Xenopus laevis (South African clawed toad)
Frogs and other amphibians have long been model systems to study embryology, because their eggs are easy to collect and observe during development. Unfortunately, because the animals themselves are hard to maintain and breed, their genetics is virtually nonexistent.
The major topic we will address with Xenopus is establishment of the vertebrate body axis.
Egg: moderately yolky. Animal hemisphere is mostly cytoplasmic, has pigment in granules located in cortex just beneath the plasma membrane. Vegetal hemisphere is yolky, with clear cortex.
Fertilization: sperm penetrates egg at animal hemisphere. Plasma membrane depolarizes, cortical granules fuse with plasma membrane, released enzymes create fertilization envelope that prevents polyspermy (fertilization by more than one sperm). Cortical contraction rotates pigmented animal cortex toward sperm entry point (s.e.p.), exposes clear cytoplasm at side opposite s.e.p. – “gray crescent.”
Cleavage: holoblastic – 1st two divisions are longitudinal. The first division passes through the s.e.p. and bisects the gray crescent. The second division is orthogonal to the first, and they intersect at animal and vegetal poles. 3rd division is equatorial. Blastomeres in animal half divide faster than vegetal blastomeres.
Blastula: hollow ball of cells. Animal half has single layer of cells forming a dome over the blastocoel cavity. This cell layer often referred to as the “animal cap.” Animal cap cells will differentiate into ectoderm. Vegetal cells will form endoderm. Cells at the margin between animal cap and vegetal cells will form mesoderm.
Gastrula: Invagination of cells begins in vegetal margin opposite the original s.e.p. – this site is called “dorsal lip of the blastopore.” Blastopore enlarges in a ring around the vegetal pole. Invagination of cells creates a new cavity, the archenteron, or primitive gut. The roof of the archenteron is initially composed of mesodermal cells from the dorsal lip. Gastrulation places the three primary germ layers, ectoderm, mesoderm and endoderm, in their final positions.
Neurula: A second infolding of ectodermal cells above the roof of the archenteron creates the neural tube, characteristic of all vertebrates. The neural tube differentiates into brain and spinal chord.
Organogenesis: Limb buds form and develop, organs and bone differentiate.
Blastomere separation: Each of the first two half-blastomeres, when separated from the other, can form a complete tadpole, and eventually, a frog. However, after the second division, the quarter blastomeres can no longer form complete tadpoles.
Spemann and Mangold’s “organizer”: In the 1920s Hans Spemann and Hilde Mangold transplanted the dorsal lip of blastopore into another gastrula stage embryo (more information in http://embryo.asu.edu/pages/spemann-mangold-organizer). The recipient embryo formed two embryonic axes. The transplanted dorsal lip differentiated into a notochord, as it would have normally, and induced the overlying presumptive ventral ectoderm to differentiate into a neural tube. Thus the dorsal lip of the blastopore, which forms mesodermal notochord, has the power to induce neural differentiation of uncommitted ectodermal cells, and subsequently “organize” an entire secondary axis.
Transplantation of the gray crescent: How and when does dorsal specification occur? A.S.G. Curtis surgically removed the gray crescent cortex from just-fertilized or 8-cell embryos. The 8-cell embryos survived and developed normally, but the just-fertilized embryos did not gastrulate. If the gray crescent cortex was implanted into the presumptive ventral margin of just-fertilized or 8-cell embryos, embryos that received the gray crescent transplants at the 1-cell stage, but not at the 8-cell stage, gastrulated at both gray crescent sites, with 2 blastopores, and developed a second embryonic axis – became twins! His reports, published in 1962, caused a flurry of research into the gray crescent cortex to identify the cytoplasmic determinants that may be located there, that could induce formation of a second dorsal lip.
UV irradiation and rotation: UV (254 nm) irradiation of the vegetal hemisphere prior to the 1st cell division also prevents gastrulation. Cell division proceeds at normal rates, but no gray crescent is formed. The result is a ventralized embryo, with no dorsal structures and no anterior structures – there is no A-P axis. Remarkably, simply tilting the egg can completely rescue UV- irradiated eggs, and restore gastrulation and normal embryo development. The eggs must be tilted prior to the second division. The direction of tilting defines the site of the dorsal lip of the blastopore. Even more remarkably, tilting non-irradiated eggs can induce twinning! Tilting can induce twinning if done between fertilization and the second division.
Q: What is the relationship between UV, tilting and the gray crescent? What does UV destroy, and how does tilting rescue UV-fried eggs?
Induction of mesoderm by vegetal cells and the Nieuwkoop center
Peter Nieuwkoop demonstrated that isolated vegetal cells induced overlying animal cap cells to form mesodermal tissues. Moreover, dorsal vegetal cells induced animal cap cells to form embryoids with axial organization and a nervous system. In effect, the dorsal vegetal cells organized the organizer. The dorsal vegetal cells were named the Nieuwkoop center.
Lithium: treatment of cleavage stage embryos with lithium results in dorsalized embryos.
Conclusions: Embryonic axis formation involves 3 signals: signal 1 is produced by vegetal cells to induce marginal animal cap cells to mesoderm fate; signal 2 is produced by dorsal vegetal cells (the Nieuwkoop center) to induce formation of the “organizer”. The “organizer” then secretes signal 3 that mediates dorso-ventral patterning.
Q: What are the molecular signals involved in induction of mesoderm, and induction of neurulation?
Q: How do these molecular signals regulate cell differentiation, particularly axis formation?
Q: what role does Notch signaling pathway play in Gastrulation and Nerulation?
Experiments have shown that Notch signaling pathway is involved in the building of the embryonic dorsal midline in X. laevis, favoring floor plate against notochord development.
What are the inductive signals from the Nieuwkoop center and Spemann’s organizer?
3 assay systems:
1. Differentiation of isolated animal cap tissue into axial mesoderm. Isolated animal cap tissue from blastula stage embryos differentiates into ectoderm. Animal cap tissue placed over isolated vegetal tissue differentiates into mesoderm. Can identify candidate mesoderm inducers, and neural inducers (look for differentiation of animal cap cells into neural tissues).
2. Secondary axis formation (twinning) or ventralization after microinjection into blastula-stage embryos – organizer activity. Injection into ventral vegetal blastomeres can identify inducers of Nieuwkoop center.
3. Rescue of UV-irradiated eggs – dorsalization of either endoderm or mesoderm.
Genes involved in signaling:
- Basic fibroblast growth factor (bFGF) family proteins induce ventral mesoderm.
- Activins and other members of the Transforming Growth Factor-beta (TGF-beta) superfamily can induce dorsal mesodermal differentiation of animal cap. Injection of RNA or protein for activinB, or activin receptor can induce partial secondary axis formation, but lacking head structures. However, activin cannot rescue UV-irradiated eggs.
Activin receptor (a transmembrane serine/threonine protein kinase) can also induce secondary axes when injected into vegetal blastomeres. Truncated activin receptor (a dominant negative mutation, blocks activin signalling) inhibits mesoderm formation and thus ventralizes embryos.
Follistatin (inhibits activin by binding activin in 1:1 molar ratio) also inhibits mesoderm formation and ventralizes embryos.
- Vg-1 is another member of the TGF-beta superfamily that can induce mesoderm in animal caps. Can rescue UV-irradiated eggs. Localized as maternal mRNA in vegetal pole cortical region of unfertilized eggs. Requires proteolytic processing for activity.
- Bmp-4, another member of the TGF-beta superfamily, ventralizes embryos and is expressed in ventral mesoderm.
- Xwnt-8 can rescue UV-irradiated eggs. Member of a family of wnt (wingless and int) proteins in Xenopus. Blastomeres injected with Xwnt-8 form dorsal vegetal cells (Nieuwkoop center) and differentiate into endoderm. But, normally expressed in ventral vegetal cells, and after the mid-blastula transition, whereas endogenous inducer must act earlier in dorsal vegetal cells. Mouse Wnt-1, Xwnt-3A and Drosophila Wg also induce axis, but not Xwnt-4, 5A or 11. Xwnt-5A can induce complete axis duplication if coinjected with mRNA for receptor; e.g., hFz5 (human frizzled 5) (He et al., 1997)
- Effects of Wnt/Fz blocked by injection of GSK-3 beta (glycogen synthase kinase-3; homolog of Drosophila zeste-white).
- beta-catenin (homologue of Armadillo in Drosophila) is a multifunctional protein, component of cell-cell adhesive junction. Normally bound to APC (adenomatous polyposis coli) protein and GSK-3beta (serine-threonine glycogen synthase kinase) and targeted for destruction. Wnt signal stabilizes free beta-catenin and causes rise in level of free beta-catenin. Beta-catenin complexes with Tcf (T-cell factor-lymphoid enhance factor: Tcf- Lef), a family of DNA binding proteins. Mis-expression of either beta-catenin or Tcf in Xenopus eggs alters D-V polarity. Mutations in beta-catenin or APC that cause elevated levels of free beta-catenin promote colon cancer, by forming active transcription complexes with Tcf-4 or Lef-1 and activating cell proliferation genes or genes that inhibit apoptosis (Peifer, 1997).
- Frzb (pronounced “frizbee”) is a secreted protein expressed in the organizer; it has homology to the Wnt binding domain of frizzled (Wnt receptor). Frzb expression is exactly complementary to Wnt8, which is expressed in horse-shoe shaped band of ventro-lateral mesoderm. Frzb antagonizes Wnt8 (Moon et al., 1997; Leyns et al. 1997; Wang et al. 1997).
Genes involved in transcriptional regulation:
Siamois, a homeobox-containing protein, can also rescue UV-irradiated eggs.
Can induce complete secondary axis. First expressed shortly after the mid-blastula transition.
Localized in dorsal endoderm of early gastrulae, just below dorsal lip. Homeobox most closely related to mix1, a Xenopus homeobox gene expressed in endoderm, and to HD1, human gene associated with facioscapulohumeral muscular dystrophy.
VegT T-box transcription factor; mRNA located in vegetal cortex, involved in mesoderm induction and dorso-ventral patterning
Genes with organizer activity (dorsalization and neural induction):
Search for genes specifically expressed in the dorsal lip of the blastopore, and which exert dorsalizing activity by forming secondary axes or rescuing UV-irradiated embryos.
Noggin is a secreted protein expressed in the dorsal lip.
Induced by activin even in the presence of cycloheximide (inhibits protein synthesis).
Not induced by goosecoid or Xnot2.
Encodes glycoprotein of 32 kD, secreted as homodimer.
Binds BMP-4 with high affinity, blocks BMP-4 binding to its receptor and thus antagonizes ventralizing activity of BMP-4 (Zimmerman et al., 1996).
Injection of Xenopus noggin into Drosophila promotes ventral development; mimics elimination of dpp activity; blocks dpp upstream of dpp receptor (Holley et al., 1996).
Goosecoid is a homeobox-containing gene, similar to Drosophila bicoid and gooseberry.
Expressed throughout the organizer.
mRNA appears shortly after the mid-blastula transition, 1 hr before gastrulation.
LiCl-treated embryos express more goosecoid mRNA, UV-irradiated embryos less.
Induced in animal cap explants by activin, even in presence of cycloheximide, but not by FGF.
Injection into ventral vegetal blastomeres induces secondary axis.
Other homeobox-containing genes: Xnot, XFH1 (HNF3 ), Xlim-1
Follistatin is a secreted protein that inactivates activin by combining with activin in a 1:1 molar ratio.
Expressed first in organizer (dorsal lip), then in notochord.
Inhibits mesoderm induction of animal cap explants by activin.
Causes direct neural induction of animal cap explants, with no expression of mesodermal markers noggin or goosecoid, nor ventral marker Xwnt-8.
Expression of follistatin is induced by activin, but not by noggin.
Chordin was identified as a gene that is differentially expressed in LiCl-treated embryos, and induced by goosecoid.
It is expressed in the dorsal lip, and later throughout the notochord and other organizer- derived tissues. Expression begins 1 hr after zygotic gsc.
Both goosecoid and Xnot2 induce chordin expression.
Rescues complete axis formation in UV-irradiated eggs, induces secondary axis formation when injected into ventral vegetal blastomeres.
Cannot by itself induce animal cap explants to differentiate into mesoderm, but can dorsalize mesoderm induced by FGF
Encodes secreted protein of 120 kD with cysteine-rich repeats.
Binds BMP-4 with high affinity, blocks binding of BMP-4 to its receptor (Piccolo et al., 1996).
Cerberus was isolated by differential screening for dorsal-specific cDNAs (Bouwmeester et al., 1996).
Second most abundant mRNA found in dorsal region of gastrula.
Encodes novel secreted protein.
Induces ectopic head formation.
Suppresses formation of trunk-tail mesoderm, defines anterior endoderm.
Inhibits Wnt signalling (Glinka et al., 1997)
Head induction by simultaneous repression of Bmp (with dominant negative Bmp receptor) and Wnt (with Wnt inhibitors) – induction of complete secondary axes, including head. Does not require siamois (Glinka et al., 1997).
Around 40 members of TGF-beta superfamily known to encode secreted proteins (Niehrs, 1996).
Homodimers and heterodimers form a large number of diverse ligands.
Type I and Type II transmembrane receptor serine/threonine kinases form diverse receptor complexes.
Ras involved in intracellular signal transduction of activin, BMP4, and FGF.
BMPs play key roles in ventralizing mesoderm, ectoderm, and possibly endoderm (reviewed by Graff, 1997). Role of dorsalizing factors such as chordin is to bind and neutralize BMP4. BMP4 is a homologue of Drosophila decapentaplegic (dpp)
Opposing actions of chordin and BMP4 determine dorso-ventral polarity in all bilateral animals, although the axes are inverted in protostomes vs deuterostomes.
Other amphibian models:
Check out another neat developmental model amphibian: the axolotl!
To learn more about the regenerative capabilities of the axolotl, click here!
Interested in the pigmentation of amphibians and how they can change colors? Click here!
How do amphibians regenerate their limbs and tails so easily? The axolotl is an organism that does this. Learn more here!
Limb Bud Regeneration
There are certain components that are important during Xenopus limb bud regeneration. A few components that are crucial to regeneration are the Hippo signaling pathway and the transcription factor, Yap1. These components help control limb bud regeneration and provide common principle of regeneration across the phyla. For more information click here!
http://bewareofthefrog.tumblr.com/ Gary McDowell, a Xenopus researcher, blogs about key papers using Xenopus as a model system.
http://studentreader.com/nieuwkoop-center/ A really excellent summary of the current model of the Nieuwkoop Center and Spemann and Mangold’s Organizer. Unfortunately, it has no citations or references.
http://www.ibioseminars.org/lectures/dev-bio-a-evolution/marc-w-kirschner.html Mark Kirschner’s series of 3 seminars on molecular embryology of the acorn worm, a hemichordate, investigating questions of chordate evolution.
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DeRobertis, E.M., 2008. Evo-devo: variations on ancestral themes, Cell 2008:185-195
DeRobertis, E.M., 2009. Spemann’s organizer and the self-regulation of embryonic fields, Mech. Dev. 126:925-941
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