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Hox基因与肢发育

                                                                Jacqueline Deschamps^*

During embryonic development, tetrapod limbs emerge from the lateral plate mesoderm at specific positions along the embryonic axis. Nascent limb buds are already endowed with anterior-posterior (AP) polarity, as evidenced by the asymmetric expression of dHand, Gli3, and some of the Hox genes, including a set of genes on the 5‘ side of cluster HoxD (see the figure). Interplay between anteriorly restricted Gli3 and posteriorly restricted dHand is thought to prepattern the limb bud (1). This prepatterning results in the formation of the zone of polarizing activity (ZPA) in the posterior of the limb bud (2), which expresses the signaling molecule Sonic Hedgehog (SHH) (3). This marks the second phase of limb development during which SHH signaling feeds back onto the early limb controller genes, reinforcing their transcription posteriorly or repressing their expression anteriorly. This second phase initiates morphogenesis of the most distal limb structures, the digits. Mouse embryos lacking Shh exhibit limb truncations suggesting that this gene is essential for digit formation. Recent work shows that Shh and Gli3 are dispensable for generating limb skeletal elements, but are required for specifying digit identity (4, 5). Inactivation of Gli3 rescues the digit phenotype in Shh-deficient mouse embryos presumably by releasing GLI3-mediated repression of the 5‘HoxD genes that play an important part during the second phase of limb development. Hox genes are typically expressed along the embryonic axis in the order in which they lie in their clusters. The expression of 5‘HoxD genes is restricted to posterior embryonic tissues. In addition, these genes are expressed in dynamic patterns in developing limb buds, and are essential for digit development (6).

On page 1669 of this issue, Zákány et al. (7) reveal that the 5‘HoxD genes contribute to patterning of limb buds much earlier than the second phase of limb bud development. Early posterior restriction of 5‘HoxD expression is essential for establishing AP polarity in the nascent limb bud. The concerted, colinear regulation of the 5‘HoxD genes is a very early determinant of limb AP asymmetry. Subsequently, the 5‘HoxD genes are involved in generating distal limb structures during the second phase of limb development.

To abolish the normal posterior restriction of endogenous 5‘HoxD expression from the earliest stage of limb development in the mouse embryo, Zákány et al. used an elegant and powerful chromosome engineering method (8). The two allelic configurations of the HoxD locus that they generated--an inversion of the cluster and deletion of its 3‘ region--caused premature expression of the 5‘HoxD genes throughout the limb bud instead of their normal restricted expression in the posterior of the limb bud. In both cases, Shh was expressed anteriorly and posteriorly, and limbs with a double set of posterior digits in mirror image developed. These experiments indicate that early posterior restriction of 5‘HoxD expression is a prerequisite for posterior localization of SHH and for correct AP patterning of the limbs. The authors propose the existence of an early limb control region (ELCR) located on the 3‘ side of the HoxD cluster that drives the progressively more posterior (colinear) regulation of HoxD genes. SHH production would act as a relay between the early phase of HoxD gene expression and the second phase mediated by the GCR (general control region) (9), that occurs in the distal limb bud (the presumptive digits) (see the figure).

The work of Zákány and colleagues sheds new light on the complex genetic cascade underlying early limb patterning. A remaining question concerns the relation between posterior restriction of 5‘HoxD expression and the early involvement of posteriorly restricted dHand expression. Is one of these two Gli3-dependent events hierarchically higher up than the other, or do they act in parallel? Early AP-restricted dHand expression accompanies limb bud emergence (10), preceding and possibly influencing early 5‘HoxD gene expression in the lateral mesoderm. Does dHAND then mediate the posterior restriction of 5‘HoxD gene expression in the limb bud? Also, it is not known whether the Hox genes of other clusters, some of which are locally expressed in the emerging limb buds, are involved in prepatterning the limb bud as well. Given the high functional redundancy of the Hox gene family, answering these questions will be a major challenge. Finally, the molecular characterization of the ELCR, and the time-dependent transition between its action and the GCR-mediated transcription of 5‘HoxD genes, should shed light on the molecular mechanisms integrating temporal and spatial information during limb development.

References

1. P. te Welscher et al., Genes Dev. 16, 421 (2002).
2. C. Tickle, Int. J. Dev. Biol. 46, 847 (2002).
3. R. D. Riddle et al., Cell 75, 1401 (1993).
4. Y. Litingtung et al., Nature 418, 979 (2002).
5. P. te Welscher et al., Science 298, 827 (2002).
6. J. Zákány et al., Proc. Natl. Acad. Sci. U.S.A. 94, 13695 (1997).
7. J. Zákány, M. Kmita, D. Duboule, Science 304, 1669 (2004).
8. Y. Herault et al., Nature Genet. 20, 381 (1998).
9. F. Spitz et al., Cell 113, 405 (2003).
10. J. Charité et al., Development 127, 2461 (2000).

Science, Vol 304, Issue 5677, 1669-1672 , 11 June 2004

A Dual Role for Hox Genes in Limb Anterior-Posterior Asymmetry

József Zákány, Marie Kmita, Denis Duboule^*

Anterior-to-posterior patterning, the process whereby our digits are differently shaped, is a key aspect of limb development. It depends on the localized expression in posterior limb bud of Sonic hedgehog (Shh) and the morphogenetic potential of its diffusing product. By using an inversion of and a large deficiency in the mouse HoxD cluster, we found that a perturbation in the early collinear expression of Hoxd11, Hoxd12, and Hoxd13 in limb buds led to a loss of asymmetry. Ectopic Hox gene expression triggered abnormal Shh transcription, which in turn induced symmetrical expression of Hox genes in digits, thereby generating double posterior limbs. We conclude that early posterior restriction of Hox gene products sets up an anterior-posterior prepattern, which determines the localized activation of Shh. This signal is subsequently translated into digit morphological asymmetry by promoting the late expression of Hoxd genes, two collinear processes relying on opposite genomic topographies, upstream and downstream Shh signaling.

Department of Zoology and Animal Biology and National Program Frontiers in Genetics, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland.

^* To whom correspondence should be addressed. E-mail: Denis.Duboule@zoo.unige.ch

Anterior-posterior (AP) asymmetry in tetrapod limbs is reflected by the anatomy of lower arms and hands. In humans, the thumb is shorter and more mobile than other digits. These differences result from the presence in the developing posterior limb bud of a zone of polarizing activity (ZPA) (1), defined by its potential both to induce supernumerary digits and to modify digit identity when transplanted anteriorly. Cells within the ZPA express the Shh gene (2), whose product propagates posterior identity in the growing bud, likely through a graded, long-range intercellular signaling mechanism (3, 4).

The effects of Shh signaling in limbs are mediated, at least in part, by posterior Hoxd genes (2, 5–9) because of the potential of SHH to prevent the production of the repressor form of GLI3 protein, which negatively regulates Hox gene transcription (10–12), likely through a global digit enhancer located near the HoxD cluster (13, 14). Models for the restriction of Shh expression in posterior limb bud cells have been proposed whereby the antagonism between the Gli3 and dHand transcription factors would initially divide the bud into anterior and posterior domains (15). Although this model is supported by genetics and experimental data (11, 12, 16–18), it falls short in explaining the spatial restriction of Shh expression.

A similar limb bud posterior specificity was observed for both Hoxa and Hoxd genes in their earliest phases of expression (19–21). Hoxd genes are activated in a collinear fashion, with Hoxd1 and Hoxd3 expressed throughout the early bud, whereas Hoxd12 and Hoxd13 are expressed posteriorly (Fig. 1A) in a domain containing future SHH-positive cells. This restriction occurs before Shh expression (5, 6, 9, 22), which suggested a role for Hox genes in AP polarity (19). In addition, ectopic expression of Hoxb8 and Hoxd12 revealed the potential of some HOX products to trigger Shh expression (23–25). Here, we use two novel genomic rearrangements to show that posterior Hoxd genes are key determinants in the early organization of limb AP asymmetry.

We engineered a loxP/Cre-dependent inversion of the HoxD cluster (Fig. 1A) and asked whether gene expression would be concomitantly modified. Because embryos carrying the inversion (Inv) did not survive, we induced the inversion in vivo, along with a wild-type chromosome, or analyzed mosaic fetuses containing both the Inv and the non-inverted alleles after recombination in vitro. When the Inv allele was present, expression of both Hoxd13 (Fig. 1, C, G, and H, and fig. S1) and Hoxd11 (Fig. 1F) was seen in the anterior domain. Expression of Hoxd1 is shown in Fig. 1D as a control. Expression of a single copy of Hoxd13 (Fig. 1B) and Hoxd11 (Fig. 1E) using the floxed configuration over a deletion of the HoxD cluster (flox/Del1-13) showed no anterior ectopic expression. We concluded that, on inversion, Hoxd13 and Hoxd11 had been brought to the vicinity of an early limb control region (ELCR) (Fig. 1), located telomeric to HoxD, that normally drives expression throughout limb bud cells.

To ascertain both the existence and the location of an ELCR, we analyzed a deletion of Hoxd1 to Hoxd10 obtained by targeted meiotic recombination (fig. S2A) [Del(1-10)]. This deficiency generated a minicluster composed of Hoxd11, Hoxd12, and Hoxd13, now located as close to a potential ELCR as in the inversion but in their native orientation and genomic order (Fig. 2A). Homozygous Del1-10 mice died soon after birth; hence specimens were analyzed either as fetuses or newborns. Homozygous newborns showed a markedly elongated thumb in forelimbs (Fig. 2, B and E, asterisks), making the AP asymmetry hardly recognizable. Furthermore, homozygotes displayed a perfectly symmetrical carpus, with bones organized in a mirror image (Fig. 2E), and the distal radius acquired the morphology of distal ulna. This bilateral symmetry, seen in the right limbs of eight animals, occurred less frequently in the corresponding left limbs. In addition, three homozygous animals had only four digits in their forelimbs (Fig. 2C), and hindlimb polydactyly was scored in both homozygous and heterozygous individuals. In all cases, digits appeared organized following a mirror symmetry, as exemplified by an additional large palmar cushion at the base of the most anterior digit (Fig. 2G).