Analysis of Biological Development (K. Kalthoff)

Updates to Topic 23: The Role of Hox Genes in Vertebrate Development


Answers to Questions in Text

Hox Genes Acting as Homeotic Genes (pp. 621)

  1. How would you test whether the lacZ-NeoR transgene has indeed become inserted into the targeted location of the Hoxc8 gene? Answer: By polymerase chain reaction (PCR, see Method 15.3, p. 387) using one primer hybridizing to the inserted transgene and the other primer hybridizing to the host genome close to the desired insertion side. The bracketed DNA segment will be present and of the predicted size only if the transgene has been inserted into the targeted location.
  2. Based on the data provided here, would you expect to find additional homeotic transformations in the area of the cervical vertebrae? Answer: No, because Hoxc8 is not expressed in this area.
  3. Homeotic transformations caused by homozygous null alleles in Drosophila tend to be complete and present in every surviving individual. In contrast, the Hoxc8-/- mice described here showed considerable variation. For instance, the ends of the 8th ribs were distant from the sternum (as in wild-type mice) in five of 14 homozygous null individuals, fused to the 7th ribs in three, and attached to the sternum (as shown in Fig. 23.13c) in six. How do you explain the weaker expressivity of homeotic transformations in mice? Answer: While Drosophila has only one Hox complex the mouse has four. The pseudo-orthologs of Hoxc8+, that is, Hoxb8+, and Hoxd8+, are very similar and partially replace the lost function of Hoxc8+. The extent of the replacement depends on the activity of surrounding enhancers, which could account for the variability of the observed mutant phenotype.

Same Genes Controlling Dorsoventral Polarity in Flies and Frogs (pp. 627-629)

  1. Injected chordin mRNA is less effective in Drosophila than in Xenopus. This could be ascribed to a less-than-perfect match between Xenopus chordin and the remaining Drosophila components of the system establishing dorsoventral polarity. Specifically, chordin and BMP-4 may both be intrinsically weaker than dpp and sog. Are there (quantitative) indications in the data reviewed here that the latter, more specific hypotheis may be correct? Can you derive a testable prediction from this hypothesis? Answer: Yes, it took 100-200 pg of sog mRNA to rescue a dorsalized Drosophila embryo, but only 25-100 pg of sog mRNA to rescue a (much larger) ventralized Xenopus embryo. A testable prediction is that BMP-4, the antagonist of chordin, has only weak dorsalizing effects in Drosophila.
  2. How would you test whether dpp from Drosophila has a ventralizing effect in Xenopus? Answer: One test, done by Holley et al. (1995, Nature 376: 249-253), is to inject dpp mRNA into each animal blastomere. This resulted in complete ventralization, as observed after UV irradiation during the first cell cycle.
  3. Assuming that dpp does have a ventralizing effect in Xenopus, what kind of embryo would you expect to develop from an 8-cell embryo in which all four animal blastomeres have been injected with dpp and one vegetal blastomere with sog? Answer: A normal, or nearly normal, embryo. See Fig. 4f in Holley et al. (1995, Nature 376: 249-253).

Comments

The progreess zone model of proximodistal pattern formation in the vertebrate limb has been called into question by new results of Dudley et al. (2002), reviewed by Saunders (2002). Within the progress zone (PZ) subjacent to the apical ectodermal ridge (AER), cells have been thought to undergo progressive distalization with the passage of time, perhaps as measure by the number of cell divisions. New data from fate mapping and transplantation experiments with chicken wing buds are not readily explained by the PZ model. Instead, they suggest that all proximodistal elements are specified early by signals from the AER, and that subsequent development involves just an expansion of different progenitor populations prior to differentiation. Thus, a better name for the limb bud zone capped by the AER may be "apical zone". Its extent may be defined by the rapid cell death that occurs after removal of the AER.

Dudley A.T., Ros M.A. and Tabin C.J. (2002) A re-examination of proximodistal patterning during vertebrate limb development. Nature 418: 539-544

Saunders J.W. (2002) Is the progress zone model a victim of progress? Cell 110: 541-543

Clarifications and Corrections

New Review Articles

Carroll S.B. (2000) Endless forms: The evolution of gene regulation and morphological diversity. Cell 101: 577-580

Ruvinsky I. and Gibson-Brown J.J. (2000) Genetic and developmental bases of serial homology in vertebrate limb evolution. Development 127: 5233-5244

New Research Articles

Chang C., Holtzman D.A., Chau S., Chickering T., Woolf E.A., Holmgren L.M., Bodorova J., Gearing D.P., Holmes W.E. and Brivanlou A.H. (2001) Twisted gastrulation can function as a BMP antagonist. Nature 410: 483-487

Oelgeschlager M., Larrain J., Geissert D. and De Robertis E.M. (2000) The evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling. Nature 405: 757-763

Ross J.J., Shimmi O., Vilmos P., Petryk A., Kim H., Gaudenz K., Hermanson S., Ekker S.C., O'Connor M.B. and Marsh J.L. (2001) Twisted gastrulation is a conserved extracellular BMP antagonist. Nature 410: 479-483

Scott I.C., Blitz I.L., Pappano W.N., Maas S.A., Cho K.W. and Greenspan D.S. (2001) Homologues of Twisted gastrulation are extracellular cofactors in antagonism of BMP signalling. Nature 410: 475-478.
These four studies investigate the Drosophila protein Twisted gastrulation (Tsg), and its homologs in zebrafish, frog, and chicken, as a new cofactor in BMP signaling. In Drosophila embryos, the BMP-type molecule Decapentaplegic (Dpp) specifies at least three elements of the dorsoventral body pattern: amnioserosa (highest Dpp), epidermis (intermediate Dpp), and neurogenic ectoderm (no Dpp). The Dpp gradient is shaped in part by Short gastrulation (Sog), which binds to and inhibits Dpp. In turn, Sog is antagonized by Tolloid, a protease that brakes down Sog and releases Dpp. The role of the new player, Tsg, seems to be two-fold. By binding to Dpp/Sog, Tsg prevents the binding of Dpp to its receptor and accelerates the diffusion of Dpp. By accelerating the breakdown of bound Sog by Tolloid, Tsg also enhances the recovery of active Dpp. The overall effect depends on the concentration of Sog. Where Sog is abundant (ventrolaterally), the effect of Tld is overpowered, and Dpp remains bound up. Where Sog is scarce (dorsally), Tld degrades Sog and releases active Dpp. This explains why the highest Dpp activity is reached at some distance from maximal Sog concentration, an effect that may also involve an inhibition of the Tld+ gene by the Brinker transcriptional inhibitor (see update 22D). The vertebrate homologs of Tsg promote the binding of chordin (Sog homolog) to BMP4, enhance the cleavage of chordin by Tld homologs, and potentiate chordin's ability to induce secondary axes in Xenopus. These observations confirm and extend the previous observations on the evolutionary conservation of BMP type signaling in the specification of the dorsoventral body pattern.

Dahn R.D. and Fallon J.F. (2000) Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling. Science 289: 438-441
The investigators used advanced microsurgical techniques to manipulate chicken limb buds and found that the identity of the digits is determined at advanced stages of development and by interdigital tissue. By splitting and transplanting digit precursors, they found that digit identity is specified by the interdigital mesoderm before the latter regresses through apoptosis. Each primordium develops according to the most posterior clues it receives. Digit identity was transformed if bone morphogenetic protein signaling was boosted or inhibited, suggesting a role for BMPs in the specification process.

Litingtung Y, Dahn RD, Li Y, Fallon JF, Chiang C. (2002) Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity. Nature 418: 979-983.
Sonic hedgehog (Shh) protein is thought to specify digit identity through dose-dependent activation of homeotic target gene expression. However, the regulatory mechanisms involved remain poorly understood. This study focuses on Gli3, a transcriptional regulator that can act as an activator or repressor dependent on Shh. The investigators report genetic analyses in mice showing that Shh and Gli3 are dispensable for formation of limb skeletal elements: Gli3(-/-) as well as Shh(-/-) Gli3(-/-) limbs are distally complete and polydactylous, but completely lack wild-type digit identities. They propose that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limb's polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities.

Yekta S., Shih I. and Bartel D.P. (2004) MicroRNA-directed cleavage of HOXB8 mRNA. Science 304: 594-596
The investigators fpound that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments indicating miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting translation.

Wellik D.M. and Capecchi M.R. (2003) Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton. Science 301: 363-367
The researchers constructed mice homozygous for loss-of-function alleles in all Hox10 or all Hox11genes. Since cluster B does not have Hox10 or Hox11 genes, there are 2x3=6 gene in each of these pseudo-orthologous groups. All of them needed to be mutated to produce a phenotype. In the absence of Hox10 function, no lumbar vertebrae were formed. Instead, ribs projected from all posterior vertebrae, extending from the thorax to beyond the sacral region. In the absence of Hox11 function, sacral vertebrae were not formed, and instead these vertebrae assumed a lumbar identity.

Web Sites

Great SEMs and drawings of mouse and human embryonic development may be found on this web site, which is slated for additional illustrations of pre-implantation and fetal development as well as the genesis of birth defects.

The Multidimensional Human Embryo is funded by the National Institute of Child Health and Human Development (NICHD) and provides a three-dimensional image reference of the Human Embryo based on magnetic resonance imaging.


Go back to Home Page



Website maintained by Dr. Klaus Kalthoff
E mail:
kkalthoff@mail.utexas.edu
Last modified: 8July 2005