1.Presumptive areas of blastula are predestined for a specific part in future development.

 Transplantation experiments: 1) autoplastic- piece of an embryo transplanted to another site in the same embryo 2) homoplastic- host (recipient) and donor individuals are of the same species 3) heteroplastic- host and donor species belong to the same genus 4) xenoplastic- host and donor species belong to different genera.  Only autoplastic and homoplastic transplants are successful in adult vertebrates.  Success in xenoplastic transplants have been achieved in invertebrates, and between embryos of salamanders and frogs, mammals and birds.

2. Presumptive areas have definite normal fates or prospective significance.  At the time of the stages used in these experiments, their fates have not been fixed 3. Ability of the parts of an early embryo to develop in more than one way is called prospective potency.  Prospective potency of epidermis and neural system areas of gastrula embryo are practically identical (includes mesodermal, endodermal tissues), in spite of their different prospective significance. Grafted material develops in conformity with its new surroundings.  An epidermis area of an early gastrula was heteroplastically transplanted into the neural system area of another embryo in the same stage of development, and vice versa.

 Transplanted epidermis became neural plate of host; reverse transplantation showed an opposite result.  Presumptive ectoderm transplanted into marginal zone of early gastrula: graft was drawn into blastopore and found in host interior

4. At the end gastrulation, prospective potencies of presumptive epidermis and neural system areas will be narrowed down to their prospective significance.  The fixing of the fate of a part of the embryo is called determination.

5. The determination of parts of the ectoderm depends on the surroundings in which the endodermal cells find themselves, and does not come from causes inherent in the ectoderm itself.  Hilde Mangold- heteroplastic transplant of the dorsal lip of the blastopore of an early gastrula to the lateral lip of the blastopore of the host early gastrula resulted to a graft which gave rise to a secondary embryo in the host.  Comparison of traits reveal that both donor and host embryos participated in formation of parts of secondary embryo.  Graft influenced development of certain parts of embryo (induction); source of influence is called inductor.

6. Contact with archenteron roof (chordomesoderm) determines development of mid-dorsal ectoderm into the neural tube.  Grafts taken from dorsal lip of blastopore and adjoining marginal zone induce neurulation in early gastrula. 7. Reacting cells must be competent, a particular state that enable them to differentiate under the influence of the inductor.  Competence of host cells to inductor is highest in the early gastrula, declines in late gastrula, and fades away at neurulation. Absence of inductor causes neural ectoderm to differentiate into epidermis. 8. Due to its ability to induce a complete 2ndary embryo when transplanted, the amphibian dorsal lip of the blastopore (also the posterior edge of blastodisc in fishes, or the anterior half of the primitive in birds) is called the primary organizer.

9. The capacity of a certain part of the egg to serve as the organization center is not fixed at the start of development, but introduced gradually.  Appearance of a definite amphibian organization center is dependent on interaction of cortical and subcortical cytoplasm of gray crescent, and the presence of yolky cytoplasm of vegetal hemisphere.  The primitive streak in birds is dependent on underlying hypoblast for its formation; discrepancies in the orientation of the epiblast and hypoblast result to primitive developing in accord with hypoblast orientation. 10.Tissues of mammalian (rabbit) gastrula have competence for neural induction with chick primitive streak as an inductor, and vice-versa.

1. Regional differentiations in the normal embryos are controlled by a balance between neuralizing and mesodermalizing substances distributed in the form of gradients. a.Regional inductions:  Archencephalic (forebrain, eyes and nose rudiments)- neuralizing (dorsalizing or activating) factor alone  Deuterencephalic (hindbrain and ear vesicles)- low mesodermalizing + neuralizing factor  Mesodermal (notochord, muscle, kidney and limbbud) mesodermalizing (caudalizing or transforming) factor alone  Spinocaudal (trunk organs and tailbud)- high mesodermalizing factor + low neuralizing factor

The dorsal lip of blastopore of early gastrula contains head inductor; same structure at late gastrula contains spinocaudal inductor. The gradient of the neuralizing substance is highest mid-dorsally and declines towards the lateral and ventral parts of the embryo. Mesodermalizing substance is concentrated at the posterior end of embryo and forms a declining gradient both in the anterior and lateral direction.

3. Any factor that affects the gradient affect the whole morphogenetic system.  Defects spread from the front end backward: nose rudiments and forebrain, eyes, posterior parts of brain, gill clefts, ear vesicles.  Raise the level of the dorsoventral gradient by treatment with sodium thiocyanate: increase in size of neural system.  Depressing the level of the dorsoventral gradient gradient at its highest level by exposing embryo to magnesium or lithium chloride or removing part of the archenteron roof beneath anterior end of nervous system results to injured embryos that develop cyclopia. Cyclopia and similar defects may result from toxicosis of mother during early gestation, e.g. contact with German measles illness or Rubella.

Babies who have too little of the Sonic hedgehog protein will be born without noses, and in the most severe cases cyclopia. An abundance of Shh produces eyes that are far apart, an abnormally wide nose, or even duplicated facial features, called diprosopus. This can range from only two noses, to a complete full second face with two mouths, two noses, and up to four eyes. Each nose has only a single nostril leading into the sinuses.

4. Gray crescent area is the center of gradient for archencephalic development:  UV irradiation of gray crescent caused microcephaly, anencephaly.  Injection of oocyte nucleus material or cytoplasm of untreated fertilized egg into the blastocoele restore embryo to normal development.

Induction is region specific. Otto Mangold transplanted 4 successive regions of archenteron roof of late gastrula newt embryos into the blastocoeles of early gastrula embryos.

Spemann and Mangold showed that the dorsal lip of the blastopore constitutes an organizer that has the ability to: initiate gastrulation movements; dorsalize ectoderm into neural ectoderm; cause neural plate to become neural tube; become dorsal mesoderm and notochord; dorsalize surrounding mesoderm into lateral mesoderm.

The field of molecular biology was refined in the late 1980s, enough to provide a starting point to understand what genes encoded the products of the organizer.

AMPHIBIAN INDUCTION

In the unfertilized egg, VegT and Wnt 11 mRNA are concentrated in the vegetal pole. Powered by microtubules and motors at fertilization, the cortical rotation displaces Wnt 11 mRNA opposite to the sperm entry point. This is crucial for giving the dorsal side regional identity, and leads to the formation of a signaling center in the vegetal region called the Nieuwkoop center.

Vegetally localized mRNA of VegT and Vg1, are endoderminducers. VegT directs synthesis of Xnr proteins which are mesoderm-inducing signals. Dorsalizing factors Wnt 11 and Xnr activate the Wnt pathway that leads to the accumulation of -catenin in the dorsal side. -catenin acts with VegT to produce higher levels of Xnr.

Translocation of the Dishevelled protein to the dorsal side of the egg during rotation stabilizes -catenin in the dorsal cells of the embryo, which establishes the Nieuwkoop center.

 F-catenin complexes with Tcf3 which activates the expression of the siamois gene.  The siamois product and a TGF- signal ((Vg1/ nodal signaling pathway) activate the goosecoid gene that specify organizer genes.  Siamois also interacts with Xnr to activate goosecoid.

 The organizer works by secreting inhibitors of BMP4, e.g., Noggin, chordin, and follistatin. They dorsalize the adjacent mesoderm by inhibiting the ventralizing signal. What is the ventralizing signal?

BMP signals are powerful ventralizing factors that induce the expression of epidermal-specific genes. Varying inductions are created by interaction of BMP4 with BMP antagonists from the organizer, e.g. low BMP doses activate muscle formation; intermediate levels instruct cells to become kidney; high levels activate blood cell and CT formation.

 In the absence of BMP signaling, neuralizing transcription factors (X1pou2, SoxD) are produced.  These factors in turn activate the neurogenin gene for the transcription factor NeuroD that cause the differentiation of the cell into a neuron.  In the presence of BMP signaling, epidermalizing transcription factors (Msx1, GATA1, Vent) are generated, leading to the activation of the pathway enabling the cell to become a keratinocyte.

AVIAN INDUCTION
1. In chick embryos, activation of various secreted factors (Vg1, Nodal, Wnt8C, FGF8 and Chordin); and transcription factors: (brachyury and goosecoid) adjacent to the site of streak formation, is required for the coordinated function of the organizer.  Removal of the hypoblast in the chick results in correctly patterned ectopic streaks, suggesting that the hypoblast may inhibit formation of the primitive streak by secreting an antagonist to Nodal. 2. BMPs do not inhibit neural induction, nor does ectopic expression of chordin in the non-neural epiblast cause neural induction. 3. Recent evidence suggests that the anterior visceral endoderm (AVE) is providing these signals for anterior neuron production.

4. FGFs produced in Hensen's node and primitive streak, appear to induce trunk and hindbrain neuronal expression in the epiblast cells.  Other posteriorizing signals (Wnt3a, retinoic acid, eFGF) can influence the anterior-posterior specification of the neural tube.  In the head region, an additional set of proteins (Cerberus, Frzb, Dickkopf) block these Wnt signal from the ventral and lateral mesoderm.

5. Ciliary beating around the primitive node displaces FGF8 to the left, causing the expression of nodal and lefty-1 on the left side of the streak, thereby activating the Pitx2 gene responsible for rotation of the gut and stomach, spleen and lung lobation. 6. The D-V axis is defined by the embryonic-abembryonic (trophoblast opposite ICM) poles. In birds, gravity is critical in determining the AP axis, while pH differences appear crucial for distinguishing dorsal from ventral.

In mammals the primitive node is an embryonic organizer. (remove HN: no neural tissue development; transplant HN to host embryo: 2nd neural axis is induced). The mammalian node is not a classic organizer because it can't act alone to induce the embryonic axis but requires other anterior germ tissues to be fully effective.

Progressive cellular interactions characterizes the organizer activity of the Hensen's node region in lower vertebrates.

In mammals, there are 2 signaling centers: one in the node, and one in the AVE (hypoblast), which becomes part of the head organizer. Prechordal plate (head organizer) pass through the node and become associated with the endoderm caudal to the oropharyngeal membrane. They are followed by the chordamesoderm (precursor of the notochord), which is the major axial signaling center of the trunk. In birds, the prechordal plate alone can induce head parts, but in mammals, the prechordal plate mesoderm sustains the induction of the anterior visceral endoderm.

 The expression of Nodal from the primitive streak drives the expression of Shh and Goosecoid.  Goosecoid- activates genes of organizer. With SHH, goosecoid activates expression of HNF-3B, which also results in the expression of Noggin and Chordin.  Noggin, chordin, follistatin- BMP antagonists from the organizer, they dorsalize mesoderm. They induce only forebrain and midbrain types of tissues.

 BMP4- secreted throughout the bilaminar disc, acts with FGF to ventralize mesoderm into intermediate and lateral plate structures. The fate of the entire ectoderm is dependent upon BMP concentrations: high levels result in epidermis formation, lower levels at the border of the neural plate and nonneural ectoderm induce the neural crest. BMP inhibition leads to neural plate induction.  HNF-3F - from the organizer, this is required for node formation, initiation of notochord function, and establishment of midline structures cranial to the node.  T /brachyury - induced by products of HNF-3F and goosecoid genes, they are necessary for normal movements of mesodermal cells through the primitive streak (T gene mutants have gross caudal body defects in humans). Brachyury also antagonizes BMP4 to dorsalize mesoderm in caudal regions of the embryo.

 To produce a head, the BMP4 and Wnt signals are blocked by transcription factors Otx-2 and Lim-1 (a homeobox gene); and the signaling molecule Cerberus-related 1 HeSXL (mutants produce headless phenotype).  WNT3a and FGF - induce caudal neural plate structures: hindbrain and spinal cord.  Retinoic acid - organize the cranial-to-caudal axis by regulating expression of homeobox genes.

Hox genes pattern the anterior-posterior axis and help to specify positions along that axis. If Hox genes are knocked out, segment-specific malformations can arise. o Causing the ectopic expressions of the same genes can alter the body axis. o Homology of gene structure and expressions between Drosophila and mammalian Hox genes suggest that this patterning mechanism is evolutionarily ancient.

Expression patterns of genes that regulate somite differentiation. SHH and noggin, secreted by the notochord and floor plate of the neural tube, cause the ventral part of the somite to form sclerotome and to express PAX1, which in turn controls chondrogenesis and vertebrae formation. WNT proteins from the dorsal neural tube activate PAX3, which demarcates the dermomyotome. WNT proteins also direct the dorsomedial portion of the somite to form epaxial (back) muscles and to express the muscle-specific gene MYF5.

The middorsal portion of the somite is directed to become dermis by neurotrophin 3 (NT-3) expressed by the dorsal neural tube. Hypaxial (limb and body wall) musculature is derived from the dorsolateral portion of the somite under the combined influence of activating WNT proteins and inhibitory BMP4 protein, which together activate MYOD expression.

 VEGF induced by Shh, this induces hemangioblasts to form hematopoietic stem cells, the precursors of all blood cells. Peripheral hemangioblasts differentiate into angioblasts, the precursors to blood vessels.  PDGF and TGF directs maturation and modeling of the vasculature until the adult pattern is established.  Notch pathway - induced by VEGF expression, this specifies arterial development and suppression of venous cell fate through expression of ephrinB2.  PROX1 - induces lymphatic vessel differentiation.  Blood vessel and nerve outgrowth appear to involve guidance factors (e.g., NGFs).

Angiogenesis is also regulated by VEGF, which stimulates proliferation of endothelial cells at points where new vessels will sprout from existing ones. Final modeling and stabilization of the vasculature are accomplished by PDGF and TGF- .

Blood vessels form in two ways: vasculogenesis (top), in which vessels arise from blood islands, and angiogenesis (bottom), in which new vessels sprout from existing ones. During vasculogenesis, FGF2 binds to its receptor on subpopulations of mesoderm cells and induces them to form hemangioblasts. Then, under the influence of vascular endothelial growth factor (VEGF) acting through two different receptors, these cells become endothelial and coalesce to form vessels.

 Chordin, activin, nodal, sonic hedgehog are also involved in the L-R asymmetry of certain body parts. The protein activin, a member of the TGF- signaling family, directs development of parts in the right axis.  There are over 24 genes currently known for L-R assymetry, and gene mutations downstream in the cascade are responsible for right-left switching for various organs (e.g., heart as seen in situs inversus, Kartageners syndrome).

The Vg1 protein activates a Nodal protein solely on the left side of the body. As in other vertebrates, the Nodal protein activates expression of Pitx2, which is critical in distinguishing leftsidedness from rightsidedness in the heart and gut tubes. Activation of transcription factor Pitx2 regulates genes involved in lateral plate mesoderm differentiation. Other factors such as Shh, FGF8, Lrd and Kif are also involved. The nodal flow model suggests that morphogens and signaling molecules involved in defining the LR axes is driven by the action of cilia that cause molecules to flow from right to left across the primitive node. Cells in the node with mutations that affect ciliary action (e.g. null mutations for Lrd which encodes ciliary dynein) alter L-R symmetry.

ERRORS OF GASTRULATION

Spinal cord Abnormalities

Little Samuel Armas was diagnosed with spina bifida and would not survive if removed from his mother's womb. His mother knew of Dr. Bruner's remarkable surgical procedure: he performs these special operations while the baby is still in the womb. 'The tiny hand of the 21-week-old fetus emerges from the mother's uterus to grasp the finger of Dr. Joseph Bruner as if thanking the doctor for the gift of life.

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