amphibians development

Question 1

amphibians and fish

Answer 1

deuterostome, verbrate, anamniotes

Question 2

amamniotes

Answer 2

amniote embryos are typically found in aquatic environments
- they have comparatively small eggs with moderate amounts of yolk
-anamniotes cleave radially, form a hollow blastula, gastrulate by invagination and epiboly
-called “anamniotes” because the lack of amnions (protective membranes) found in higher vertebrates

Question 3

rotation of the cortical cytoplasm

Answer 3

• sperm typically fuse in the pigmented animal region –> sperm centriole sets up microtubules tracks that promote cortical cytoplasm to rotate relative to central yolky cytoplasm
  -cortical rotation is up to 30 degrees and creates a “gray crescent” where darker yolk pigment can be seen through the clearer cortex
  -this sets up the dorsal/ventral axis with the sperm entry site becoming ventral
• dorsal will form directly opposite and become the site where gastrulation begins

Question 4

frog cleavage

Answer 4

radial holoblastic cleavage
- because of the dense vegetal yolk, initial divisions are asymmetric with regard to size
- dense yolk also slows the division process, so more cells form in the animal region
-by the radial 8 cell stage, the 4 animal blastomeres are called micromeres and 4 vegetal macromeres
-embryos from 16 to 64 cells are often morulas
- by 128 cells, the blastula is formed containing a large blastocoel in the animal half

Question 5

frog fate map

Answer 5

-animal regions become ectoderm and vegetal endoderm
-mesoderm forms from cells between endoderm and the blastocoel
-the blastocoel helps keep animals cells undifferentiated; if they contact the blastocoel bottom, they become mesoderm

Question 6

gastrulation movements in Xenopus

Answer 6

1. blastula; marginal cells set the boundary for presumptive ectoderm from vegetal endo/mesoderm
2. the bottle cells undergo apical constriction to begin invagination to form dorsal blastopore lip; upward vegetal rotation into the dorsal blastocoel begins involution and the differentiation of mesoderm
3. as involution continues, mesoderm and endoderm continue to form against the overlying ectoderm; this process simultaneously creates archenteron (gut) while collapsing the blastocoel; ectoderm begins to extend downward by epiboly, shifting the A/P pole apex
4. involution continues, forming mesoderm and endoderm as cells migrate along the ectoderm; ventral bottle cells form via apical constriction and invagination creates ventral blastopore lip; limited ventral involution creates some local ventral mesoderm
5. the blastocoel collapses completely; dorsal and ventral blastopore lips converge due to epiboly, creating a yolk plug of remnant vegetal cells
6. anterior and ventral mesoderm connect, and the remaining endoderm is completely internalized; dorsal mesoderm forms the notochord, while other areas form mesenchyme; the blastopore closes, eventually the gut endoderm near the site of the blastopore fuses with overlying ectoderm to form the anus

Question 7

germ layer specification in frog embryo

Answer 7

ENDODERM
-vegetal cells become endoderm through the action of maternal determinants including vegT and Vg1 mRNAs –> soon after fertilization VegT mRNA is translated and functions as an activator to express zygotic Sox17, a critical endodermal determinant
- VegT also activates Nodal expression by endoderm, and dorsal endoderm translates Vg1 mRNA

MESODERM
- Nodal and Vg1 = TGF - B ligands that signal to equatorial to become mesoderm –> signaling activates Smad2 –> activates expression of Eomes genes –> mesoderm formation
- ectoderm is formed from animal cap cells in the absence of these signals

Question 8

Dorsal/Ventral B-catenin activity

Answer 8

-B-catenin is made throughout, but protein is degraded rapidly
-Wnt11/Dsh/GBP are stabilizers of B-catenin, and are localized in vegetal cortical cytoplasm
-microtubule transport and cortical rotation moves these factors to the site of future dorsal, opposite the sperm entry site
-these factors inhibit GSK3, which allows B-catenin accumulation and the specification of dorsal identities
- loss of B-catenin causes loss of dorsal structures = centralization
- blocking GSK3 ventrally causes dorsalization = twinning

Question 9

mesoderm types are created by gradient of TGF-B signaling

Answer 9

• recall that Nodal and Nodal-like Vg1 specificy equatorial mesoderm
• vegetal expression of Nodal related genes (Xnr) that also functions as signals
  -B-catenin also activates Xnr, resulting in an Xnr gradient with highest levels dorsally
• the combination of Nodal, Nodal like Vg1, and Nodal related Xnr results in TGF-B signaling/Smad activation dorsally, contributing to organizer formation

Question 10

induction of neural ectoderm by inhibition of BMP signaling

Answer 10

• BMP4 are secreted by mesoderm and ectoderm (essentially everywhere)
  -BMP signaling induces ectoderm to form epidermis
  -however, in the dorsal region, BMP signaling is blocked, so epidermal specification is also blocked
  -in the absence of BMP signals, ectoderm differentiates into neural tissues
  -thus, the dorsal organizer blocks BMP signaling by expressing and secreting BMP antagonists (Chordin, Noggin, and Follistatin)
  -thus, neural development is the “default” state of ectoderm; BMP signaling induces epidermis

Question 11

Noggin and dorsalization

Answer 11

• UV irradiation blocks cortical rotation in early embryos, resulting in a “ventralized” phenotype
  -injection of noggin mRNA into such embryos restores dorsal development in a concentration dependent manner
  -when levels become too high, only dorsalized “noggins” are produced