Outgrowth of the limb is proximal→distal, meaning proximal structures develop first and distal structures last.
In the early limb bud most mesenchymal cells are in the Progress Zone. However, as cells divide and the limb grows, more and more cells are found outside of the Progress Zone. They only begin to differentiate once outside the Progress Zone. This is similar to the nervous system, where progenitor cells only differentiate after leaving the ventricular proliferative zone.
Experiment	Overview
Labeling	Different cells are labeled with different markers. This reveals that as development proceeds, cells in the Progress Zone proliferate. As they proliferate, some cells are pushed out of the Progress Zone. Cells pushed out first become proximal structures. As limb development proceeds, cells i the Progress Zone continue to proliferate and additional cells are pushed out.

Progress Zone, Part II
Since the amount of time spent in the Progress Zone fates a cell along the proximal/distal axis, the labeling experiment explains why removing the AER at an early stage leads to greater limb truncations.
Transplantation	An early Progress Zone was transplanted onto a late limb bud. Many cells had already left the host’s late Progress Zone. Grafting a new Progress Zone onto the tip of the limb caused a new population of cells to proliferate and leave the Progress Zone as the host cells had. The result is duplication of structures.
Transplantation	An early limb bud Progress Zone was removed and replaced with a late limb bud Progress Zone. Only the most proximal structures had time to leave the host Progress Zone. The donor Progress Zone was only left with the most distally determined cells. This resulted in a loss of intermediate limb elements.

The Apical Ectodermal Ridge (AER) produces FGF-4 and -8. The FGFs act on the Progress Zone and maintain the Polarizing Region (ZPA).

The Polarizing Region (ZPA) produces Shh, and this Shh gradient establishes the anterio-posterior axis. Furthermore, Shh acts on the Progress Zone and AER. Thus, Shh is required for proximal→distal outgrowth.

The Progress zone maintains the Apical Ectodermal Ridge (AER). The Progress Zone produces FGF10.

Limb buds arrive at precise locations along the anterio-posterior axis via Hox genes. Hox gene expression patterns determine specific locations where FGF10 becomes expressed in the lateral plate mesoderm.

FGF10 is a signal that induces formation of the Apical Ectodermal Ridge. As soon as the AER forms, it immediately begins its function of controlling limb bud formation and distal outgrowth.

Later, Shh expression is induced by FGFs produced in the AER. Evidence that FGFs induce limb formation: FGF protein induces ectopic limb formation; and FGF10^-/- mutants lack limbs.

Tetrapod forelimbs and hindlimbs develop in different ways. Thus, there must be differences in gene expression that give rise to these differences in structure. First of all, regions where forelimbs and hindlimbs develop have different sets of Hox genes. Thus, Hos genes must in some way activate different gene expression patterns in forelimbs and hindlimbs.

As again in development of ectodermal appendages and the endoderm, it is the mesoderm that confers regional specificity. This is indicated by tissue transplantation experiments. Wing bud (forelimb) mesenchyme was replaced with leg bud (hindlimb) mesoderm. If the mesenchyme controls limb identity, then the forelimb ought be replaced by a hindlimb.

As expected, mesoderm (mesenchyme) controls limb identity via regional specificity of induction. This is much like the experiments on epithelial-mesenchymal interactions during ectodermal appendage formation and also in studies of endodermal organ formation. The mesoderm determines the ectodermal appendage or endodermal organ formed.

Hox genes are expressed along the anterio-posterior axis. Hindlimbs and forelimbs appear at different places along the anterio-posterior axis. Furthermore, hindlimbs and forelimbs have different identity. Hox gene expression is responsible for this, as it induces various Tbx genes which the mesodermal mesenchyme conferring regional specificity to the overlying ectoderm.

The Primary Organizer dorsalizes the mesoderm, resulting in dorsal mesoderm (gives rise to somites) and intermediate mesoderm (gives rise to kindey and gonad). Intermediate mesoderm is located between the paraxial mesoderm and the lateral plate. It develops paired epithelial thickenings.

These paired epithelial thickenings are known as genital ridges, from which primordial gonads arise. During the indifferent stage, the genital ridge epithelium proliferates and extends into the loose mesenchymal tissue above it. The epithelium that proliferates into the mesenchyme forms the primitive sex cords.

The primary sex characteristic is the sex of the gonad: in males, it is the testes; in females, it is the ovary. Secondary characteristics are all other differences between males and females.

These are referred to as secondary, because the primary sexual characteristic sets in motion all of the events associated with the development of the secondary sex characteristics.

Secondary sex characteristics can be internal or external. Internal secondary sex characteristics include the accessory organs used to transport gametes to the site of fertilization. External secondary sex characteristics include differences in overall size, musculature and bone shape, mammary glands, hair-growth patterns, etc.

In many organisms, behavioral differences are also secondary sex characteristics. For example, in song birds, only the males sing, and this is due to the effects of testosterone on development of specific neurons in the brain. In humans, social factors cloud what behaviors are or are not secondary sexual characteristics.

1. the mesonephric (Wolffian) ducts regress
2. the Müllerian ducts differentiate into:
   • oviducts (Fallopian tubes)
   • uterus
   • upper vagina

1. the Mullerian ducts regress
2. the Wolffian ducts differentiate into:
   • epididymis
   • vas deferens
   • seminal vesicle

1. genital tubercle forms clitoris
2. genital fold forms labia minora
3. genital swelling forms labia majora

1. genital tubercle forms head of penis
2. genital fold forms shaft of penis
3. genital swelling forms scrotum

A series of experiments in fetal rabbits led to the conclusion that the sex of the gonad controlled the development of all other sexual characteristics. These experiments showed:
Removed	Overview
Testes	Removing testes from a male fetus will cause it to develop a female phenotype without gonads.
Ovaries	Removing ovaries from a female fetus will cause it to develop a female phenotype without gonads.
Conclusion: The ground state is female. Testis are required for male differentiation. Without testis then the phenotype is female.
These results led to the hypothesis that a substance produced by the male gonad causes male differentiation. We now know that the key products of the testes that allow male development of secondary sexual characteristics are hormones.

Injection	Overview
Testosterone	Testosterone injected into female fetuses will cause them to develop as males, except that the ovary does not differentiate into a testis and produce sperm, and the paramesonephric (Mullerian) ducts do not degenerate.
Estrogen	Estrogen injected into fetuses will not cause any change in the fetus’ development. Males still develop as males and females as females.
AMH	Regression of the Mullerian ducts in the male is under the control of anti-Mullerian duct hormone (AMH, a TGF-β) secreted by testis’ Sertoli cells.

Conclusion: The development of the male phenotype in mammals depends on the development of the testis, which then controls secondary sexual development by producing testosterone and AMH. As a side-note, testosterone is secreted by the Leydig cells of the testis and estrogen is produced by the ovary.

Amphibian primordial germ cells (PGCs) originate in the endoderm of the gut. Mammal and bird PGCs originate in the epiblast and then migrate to the yolk sac. Later, PGCs perform directed cell migration to the genital ridge.

Stromal-derived factor-1 (SDF-1) guides PGCs in many vertebrates. SDF-1 is expressed along the PGC pathway, and PGCs express the SDF-1 receptor. In SDF-1^-/- or the SDF-1-receptor^-/- mutants, germ cells do not reach the genital ridge.

The PGCs invade both the primitive sex cords (medulla) and cortex of indifferent gonad (at week 6 in humans). The different fates of the cortex, medulla and PGC’s in males and females is determined by the sex of the gonad.

The gene SRY has been cloned from the human Y chromosome and is required to confer maleness in mammals. This gene has a close homolog in mice, is expressed in gonad at the time of testis differentiation, is not expressed in ovaries, and is a DNA or RNA-binding protein. Transgenic SRY mice always had a male phenotype even when they had an XX genotype. However, male XX^SRY were incapable of spermatogenesis since sperm cell differentiation requires additional Y chromosome proteins. This shows that the presence of the SRY gene is sufficient to drive almost all aspects of male sexual differentiation.

Sox9 is an autosomal gene necessary for maleness in all vertebrates. Sox9^-/- causes a female phenotype regardless of genotype. The current hypothesis is that in mammals and marsupials, SRY controls the expression of Sox9. The role of Sox9 as a determinant of testes formation appears to be conserved among all vertebrates. This is true even in vertebrates other than mammals, which do not have the Sry gene. It is not known why expression of Sox9 came under the control of Sry in mammals. In vertebrates such as fish and reptiles, where sex is determined by hormones or the temperature at which the embryo develops, it is thought that Sox9 activity is influenced by sex hormones.

Researchers have not deciphered how sex is determined in animals that use a ZW system (including birds). This is largely due to the impossibility genetic screens or other genetic manipulations in birds. However, RNAi has revealed that the DNA-binding protein DMRT1 is sex-determining. DMRT1 is required for the gonad to differentiate as a testis in birds. Interestingly, DMRT1 is highly conserved. It is expressed in mammals and in Drosophila its homolog (Doublesex) determines sex in flies. As a side-note, monotreme mammals (egg layers such as platypus and echidna) use a ZW system, indicating that the XY system evolved rather recently.

The female phenotype is not simply a default state. The development of secondary sexual characteristics in females requires estrogen and other hormones. Also, the Wnt4 gene is required for the development of the female gonad as an ovary.

In female mice lacking Wnt4, the gonad develops male characteristics (Sertoli cells) and expresses genes involved in testosterone production. Wnt is a member of the Wnt family of genes related to the Drosophila wingless (wg) gene.

Experiments Demonstrating Role
1	Marginal zone cells do not form mesoderm when isolated and cultured until after the 64-cell stage.
2	Removing the marginal zone and recombining animal and vegetal caps, dorsal mesoderm arises from animal cap cells nearest the vegetal cap, and vegetal cells opposite the SEP.
3	Recombining animal caps with various vegetal blastomeres: dorsal vegetal blastomere cells induced dorsal mesoderm; ventral vegetal blastomere cells induced ventral mesoderm. Also, dorsal mesoderm induced ventral mesoderm to become lateral mesoderm. Thus, signaling must be involved in dorsal and central cell fates.
4	Fertilized eggs did not form a D/V axis when irradiated at the vegetal pole, but could be restored by a single vegetal- and dorsal-most cell. Thus, this vegetal- and dorsal-most structure must induce other regions to become dorsal mesoderm. This vegetal-most and dorsal-most region first induces the primary organizer (which then induces other tissues) and was called the Nieuwkoop center.
5	To determine which part of the embryo acts to organize the mesoderm into dorsal structure and to induce neural tube formation, the dorsal lip of the blastopore of an early gastrula from a light-colored newt was transplanted into an early gastrula of a dark-colored newt. The donor tissue formed a second embryonic axis. The notochord of this second embronic axis was composed entirely of graft (donor) tissue, while the neural tube and somites were composed only partly of graft (donor) tissue and the kidney tubules and gut of the new axis were composed entirely of host tissue. Spemann and Mangold concluded that the graft tissue induced a new embryonic axis. This structure is named Primary organizer.

The Nieuwkoop Center is the dorsal- and vegetal-most cell of the early blastula. It gives rise to the Spemann Organizer, which is the dorsal lip of the blastopore. The Spemann Organizer has a dorsalizing effect, and together with the Sperm Entry Point (SEP) gives rise to the dorsal/ventral axis.

The first signals are the vegetal maternal mRNAs Veg1 and VegT. The second signal is the dorsal-most protein β-catenin, which accumulates via cortical rotation. This induces formation of the Spemann Organizer and the Nieuwkoop Center. The Spemann Organizer is responsible for the third signal by expressing Noggin, Chordin and Follistatin, which bind and inhibit the ventralizing factors BMP-4 and Frzb.

Drosophila Dpp is homologous to BMP-4. Dpp activity is highest at the dorsal region. Drosophila Sog is homologous to Chordin. [Sog] is highest at the ventral region, where it binds and inactivates Dpp. These Drosophila genes can be replaced by their Xenopus homologs, but their activity along the dorsal-ventral axis is inverted. This switching occurred in a vertebrate ancestor. Also, Drosophila Wingless is homologous to Wnt-8.

Starting at the ventral end, the following structures comprise the spinal cord: floor plate; cell bodies of somatic motor neurons; cell bodies of commisural neurons; roof plate. The floor plate guides axons that grow into it, and the motor neurons send out axons that innervate muscles.

The notochord patterns the neural tube’s dorso-ventral axis. An additional notochord transplanted underneath a closing neural tube in the chick embryo induces an additional floorplate with adjacent motor neurons. Removing the notochord from the neural tube results in absence of a floor plate and motor neurons. Thus, the notochord induces at least the most dorsal neural tube structures. Furthermore, both the notochord and floorplate induce adjacent somite tissue into sclerotome, which gives rise to the vertebrae and ribs.

Vertebrate Sonic hedgehog protein (Shh, homolog of Drosophila Hedgehog) is localized to the notochord and floorplate of the spinal cord. Perhaps Shh is the notochord signal that patterns the neural tube in dorso-ventral axis, and that patterns the somites. Addition of cells producing Shh has a similar effect to adding an additional notochord: an ectopic floorplate and motor neurons. The floorplate differentiates where the level of ectopic Shh is highest and the motor neurons where the level is lower. This indicates that a gradient of Shh released from the notochord patterns the neural tube.

The key role of Sonic hedgehog was revealed by the phenotype of Sonic hedgehog knockout mice. Shh^-/- mice die early and are extremely defective. The defects observed are consistent with a role of Shh in inductions:

Floorplate	Shh form the notochord induces the floorplate. Shh-/- fetuses have severe ventral midline defects, i. e., the ventral midline seems to degenerate. This results in a fusion of the eye and nose primordia in the ventral midline (cyclopia). This defect is consistent with the known role of Shh in inducing the formation of the floorplate and other ventral cell types in the neural tube.
Sclerotomes	Shh from the notochord and floor induces sclerotomes. Another defect is the virtual absence of the vertebrae and the ribs. Again, this is consistent with the role of Shh in giving a more ventral identity (sclerotome) to a portion of each somite. We will discuss this aspect of Shh signaling in a future lecture.

The posterior neural tube gives rise to the spinal cord. The anterior neural tube gives rise to the brain. The neural tube begins subdividing into a complex series of bulges (later forming the brain) before it has even completely formed at the posterior. First, three primary vesicles are formed. Starting at the anterior: the forebrain (prosencephalon), the midbrain (mesencephalon) and the hindbrain (rhombencephalon). The prosencephalon then becomes subdivided into the telencephalon at the anterior and the diencephalon at the posterior. The telencephalon will give rise to the cerebral hemispheres. The diencephalon evaginates to form the optic cup (which will form much of the eye), and also will give rise to the thalamic and hypothalamic regions of the brain.

The rhombencephalon is the only part of the brain that appears to be segmented into separate bulges, referred to as rhombomeres. Each rhombomere is a morphologically distinct compartment. A compartment is a region of cell lineage restriction. Once boundaries between individual compartments form, cells are confined to their respective compartments and cannot cross from one side of a boundary to another. This implies that the cells within each compartment have differential affinities for each other, but not for cells from neighboring compartments. This is similar to the segments of the Drosophila embryo. Different types of appendages arise from different Drosophila segments. Similarly, distinct cranial nerves arise from different rhombomeres.

The strongest evidence that rhombomeres represent metameric units analogous to the segments of the Drosophila embryo is that their identities are controlled by Hox genes. Anterior boundaries of expression of individual Hox genes mark boundaries between rhombomeres. Knockouts of certain Hox genes in mice (for example Hoxa-1 and Hoxa-3) result in abnormal development of the anteriormost rhombomere in which the gene is normally expressed. As with Drosophila segments, loss of Hox genes leads to transformations to a more anterior identity. Hox genes thus appear to play similar roles in rhombomere identity, vertebral identity and Drosophila segment identity.

Hox genes are not expressed in the midbrain and forebrain. These regions are probably also divided up into subunits by the spatially localized expression of various other transcription factors. There are at least 25 homeobox-containing genes that are expressed in regionally restricted patterns in the forebrain. Many of the genes expressed in these overlapping patterns (Emx, Otx, Dlx) are homologous to genes (ems, otd, Dll) expressed in segmental patterns in the embryonic fly head. The few mutations observed so far have been instructive. For example, Drosophila otd (and its murine homolog Otx) mutants lack the anterior portion of the brain.

A key feature of cells located at the boundaries of compartments is that they often serve as signaling centers. We saw this when we considered the segment polarity genes of Drosophila. We saw that the cells on either side of the compartment boundary expressed either Hedgehog or Wingless proteins.

These proteins then diffused away from their sources and influenced the development of neighboring cells in the compartments. Similarly, boundaries of lineage restriction in the developing midbrain and forebrain act as signaling centers. For example, a signaling center forms at the boundary between the midbrain and hindbrain.

The cells at this boundary not only act as a barrier of cell lineage restriction, but also secrete extrinsic signals, such as FGF8 and Wnt1 (a homolog of Wingless). These molecules are thought to form gradients that pattern adjacent regions. Roles for FGF8 and Wnt1 are demonstrated by the fact that tissue-specific loss of these genes in neural tissue leads to loss of the midbrain. Other signaling centers have been found in the forebrain, but their functions are less well understood.

Injecting Sonic hedgehog protein (Shh) → extra floor plate. Sonic hedgehog is expressed in notochord and floorplate. Shh is a homolog of Drosophila hedgehog. High levels of Shh → floorplate. Low levels of Shh → motor neurons.

As the primitive streak regresses posteriorly, cells furthest from the node are no longer under the influence of FGF. Once outside the influence of FGF, paraxial mesoderm cells begin to compartmentalize into somites. Cells within an individual somite compact by increasing cadherin expression. These changes in cell adhesion cause the newly forming somite to separate from the rest of the paraxial mesoderm.

The region separating a newly formed epithelial somite from neighboring unsegmented paraxial mesoderm is a boundary. Boundaries arise in vertebrate brain rhombomere compartments and Drosophila embryo segments. Compartments are kept separate due to differences in cell adhesion. The same is true for cells in somites and in the unsegmented paraxial mesoderm.

The advantage of dividing the paraxial mesoderm into separate somites, each of which is an individual compartment, is that this allows the formation of repetitive structures such as vertebrae to use a common genetic pathway in each somite. The Notch pathway is critical for formation of a boundary between the newly formed somite and unsegmented mesoderm, and between rhombomeres.

The most apical (facing the lumen of the neural tube) zone is the ventricular zone; this is where neuronal progenitors undergo cell division. This can be seen as the region where 3H-thymidine is incorporated. Once cells are postmitotic (undergone their last division) they migrate away from the ventricular zone to differentiate. The identity of the newly born neuronal or glial cell is specified before it starts to migrate.

Asymmetric cell division may be involved in the formation of neuroblasts (cells committed to terminal differentiation) from neural progenitor cells in the ventricular zone. In Drosophila, asymmetric cell divisions lead to the segregation of cytoplasmic determinants that affect the Notch pathway. There is some evidence that similar mechanisms operate in parts of the vertebrate nervous system.

In order to generate a functional nervous system, the correct numbers of neurons must be generated, and they must differentiate into the correct cell types. How is the number of neurons regulated? Due to lateral inhibition, only one cell in each proneural cluster becomes a neuroblast. Similarly, in vertebrates, lateral inhibition controls the rate at which neuroblasts are specified from the cells in the ventricular zone.

Lateral inhibition is mediated by the Notch signal transduction system. Notch and Delta are transmembrane proteins. Notch is a receptor that interacts with specific ligands including Delta. Therefore, the Notch-Delta interaction requires direct cell-cell contact. Initially, all cells in the proneural cluster express low levels of Notch and Delta. Activating the Notch receptor inhibits expresion of proneural genes, and Notch-Delta interaction downregulates Delta expression. Through an unknown mechanism, one cell in each proneural cluster gains a slight advantage over the others by expressing slightly more Delta.

In a likely schochastic (random) process, one cells expresses slightly more Delta than its neighbors. This cell thus engages more Notch receptors on neighboring cells, downregulating their expression of proneural genes and Delta whilst retaining their Notch expression. Thus, these neighboring cells lose the ability to differentiate as neurons (due to decreased proneural gene expression) and also the ability to inhibit proneural expression in other cells (due to decreased expression of Delta). As a result the single cell that expressed slightly more Delta is the only cell in which proneural gene expression is maintained.

The importance of Notch-Delta signaling can be seen from analysis of mutants in this pathway. A clear example is provided by the sensory receptor cells of the inner ear. These cells are called hair cells for the characteristic cilia on their apical surfaces. These cilia are deflected by mechanical forces created by sound waves, and the signal is transmitted along axons to the brain. Hair cells are surrounded by various types of support cells.

pattern of hair cells in the inner ear is highly invariant, and this regular pattern is required for proper sound reception. In Delta^-/- mutants, lateral inhibition is defective, and all cells, including the support cells, develop as hair cells. Thus, lateral inhibition normally regulates the number of hair cells that develop, ensuring that there is a correct balance between hair cells and supporting cells.

Neural crest cells form at the anterior end of the neural plate, at the border of the epidermis and neural plate. This is where BMP levels are intermediate. Sensory placodes arise from this region and are induced by the same signals. Neural crest cells migrate through the anterior half of the somite but avoid the posterior half due to repulsive ephrin signals from somite’s posterior half.

Neural crest cells form dorsal root ganglia (sensory neurons) and melanocytes (pigment cells). Dorsal root ganglia form the adrenal medula and also sympathetic ganglia in the gut. Other neural crest derivatives are head mesenchyme and Schwann cells. Head mesenchyme includes bones, connective tissue and dental papilae (tooth pulp). Schwann cells myelinate peripheral neurons.

Neural crest cells were transplanted from one position in the body to another position. They developed into neural crest derivates from their new position. Neural crest cells are apparently pluripotent, as they give rise to the cell types expected from the position to which they have been transplanted.

For example, any neural crest cell can give rise to parasympathetic ganglia if transplanted to a certain position. Thus, neural crest cells must respond to environmental cues during their migration and subsequent differentiation. These environmental cues are often identical to the cues used by axons.

When Neural Crest Cells begin to migrate, they lose their adhesion to the neural tube and adjacent epidermis. This involves losing expression of both N-cadherin and E-cadherin.

Neural Crest Cells are guided by similar mechanisms as axons, consisting of short-range (cell-surface) and long-range (diffusible) signals of attraction and repulsion.

Short-range attractive cues include cadherins; short-range repulsive cues include ephrins. Ephrins are recognized by neural crest ephrin receptors. The posterior half of each somite expresses ephrin, relegating neural crest cells to migrate through the anterior half of each somit.

Long-range attractive cues include netrins. Long-range repulsive cues include semaphorins.

The extracellular matrix is the substrate over which a neural crest cell migrates. The cells bind primarily to laminin and secondarily to fibronectin and collagen. Cues guide neural crest cells across the extracellular matrix.

Ephrins are short-range repulsive cues to prevent cell mixing between adjacent somites. The posterior half of the somite produces a repulsive signal called ephrin. Loss of Ephrin-B1 induces NCC migration defects.

Retinal neurons send a bundle of axons (the optic nerve) to precise locations in the tectum (a region of the brain). The precisely ordered connections are known as a retino-tectal map. This map is broadly established by a gradient of ephrin and ephrin receptors, and refined by additional interactions.

Retinal neurons express the ephrin EphA3 in a graded fashion, with the highest levels on the side furthest from the nose. A corresponding gradient of ephrins A2 and A5 is seen in the tectum, where the lowest [ephrins] is at the anterior side and the highest [ephrins] is at the posterior side.

Retinal neurons from the nasal side of the retina express low levels of EphA3, and are thus insensitive to the repulsive effects of ephrins A2 and A5. They thus project only to the anterior side of the tectum. In contract retinal neurons furthest from the nose will project to the posterior side of the tectum to avoid repulsion.

If the optic nerve is severed and the eye rotated 180°, the retinotectal map will still form properly. This indicates that the retina and tectum release certain signals which define the retinotectal map, regardless of orientation.

The extracellular matrix is a substrate over which axonal growth cones can migrate. Laminin is the primary active component in the extracellular matrix, followed by fibronectin and collagen.

Netrins are long-range chemoattractants. They are secreted by floorplate cells, thus attracting neurons to the midline. Commisural neurons first extend ventrally, then project to netrin^+/+ floorplate cells.

Floorplate or floorplate extract can be placed on agar. Axons within 250µm of this will reorientate their growth toward it. In netrin^-/- knockouts, commisural axons fail to grow to the floorplate (midline).

Slit and semaphorin are repulsive cues. Netrin and slit signaling systems play opposing roles during positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryos. Slit is present outside of the midline, and is taken up in a Roundabout (Robo) independent manner along the commissural tracts into the longitudinal connectives. Guidance is partially or fully ablated in null mutants of Slit or Robo.

Netrin draws commissural neuron growth cones toward the floorp-plate/midline. Then Slit (secreted by ventral midline) and Semaphorin (secreted by neural tube) divert the commisural neuron growth cones toward the brain. Thereby nerves extend themselves from the body to the brain. First they reach the midline due to the attractant Netrin, then they travel up to the brain along a narrow path defined by the repellents Slit and Semaphorin.

A placode is a thickened region of the ectoderm, and are induced at the anterior end of the neural plate where BMP levels are intermediate (this same region gives rise to neural crest cells). Ectoderm is epithelial, while the mesoderm contains both mesenchymal and epithelial cells.

Sensory placodes and their determining signals: nasal placodes (Pax6); lens placodes (Pax6); trigeminal nerve (Pax3/8); facial nerve (?); ear (Pax2); glosso-pharyngeal nerve (?); vagus nerve (?). Pax6 expression is inhibited by high concentrations of Shh and BMP, causing holoporsencephaly.

The optic vesicle induces the lens placode; the lens vesicle induces the optic cup. Competence is the ability to respond to a signal. Only anterior ectoderm is competent to respond to the inductive signal from neural tissue. Transplanting an optic vesicle outside the anterior ectoderm will not result in ectopic eye formation.

Human Pax6 is needed for eye formation; its homologs in other species are murine Smalleye and Drosophila Eyelesss. In Drosophila, ectopic expression of human Pax6 is sufficient to induce ectopic eye formation! In mice and humans, Pax6^-/- mutants never even develop an optic cup, and wind up with eyes nor a nose.

Pax6 is expressed in the optic vesicle (neural tissue) and in the lens vesicle it induces (an ectodermal placode). Researchers wondered whether Pax6 is required in both or only tissue to induce the lens. Tissue recombination experiments between Pax6^+/+ and Pax^-/- embryos answered this question.

Epithelial-mesenchymal interactions that induce cutaneous structures are described as regionally specific. Skin is composed of two main tissues: an outer epidermis (ectoderm-derived epithelium); and an inner dermis (mesoderm-derived mesenchyme). Chick epidermis secretes Shh and TGF-β, which induces underlying dermis to condense; this condensed dermal mesenchyme in turn secretes factors that cause the epidermis to form regionally specific cutaneous structures.

Mesoderm specifies the identity of regional overlying structures. Researchers separated embryonic epithelium and mesenchyme from each other; the embryonic epithelium and mesenchyme was recombined in various ways. In every case, the a given mesenchyme imparted the same identity to any of the overlying embryonic epithelia. For example, chick leg mesenchyme transplanted onto wing embryonic epithelium, will induce leg feathers in the wing embryonic epithelium.

Epithelial-mesenchymal interactions that induce cutaneous structures are described as genetically specific. Mesenchyme instructs epithelium to activate certain genes in a regionally specific manner. However, the epithelium responds only as its genome permits. This was discovered by transplanting together tissues from different species.

Spemann and Schotté transplanted frog flank ectoderm to the oral region of a newt gastrula. Similarly, they transplanted newt flank ectoderm to the oral region of a frog gastrula. The newt gastrula developed a froglike mouth; the frog gastrula developed a newtlike mouth. In other words, the mesoderm instructed the ectoderm to make a mouth but the foreign ectoderm did so according to its own genome.

This reveals that mesenchymal tissue can induce overlying ectoderm across species barriers, but that the epithelium responds in a species-specific manner. The type of organ induced is controlled by mesenchyme, but species specificity is controlled by the responding epithelium.

Ectodermal Dysplasia is the abnormal development of cutaneous structures: fewer, smaller teeth, sparse hair, small fingernails and toenails, few sweat glands and epithelial placode retardation. Edar is expressed in ectodermal placodes. Eda is expressed in the surrounding epidermis. BMP-4 is a placode inhibitor.

Foxi3 is transcription factor involved in appendage formation. Foxi3 mutants have defects in hair, teeth, sweat glands. Thus, studies of ectodermal dysplasia have revealed that ectodermal appendage development requires expression of Foxi3. Without Foxi3, ectodermal appendages cannot develop properly.

Hensen’s Node is present in birds and mammals. The Spemann Organizer is present in Xenopus. The Node establishes l

Discovery
Transplant	Transplanting a small group of cells from a region that will eventually form a somite boundary, into a region of unsegmented paraxial mesoderm that normally would not be part of a boundary.
Result	Transplanted cells instruct the cells anterior to them to undergo a mesenchymal-epithelial transition and to separate from the unsegmented mesoderm.
Boundaries are signaling centers, and the somite boundary instructs neighboring cells to undergo a mesenchymal-epithelial transition. Nonboundary cells can acquire this ability to induce boundary formation if the Notch pathway is activated in them, for example, by introducing an activated Notch receptor.
Mutations in Notch signaling lead to defects in somite formation. For example, mice lacking the Notch ligand Delta-like 3 (Dll3) have serious vertebral and rib defects. As we will see below, these structures are derived from somites. In Dll3-/- mice, somite formation is irregular and delayed. As a result the structures that form from the somites are abnormal.

Notch controls somite size and segmentation in a negative feedback pathway. The Notch pathway establishes an oscillating pattern in somites, and one cycle of the oscillation corresponds to the budding off of one somite from the unsegmented paraxial mesoderm. Notch signaling activates a transcription factor (RBJ) that activates the expression of Hes.

Hes is a transcriptional repressor that has two functions. First, it represses expression of itself. This limits the duration of the Notch response. Second, Hes represses expression of an inhibitor of the Notch receptor, lunatic fringe (Lfng). Thus, this activity of Hes would serve to activate the Notch pathway. The oscillations are created because Hes has a very short half-life, leading to transient repression of Notch.

Isolated ectodermal cells become neural. However, entire ectodermal explants do not. This is because BMP4 is produced in the ectoderm. As in the mesoderm, BMP is a ventralizing signal. Ventral ectoderm becomes epidermis. Hence, BMP produced by the ectodermal explants induces the entire explant into epidermis.

In the embryo, BMP antagonists secreted by the organizer (dorsal mesoderm) to overlying ectoderm inhibit the ventral fate. Furthermore, Isolated ectodermal cells produce BMP4, but it diffuses away and the ventral fate is thus not induced. Unless overridden by BMP4, ectodermal cells by default differentiate as neural even in the absence of chordin and noggin.

Injecting morpholinos against BMPs into frog embryos will cause excess neural tissue to form. This reinforces the theory that BMPs induce ectodermal cells to become epidermal, and that BMP antagonists prevent this to allow cells to default to a neural state.

Ectoderm appears to undergo a neural path by default, but in fact they have already received neural signals. For example, treatment of embryos with FGFs (fibroblast growth factors) stimulates neural development, and FGFs are produced throughout the embryo.

In invertebrates, neural precursor cells segregate from a specialized region within the ectoderm, called neurectoderm. The neurectoderm contains cells that will give rise to epidermis (the outer covering of the insect) and cells that will give rise to the nervous system. The neural precursors delaminate individually from the neurectoderm into the interior of the embryo, where they begin to proliferate and are referred to as neuroblasts.

This delamination involves a number of the cytoskeletal and cell adhesion mechanisms that we discussed previously. The delaminating cells lose their connections, via tight junctions and adherens junctions, to cells of the ectodermal epithelium; this is an epithelial to mesenchymal transition. In addition, the delaminating cells constrict at their apical surfaces via microfilaments, and elongate via microtubules.

Neuroblasts in insects are determined in a two-step mechanism.

Proneural

Proneural clusters are groups of neuroectoderm that become competent to give rise to neuroblasts. This is due to their activation of proneural gene expression. Proneural genes a group of bHLH (basic helix-loop-helix) transcription factors encoded by the Achaete-scute complex. The requirement for the proneural genes is shown by the fact that many neuroblasts do not appear in Achaete-scute-/- embryos.

Inhibition

One of the cells that has delaminated from the proneural cluster inhibits other cells of the cluster from becoming neuroblasts. This process is called lateral inhibition, and it is important for assuring that the correct number of cells become neuroblasts.

Lateral inhibition is mediated by Notch signal transduction. Notch is a transmembrane receptor that interacts specifically with Delta, a transmembrane protein. Therefore, Notch-Delta interaction requires direct cell-cell contact. Initially, all cells in the proneural cluster express low levels of Notch and Delta. One cell in each proneural cluster gains a slight advantage over the others by expressing slightly more Delta. This is likely stochastic (random). This cell thus engages more Notch receptors on neighboring cells, causing them to downregulate proneural gene and Delta expression whilst retaining Notch expression. These cells thus lose the ability to differentiate as neurons (due to decreased proneural gene expression) and also lose the ability to inhibit proneural expression in other cells (due to decreased expression of Delta). As a result the single cell that expressed slightly more Delta is the only cell that maintains proneural gene expression.

Genes involved in this inhibitory process are called neurogenic genes; their required role in this repression is demonstrated by the fact that, in an embryo homozygous for a null mutation in a neurogenic gene, the entire neurectoderm develops as neuroblasts (since all of the cells in the proneural clusters, which constitute the neuroectoderm, now develop as neuroblasts).

Notch and Delta are extremely important neurogenic genes in Drosophila. The Notch receptor and its various ligands have been found to play multiple roles in cell signaling and cell fate determination not only in Drosophila, but also in vertebrates. Moreover, their role in lateral inhibition is largely conserved in vertebrates.

Recall that in amphibian gastrulation, the mesodermal and endodermal cells must enter the interior of the embryo in order to correctly arrange the three germ layers. This is initiated through an invagination, followed by involution. The dorsal lip of the blastopore is the site where this process is initiated, and it forms opposite the site of sperm entry on the dorsal side of the embryo. Spemann and Mangold wondered if all cells are undetermined at the beginning of gastrulation. They transplanted the dorsal lip of the blastopore of an early gastrula from a light-colored newt into an ectopic region of a dark-colored newt early gastrula.

Spemann and Mangold observed that the donor tissue (dorsal blastopore lip) invaginated into the interior and a second embryonic axis developed. The notochord of this second embryonic axis was composed entirely of graft donor tissue, while the neural tube, and somites (a mesodermal tissue that we will discuss in a later lecture) were composed only partly of graft (donor) tissue. The notochord is the most dorsal mesodermal derivative. All other tissues of the new axis (eg, kidney tubules and gut) were entirely of host tissue. Spemann and Mangold concluded that the graft tissue induced a new embryonic axis.

The organizer (induced by the Nieuwkoop Center) dorsalizes the adjacent mesoderm by secreting Noggin, Chordin, Follistatin and Frzb. These secreted proteins induce the neural plate by antagonizing the ventralizing proteins BMP-4 (noggin, follistatin, and chordin) and Wnt-8 (frzb). Recall that the Organizer organizes the mesoderm around it into an axis by dorsalizing the mesoderm so that dorsal derivative like somites (future trunk muscle and bones) and intermediate derivatives (kidney and gonad) can form. In vertebrates, the most dorsal mesoderm forms the notochord; this requires the dorsalizing Organizer secretions.

Genes encoding Noggin, Chordin, Follistatin and Frzb are able to induce a second axis. Similarly, the major component of the Nieuwkoop center (β-catenin) can also induce a second axis. Thus, the major role of the Nieuwkoop center is to induce the organizer, which in turn produces BMP and Wnt antagonists. For example, injection of mRNA of noggin or chordin into an early embryo can induce a second axis. This second axis is due to the resultant protein’s ability to dorsalize ventral mesoderm (resulting in the notochord and somites), and to dosalize the overlying ectoderm (resulting in the neural plate).