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...

The 'progress zone' model provides a framework for understanding progressive development of the vertebrate limb. This model holds that undifferentiated cells in a zone of fixed size at the distal tip of the limb bud (the progress zone) undergo a progressive change in positional information such that their specification is altered from more proximal to more distal fates. This positional change is thought to be driven by an internal clock that is kept active as long as the cells remain in the progress zone. However, owing to cell division, the most proximal of these cells are continually pushed outside the confines of the zone. As they exit, clock function ceases and cells become fixed with the positional value last attained while within the zone. In contrast to this model, our data suggest that the various limb segments are 'specified' early in limb development as distinct domains, with subsequent development involving expansion of these progenitor populations before differentiation. We also find, however, that the distal limb mesenchyme becomes progressively 'determined', that is, irreversibly fixed, to a progressively limited range of potential proximodistal fates.

The bHLH transcription factor dHAND is required for establishment of SHH signaling by the limb bud organizer in posterior mesenchyme, a step crucial to development of vertebrate paired appendages. We show that the transcriptional repressor GLI3 restricts dHAND expression to posterior mesenchyme prior to activation of SHH signaling in mouse limb buds. dHAND, in turn, excludes anterior genes such as Gli3 and Alx4 from posterior mesenchyme. Furthermore, genetic interaction of GLI3 and dHAND directs establishment of the SHH/ FGF signaling feedback loop by restricting the BMP antagonist GREMLIN posteriorly. These interactions polarize the nascent limb bud mesenchyme prior to SHH signaling.

Pitx2, a member of the bicoid-related family of homeobox-containing genes, is asymmetrically expressed in the left lateral plate mesoderm and derived tissues during chick and mouse development. Modifications of Pitx2 pattern of expression in the iv mouse mutation correlate with the situs alterations characteristic of the mutation. Misexpression experiments demonstrate that Shh and nodal positively regulate Pitx2 expression. Our results are compatible with a Pitx2 function in the late phase of the gene cascade controlling laterality.

Sirenomelia, also known as sirenomelia sequence, is a severe malformation of the lower body characterized by fusion of the legs and a variable combination of visceral abnormalities. The causes of this malformation remain unknown, although the discovery that it can have a genetic basis in mice represents an important step towards the understanding of its pathogenesis. Sirenomelia occurs in mice lacking Cyp26a1, an enzyme that degrades retinoic acid (RA), and in mice that develop with reduced bone morphogenetic protein (Bmp) signaling in the caudal embryonic region. The phenotypes of these mutant mice suggest that sirenomelia in humans is associated with an excess of RA signaling and a deficit in Bmp signaling in the caudal body. Clinical studies of sirenomelia have given rise to two main pathogenic hypotheses. The first hypothesis, based on the aberrant abdominal and umbilical vascular pattern of affected individuals, postulates a primary vascular defect that leaves the caudal part of the embryo hypoperfused. The second hypothesis, based on the overall malformation of the caudal body, postulates a primary defect in the generation of the mesoderm. This review gathers experimental and clinical information on sirenomelia together with the necessary background to understand how deviations from normal development of the caudal part of the embryo might lead to this multisystemic malformation.

Growth of limb cells in culture conditions with subsequent in vivo transplantation allows the dissection of limb patterning.

We have analyzed a new limb mutant in the chicken that we nameoligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod(ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that theozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. NeitherPtc1 nor Gli1 are detectable in mutant limb buds. However,Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation ofHoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II,and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern toozd limbs; however, retinoic acid fails to induce Shh in ozdlimb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/-mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

The Apical Ectodermal Ridge (AER) is one of the main signaling centers during limb development. It controls outgrowth and patterning in the proximo-distal axis. In the last few years a considerable amount of new data regarding the cellular and molecular mechanisms underlying AER function and structure has been obtained. In this review, we describe and discuss current knowledge of the regulatory networks which control the induction, maturation and regression of the AER, as well as the link between dorso-ventral patterning and the formation and position of the AER. Our aim is to integrate both recent and old knowledge to produce a wider picture of the AER which enhances our understanding of this relevant structure.