Analysis of Biological Development (K. Kalthoff)

Updates to Topic 09: Axis Formation and Mesoderm Induction


Answers to Questions in Text

Cortical Rotation and Microtubules in Xenopus Eggs (pp. 207-209)

  1. If the investigators had not removed the perivitelline fluid from the eggs observed, at which level relative to the plasma membrane should they have seen the fastest movements of cytoplasmic components? Answer: Between 0 and 4Ám, because now the cortex rotates around the core of the egg, with the latter keeping its position under the influence of gravity. However, the investigators did not actually use such conditions because the floating egg does not lend itself to microscopy.
  2. Remarkably, Larabell and her colleagues did not find oriented microtubules until 0.4NT, which is well after the onset of slow yolk platelet movements, and bundled microtubules were first observed between 0.5 and 0.55 NT, when cortical rotation has already reached plateau speed. How do you interpret these observations? Answer: These measurements indicate that cortical rotation is initiated either independently of microtubules or by an undetectably small number of oriented microtubules. Once initiated, the shearing movement of the cortical rotation orients more micotubules in the direction of the rotation while those microtubules that are already oriented drive the cortical rotation in the same direction.

Role of Dorsal Vegetal Blastomeres in Axis Formation (p. 211/212)

  1. The fate of the D1/D1' blastomeres in normal embryos is to form endoderm. Therefore, the blastomeres transplanted in the experiment of Gimlich and Gerhart most likely did not form the dorsal mesodermal structures in the rescued embryos but rather induce them. How would you test this hypothesis? Answer: By transplanting D1/D1' blastomeres from a labeled donor, such as a donor injected with a fluorescent dye before cleavage. Indeed, the transplanted blastomeres form endoderm in the recipient, as would have been their fate in the donor.
  2. How do you interpret the blastomere transplantation experiments of Gimlich, Gerhart, and Kageura in the light of the subsequent cytoplasmic transplantation experiments of Yuge, Fujisue, Holowacz, and their colleagues? Answer: The dorsalizing cortical activity that is first present near the vegetal pole and shifts from the vegetal pole to a dorsal subequatorial position during the first cell cycle is segregated into the same blastomeres that display dorsalizing activity later.

Comments

Ken Robinson of Purdue University pointed out to me that several ideas about the polarization of fucoid algae are more controversial than I had realized. The areas of disagreement are explored in the review article by Robinson et al. (1999) quoted below. Also of interest is the research article by Pu and Robinson (1998).

Clarifications and Corrections

p. 210, legend to Fig. 9.13, last sentence should read: "Note that the thickness of the cortex including the shear zone (about 8 Ám ) is exaggerated relative to the egg radius (about 650 Ám)."

p. 216, left column, 2nd line from below should read: "test their hypothesis, they combined..."

New Review Articles

Munoz-Sanjuan I. and H.-Brivanlou A. (2001) Early posterior/ventrl fate specification in the vertebrate embryo. Devel. Biol. 237: 1-17

Nishida H. (2002) Patterning the marginal zone of early ascidian embryos: localized maternal mRNAs and inductive interactions. BioEssays 24: 613-624

Robinson K.R., Wozniak M., Pu R. and Messerli M. (1999) Symmetry breaking in the zygotes of the fucoid agae: Controversies and recent progress. Curr. Topics Devel. Biol. 44: 101-125

Sharpe C., Lawrence N. and Martinez Arias A. (2001) Wnt signalling: a theme with nuclear variations. BioEssays 23: 311-318

Sokol S.Y. (1999) Wnt signaling and dorso-ventral axis specifcation in vertebrates. Curr. Opin. Genet. devel. 9: 405-410

New Research Articles

Bubunenko M, Kress TL, Vempati UD, Mowry KL, King ML. (2002) A consensus RNA signal that directs germ layer determinants to the vegetal cortex of Xenopus oocytes. Devel. Biol. 248: 82-92

Heasman J, Wessely O, Langland R, Craig EJ, Kessler DS. (2001) Vegetal localization of maternal mRNAs is disrupted by VegT depletion. Devel. Biol. 240: 377-386

In Xenopus, the correct localization of the RNA encoding the T-box transcription factor VegT is essential for the correct spatial organization and identity of endoderm and mesoderm (see pp. 202/3 of text). To investigate possible common features between signals that direct VegT mRNA and other RNAs to the vegetal oocyte cortex, Bubunenko et al. compared the uncharacterized VegT mRNA localization signal with the well-studied Vg1 localization signal. Their results suggest that the twomRNAs use the same cis-acting protein-binding motifs and trans-acting factors. They propose that what characterizes RNA localization signals in general is not thenucleotide sequence or secondary structure per se, but the critical clustering of specific redundant protein-binding motifs.
Heasman et al. found that depletion of VegT mRNA caused the release of Vg1 mRNA from the vegetal cortex and a reduction of Vg1 protein, without affecting the total amount of Vg1 transcript. Furthermore, they report that Bicaudal-C and Wnt11 mRNAs were also dispersed, but not degraded, by VegT depletion. In contrast, the localization of Xcat2 and Xotx1 mRNAs was unaffected. This effect was specific to the loss of VegT mRNA and not VegT protein, since an antisense RNA against VegT, which blocked translation without degrading mRNA, did not disperse the vegetally localized mRNAs. Therefore, a subset of localized mRNAs is dependent on VegT mRNA for anchoring to the vegetal cortex, indicating a novel function for maternal VegT mRNA.

Essner JJ, Vogan KJ, Wagner MK, Tabin CJ, Yost HJ, Brueckner M. (2002) Conserved function for embryonic nodal cilia. Nature 418: 37-38

Nonaka S, Shiratori H, Saijoh Y, Hamada H. (2002) Determination of left-right patterning of the mouse embryo by artificial nodal flow. Nature 418: 96-99

The left right handedness of the developing mouse embryo is thought to arise from an extracellular flow (nodal flow) generated by dynein-dependent rotation of monocilia on Hensen's node (see p.220 of text). Essner et al. show that the existence of node monocilia as well as the expression of a dynein gene implicated in ciliary function are conserved among mouse, chicken, Xenopus, and zebrafish. Thus, a similar ciliary mechanism may establish handedness in all vertebrates. Nonaka et al. used a system in which mouse embryos are cultured under an artificial fluid flow to examine how flow affects L-R patterning. An artificial rightward flow that was sufficiently rapid to reverse the intrinsic leftward nodal flow resulted in reversal of situs in wild-type embryos. The artificial flow was also able to direct the situs of mutant mouse embryos with immotile cilia. These results provide the first direct evidence for the role of mechanical fluid flow in L-R patterning. The nature of the critical molecule(s) whose distribution is skewed by the ciliary rotation remains to be established.

Marrari, Y., Terasaki M., Arrowsmith V. and Houliston E. (2000) Local inhibition of cortical rotation in Xenopus eggs by an anti-KRP antibody. Devel.Biol. 224: 250-262

The investigators injected beneath the vegetal cortex of Xenopus eggs an antibody binding to several kinesin-related proteins (KRPs). Their observations suggest that KRPs restrict the movement of subcortical microtubules, and they support the notion proposed earlier by others that cortical rotation and microtubule alignment may reinforce each other.

Pu R. and Robinson K.R. (1998) Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid ala Pelvetia compressa. J. Cell Sci. 111: 3197-3207

The investigators used a calcium indicator, Calcium Crimson, to observe the formation of a cytoplasmic Ca2+ gradient within one hour of the exposure of the cells to unilateral light. The region of high Ca2+ predicted the site of rhizoid formation. These data provide direct evidence for the existence of a Ca2+ gradient during photopolarization. The injection of ungerminated zygotes with antibodies against calmodulin inhibited germination, indicating that this Ca2+-dependent signal protein acts as a mediator of the Ca2+ gradient.


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