13.2.      Unequal radial holoblastic cleavage

Holoblastic and radially symmetrical cleavage takes place in frog and salamander embryos, just similar to echinoderm cleavage. In the amphibian egg, the yolk is concentrated in the vegetal hemisphere, which is a barrier to cleavage. As a result, the first division starts at the animal pole and gradually takes place into the vegetal region.

The first cleavage division in salamanders and frogs involves bisection of the grey crescent (the region opposite the point of sperm entry). Furrow is formed during the frog egg's first cleavage, between the animal and the vegetal hemispheres. Furrow completely divide the animal cytoplasm but division at the vegetal pole is not completed, at this time, the second cleavage, which is meridional (at right angles to the first one) get starts at animal pole. 

As the cleavage, furrow cleaves the yolky cytoplasm of the vegetal hemisphere. The second cleavage is then already started near the animal pole. This cleavage is (but at the right angle to the first one) meridional. The third cleavage is equatorial but due to concentrated yolk present at vegetal pole, the third cleavage furrow is not really at the equator but is the shift toward the animal pole. As a result formation of four large blastomeres (macromeres) in the vegetal region and four small animal blastomeres (micrometres) in the animal region into the embryo.

Up to the twelfth cell, cycle blastomeres continue to divide at the same rate in spite of their unequal sizes. As cleavage progresses, the small number of large, yolk containing macromeres appear to be present at vegetal region but animal region turn into the crowded region with numerous small cells. The stage at which amphibian embryo contain 16 to 64 morula stage, when cell no reaches up to 128, at that stage blastocoel becomes clear and the stage known as a blastula. Two major functions are performed by blastocoel,

It allows cell migration through gastrulation.

It prevents the premature interaction of cell present above to its position and cell present beneath to its position.

If premature interaction occurs when the expression of the cell gets affected. Due to this prevention, the vegetal cells destined to become endoderm do not interact with those cells in the ectoderm fated to give rise to the skin and nerves. Because when embryonic new cells from the roof of the blastocoels in the animal hemisphere (a region often called the animal cap) get placed by Nieukoop next to the yolky vegetal cells which form the base of the blastocoel, as a result, the animal cap cells differentiate into mesodermal tissue in spite of ectoderm. Blastomeres are kept together by many cell adhesion molecules, most important of these is EP-cadherin if EP-cadherin not present due to any reason, it causes dramatically reduced adhesion between blastomere in turn cause destruction of the blastocoels.

13.3.      The mid-blastula transition: Preparing for gastrulation

Activation of the genome is prerequisite for gastrulation. During early cleavage, a few genes appear to be transcribed. Up to late of the twelfth cell cycle, many nuclear genes are not activated. During this time, the embryo experiences the occurrence of mid-blastula transition (MBT). At this time blastomere become motile, the cell cycle attains gap phases and transcription of different gene takes place. During mid-blastula transition alteration of the ratio of chromatin to the cytoplasm in the cell takes place. Chromatin modification is one of the events that trigger the mid-blastula transition.

First, particular set promoters are demethylated, permitting the transcription of these genes. However, loss of methylation on the promoters of genes during the time of late blastula stages takes place, in turn, cause activation of the gene at mid-blastula transition. Methylation of the fourth lysine of Histone H3(H3K4) cause activation of that respective gene.

A crucial role is played by nucleosomes associated with the promoter in controlling the timing of gene expression at the midblastula transition, after chromatin remodelling  various transcription factors, for example, VegT protein (protein form by maternal mRNA which is localized in vegetal cytoplasm) induce the vegetal cell to form endoderm and start releasing factors that cause induction of the cells placed above to them to become the mesoderm.

Gastrulation in the process of highly integrated, Co-ordinated

Precise cell migration of prospective endodermal to mesodermal areas to their definite position in the embryo. The movement of cell is called a morphogenetic movement. This creates a triploblastic embryo.

There are three types of morphogenetic movement during gastrulation.

The invagination-the process of invagination is initiated at the future dorsal surface of the embryo. Invagination in inward movement of cells.

Bottle cells appear to function by creating a local invagination.

13.4.      Amphibian Gastrulation

The most detailed investigations had been carried out on Xenopus larva in recent years, so LET'S TALK about the mechanism of gastrulation in that species.

13.4.1.   Vegetal rotation and the invagination of the bottle cells

Amphibian blastula brings cells containing those areas inside the embryo fated to form the endodermal organs, surround the embryo with those cells which destined to form ectoderm and place some cell between them incorrect location destined to form mesoderm. Through fat mapping, we identify that the cells have different fate depend on their residing location like superficial layer of cell give rise to endoderm and ectoderm and deep layer of cells give rise to mesoderm, mesodermal precursor positioned at that area which is placed underneath the surface in the equatorial (marginal) region of the embryo.

Gastrulation movements in frog embryos act to position the mesoderm between the outer ectoderm and the inner endoderm. Movements which are responsible for the positioning of mesoderm begin to initiate on the future dorsal side of the embryo which is present within the region of the grey crescent located just below the equator. Within the region of the grey crescent or appropriately the region of marginal zone (the region surrounding the equator of the blastula and meeting of where the animal and vegetal hemispheres take place) of the grey crescent a slitlike blastopore is formed due to invagination of the cell of that area. These cells are called as bottle cells. During the early gastrulation, inward movement of the bottle cell of the margin takes place, as a result, the dorsal lip of the blastopore form, and then the involution of the mesodermal precursors under the roof of the blastocoel also occur.

The shape of those cell changes dramatically, which is required to initiate gastrulation. The shape of cells get changes in a manner, the displacement towards the inside of the embryo of the main body of each cell takes place and also each cell through the way of slender neck maintaining the contact with the outside surface of the embryo and due to this condition of cells blastopore form, bottle cell line. The archenteron (primitive gut) is formed by the invagination of those cells. At marginal zone, the endodermal cells are not as big or as yolky as they present at the most vegetal blastomeres. When the subsurface marginal cells are carried to get in touch with the basal region of the surface blastomeres, the subsurface marginal cells start to involute on the extracellular matrix secreted onto the basal region of bottle cell. In the progress of such involution movements, bottle cells are no longer necessary. After removal of bottle cell at that time involution along with blastopore formation and closure carry on. Thus, in Xenopus,  involution of the subsurface cells are the major factor rather than the movement of superficial marginal cells.

13.4.2.   Involution at the Blastopore Lip

In the next phase, marginal zone cells involute, whereas the animal cells undertake epiboly and meet at the blastopore. While the migrating marginal cells arrive (and become) the dorsal lip of the blastopore, move along the inner surface of the outer animal hemisphere cells (i.e., the blastocoel roof) by turning inward. Thus, the lip of the blastopore containing cells is constantly changing.

13.4.3.   Progressive Determination of the Amphibian Axes

The unfertilized amphibian egg has polarity along the animal-vegetal axis present within the unfertilized egg. Thus, even before fertilization, the germ layers into the oocyte can be mapped. The cells of the ectoderm (skin and nerves) are formed by the animal hemisphere blastomeres. The cells of the gut and related organs (endoderm) is formed by the vegetal hemisphere cells. Mesodermal cells are formed by the internal cytoplasm around the equator. The vegetal cells have two major functions perform by vegetal cells. Vegetal cells are differentiated into endoderm. The other cells cause the formation of mesoderm by induction of the cells immediately above the vegetal cell.

Transcription factor VegT encoding mRNA is anchored to the vegetal hemisphere’s cortex and is allocated to the vegetal cells during cleavage. When VegT transcripts are destroyed by any mean, the entire embryo develops into epidermis only. Zygotic genes transcription is activated by VegT, which includes gene of the family of TGF-\beta paracrine factor, paracrine factor Vgl and at least six Nodal-related genes. Mesoderm induction hardly occurs or does not occur on the blocking of either Nodal or Vgl signalling. Which cells from which germ layer, this indication is provided by the animal-vegetal polarity events that are triggered at fertilization and recognized during gastrulation specified the anterior-posterior, left-right and dorsal-ventral axes. Formation of the dorsal-ventral axis is linked to the formation of the anterior-posterior axis. Sperm entry defines the dorsal-ventral axis and the anterior-posterior axis is established by the movement of the involuting mesoderm.

Migration of first endo mesoderm over the dorsal blastopore will induce the ectoderm on top of it to produce anterior structures, like forebrain. Involution of mesoderm later (through the dorsal blastopore lip) permits the ectoderm to form more posterior structures, like spinal cord and hindbrain Primary embryonic induction is the process under which formation of central nervous system occurs through interaction with the underlying mesoderm, it is the principal ways that the vertebrate body organization takes place. Descendants of dorsal blastopore and dorsal blastopore themselves are called "the Organizer," and this region is diverse from all the other parts of the embryo.

13.4.4.   Molecular Mechanisms of Amphibian Axis Formation

The experiments of Spemann and Mangold showed that the dorsal lip of the blastopore, dorsal mesoderm and pharyngeal endoderm constituted an "organizer" and the formation of embryonic axes was carried out by the instruction of this organizer.   

Formation of organizer

Unexpected answer is given by the resent research that at the right time the position of these is at the right place, it is the point where two signals come together. The first signal defines that the cells are dorsal. The second signal defines that the cells are mesoderm.

13.5.      The dorsal signal: \beta-Catenin

The mesoderm cells of organizer become specific due to the presence of the special group of vegetal cells. Ectoderm beneath the organizer provides the organizer its properties. When the animal cap (presumptive ectoderm) and vegetal cap (presumptive endoderm) produced were recombined, then the induction of animal cap cells takes place as a result of the formation of mesodermal structures such as notochord, muscles, kidney cells and blood cells. Endodermal (vegetal) fragment was taken from the dorsal or the ventral side. It defines the polarity of induction that the dorsal mesoderm or ventral mesoderm) form by the animal cell.

Ventral (mesenchyme, blood) and intermediate (kidney) mesoderm is induced by ventral and lateral vegetal cells (those closer to the side of sperm entry). Dorsal mesoderm components (somites, notochord) and also including the organizer properties. Organizer gets induced by the dorsal-most vegetal cells of the blastula. The group of those dorsal-most vegetal cells is called as the Nieuwkoopcenter. Some experiment related to transplantation and recombination.

Transplantation of the dorsal most vegetal blastomere from one blastula into the ventral vegetal side of another blastula, two embryonic axes are formed.

During the blastula stage, the vegetal hemisphere releases a dorsal signal by the Nieuwkoop centre and a ventral signal by lateral-ventral blastomeres.

When recombine uppermost animal tier of a fluorescently labelled embryo of 32-cell Xenopus with single Vegetal blastomeres from a 32-cell Xenopus embryo, as a result, Induction of the animal pole cells to become dorsal mesoderm done by the dorsalmost vegetal cell, as expected. Induction of the animal cells to produce either intermediate or ventral mesodermal tissues done by the remaining vegetal cells.

Cortical cytoplasm from the dorsal vegetal cells of the 16-cell Xenopus embryo injected into ventral vegetal cells cause the formation of secondary axes Animal cells get induced by dorsal vegetal cells to become dorsal mesodermal tissue. Nieuwkoop center within these vegetal cells form by the β-catenin, a Wnt signalling induced nuclear transcription factor or perform as an anchor for cell membrane cadherins. In dorsal tissues formation, β-catenin is a key player and β-catenin mutant lack dorsal structures. A secondary axis gets produced by the injection of β-catenin at the ventral side.

Micromeres of the sea urchin embryo also get specify by β-catenin. Through maternal mRNA β-catenin is primarily synthesized throughout the Xenopus embryo. During the cytoplasmic movements of fertilization, β–catenin is started to accumulate in the dorsal region and throughout early cleavage get accumulated preferentially at the dorsal side. Within the nuclei of the dorsal cells β-catenin accumulation gets to visualize and shows to cover both the organizer regions and Nieuwkoopcenter.

VegT (maternal TF) & Vg1(maternal TGF- ß member) in lateral-ventral blastomeres activates low-level zygotic expression of the TGF-ß family member, xnr1,xnr2, and xnr4 and the higher level expression in Nieuwkoop centre occurs because of ß-catenin. ß-catenin accumulates on the dorsal side of the embryo and activates transcription of dorsal genes like siamois, twin, and xnr3 by forming complexes with the transcription factor XTcf3. Siamois and twin expressing dorsal-vegetal cells form a region that is known as Nieuwkoop centre. The Nieuwkoop Center is the dorsal- and vegetal-most cell of the early blastula. It gives rise to the primary organizer, which is the dorsal lip of the blastopore (DLB).

The Xnrs high levels induce dorsal-specific genes (e.g. goosecoid) to form dorsal mesoderm and low level induces ventral-specific genes (e.g.xbra) to form ventral mesoderm. The cells from dorsal mesoderm can induce the other group of cells to influence their fate, so the region of such inductive cells called as the organizer or Spemann organizer. The organizer produces the dorsalizing signals like noggin, chordin and follistatin that inhibits the BMP that would otherwise ventralize the mesoderm and activate the epidermal genes in the ectoderm.

The organizer consists of pharyngeal endoderm, head mesoderm, notochord, and dorsal blastopore lip. The BMP4 is a ligand of the TGF-b family that induces ventral mesoderm and epidermal fates like blood, kidneys, muscles and also suppresses neural ones. In the head region, an additional set of three proteins (Cerberus, Frzb, Dickkopf) block the Wnt signal from the ventral and lateral mesoderm.

They have only weak dorsalizing activity but they neuralized animal caps, by cooperating with BMP inhibitors. The neurogenin is also a protein that acts as a transcription factor and activates a series of genes that helps in neural differentiation of ectoderm.

The organizer is situated at dorsal pore lip induce the neural tube and it also specifies the regions of the neural tube. Some transplantation experiment are given below indicating the potent induction via organizer, where the results from Blastopore transplants vary with time

If dorsal blastopore lip is grafted to the ventral side of the marginal zone, it leads to the twinned embryo in which the second embryo can have the head, trunk and, sometimes and a tail but will be joined to primary embryo along the axis. (Early gastrula induces a full 2nd embryo).

Mid-gastrula induces trunk and tail.

Late gastrula induces only tail.

The factors responsible for the posterior specification of Xenopus embryo are eFGF, RA and Wnt3a that also activate the expression of posterior HOX genes, which regulates the specification of body segments along the anterior-posterior axis. The Wnt3 may suppress the anterior genes and allowing the FGF and RA to help in the patterning of the spinal cord and hindbrain respectively. The right-left axis specification among all vertebrates is critically influenced by Nodal gene expression, that is Xnr-1(Xenopus nodal-related) in LPM of left side only and expressed by through the activation by Vg 1 protein, Xnr1 also activates the expression of protein pitx-2 helping in left axis specification. Such tadpole embryos hatch out from the egg in the form of larva and then develop in aquatic medium to transform them into the adult.

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