## MOLECULAR MECHANISMS OF AMPHIBIAN AXIS FORMATION

### MOLECULAR MECHANISMS OF AMPHIBIAN AXIS FORMATION

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 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.O rganizer 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 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 get 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.