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
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Hox Genes in the Limb: A Play in Two ActsJacqueline 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).
Patterning the budding limb. Biphasic regulation of the 5'HoxD gene cluster during limb bud outgrowth. (Left) The early overlapping expression domains of HoxD genes--from 3' (HoxD1, white) to 5' (HoxD11 to D13, yellow to dark orange)--are progressively restricted posteriorly in the nascent limb bud. E9.0, embryonic day 9; ELCR, hypothetical early limb control region. At E9.5, localized Shh expression arises within the expression domain of the 5'HoxD genes. (Right) The Shh expression domain subsequently extends and is displaced distally, engaging the 5'HoxD genes in a second phase of concerted regulation in the distal limb bud (the presumptive digits). This phase is under the control of a different general control region (GCR) in a 5' location. The colinear response of 5'HoxD genes becomes reversed: HoxD13 (dark orange) is expressed at the highest level (thick arrow) and in a domain extending the most anteriorly. The expression of Gli3 (o) and dHand () shows a complementary anterior-posterior distribution at the different developmental stages.
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.
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