The formation of repetitive structures (such as stripes) in nature is often consistent with a reactiondiffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton ‡ To whom correspondence should be addressed. marian.ros@unican.es (M.A.R.); james.sharpe@crg.eu (J.S.); marie.kmita@ircm.qc.ca (M.K. Digit patterning has commonly been interpreted in the context of a morphogen gradient model (1, 2). The proposed morphogen Sonic hedgehog (Shh) emanates from the zone of polarizing activity (a cluster of mesodermal cells in the posterior border of the limb bud) and establishes a gradient with maximum levels posteriorly. Gli3 is the major mediator of Shh signaling in limb development and a genetic cause of polydactyly (2). Because Shh prevents the processing of Gli3 to its repressor form (Gli3R), the Shh gradient is translated into an inverse gradient of Gli3R (3, 4). The surprising finding that mouse Gli3 and Shh;Gli3 null mutants display identical polydactylous limb phenotypes demonstrates that an iterative series of digits can form in the absence of Shh (4, 5). Rather than supporting a gradient model, this observation is consistent with a Turing-type model for digit patterning (6-11) in which dynamic interactions between activator and inhibitor molecules determine the wavelength of the specific pattern and produce periodic patterns of spots or stripes. This pattern has been hypothesized to act as a molecular prepattern for chondrogenesis. According to one of the specific predictions of the model, the digit period or wavelength, defined as the combined thickness of both digit and interdigital region, should be subject to modulation by perturbing the correct parameter of the gene network. This should lead to autopods with digits varying in thickness and number, which has never been clearly observed to date.Although the core molecules of a self-organizing mechanism remain unknown, potential candidates for molecular modulators of the system include the Hox genes (10, 12). Distal Hoxa and Hoxd genes have a well-documented impact on digit number (13), though their specific role remains unclear, possibly due to their various interactions with the Shh-Gli3 pathway. These interactions include the mutual transcriptional regulation between Hox genes and Shh and the binding of Hoxd12 to Gli3R, resulting in a blockage of Gli3R repressor activity (14-16). In general, gain-and loss-of-function experiments suggest a positive relation betwee...

During vertebrate development, clustered genes from the Hox family of transcription factors are activated in a precise temporal and spatial sequence that follows their chromosomal order (the "Hox clock"). Recent advances in the knowledge of the underlying mechanisms reveal that the embryo uses a variety of strategies to implement this colinear process, depending on both the type and the evolutionary history of axial structures. The search for a universal mechanism has likely hampered our understanding of this enigmatic phenomenon, which may be caused by various and unrelated regulatory processes, as long as the final distribution of proteins (the HOX code) is preserved.

Vertebrate HoxA and HoxD cluster genes are required for proper limb development. However, early lethality, compensation and redundancy have made a full assessment of their function difficult. Here we describe mice that are lacking all Hoxa and Hoxd functions in their forelimbs. We show that such limbs are arrested early in their developmental patterning and display severe truncations of distal elements, partly owing to the absence of Sonic hedgehog expression. These results indicate that the evolutionary recruitment of Hox gene function into growing appendages might have been crucial in implementing hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages. Accordingly, these mutant limbs may be reminiscent of an ancestral trunk extension, related to that proposed for arthropods.

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.