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...

Congenital malformations of the limbs are among the most frequent congenital anomalies found in humans, and they preferentially affect the distal part--the hand or foot. The presence of extra digits, a condition called polydactyly, is the most common limb deformity of the human hand and is the consequence of disturbances in the normal program of limb development. However, despite the extensive use of the developing limb as a classical developmental model, the cellular and genetic mechanisms that control the number and identity of the digits are not completely understood. The aim of this review is to introduce the reader to the current state of knowledge in limb development and to provide the necessary background for an understanding of how deviations from the normal developmental program may lead to polydactyly.

During limb development, Sonic hedgehog (SHH) and HOX proteins are considered among the most important factors regulating digit number and identity. SHH signaling prevents the processing of GLI3 into a short form that functions as a strong transcriptional repressor. Gli3 mutant limbs are characterized by a severe polydactyly and associated ectopic anterior expression of 5'Hoxd genes. To genetically determine the involvement of 5'Hoxd genes in the polydactyly of Gli3 mutants, we have generated a compound mutant that simultaneously removes the three most 5'-located Hoxd genes and Gli3. Remarkably, the limbs that form in the absence of all four of these genes show the most severe polydactyly so far reported in the mouse. The analysis of gene expression performed in compound mutants allows us to propose that the increase in the number of digits is mediated by the gain in function of Hoxd10 and Hoxd9. Our results also support the notion that an adequate balance between positive and negative effects of different Hoxd genes is required for pentadactyly.

SUMMARYLimb development relies on an exquisite coordination between growth and patterning, but the underlying mechanisms remain elusive. Anterior-posterior and proximal-distal specification initiates in early limb bud concomitantly with the proliferative expansion of limb cells. Previous studies have shown that limb bud growth initially relies on fibroblast growth factors (FGFs) produced in the apical ectodermal ridge (AER-FGFs), the maintenance of which relies on a positive-feedback loop involving sonic hedgehog (Shh) and the BMP antagonist gremlin 1 (Grem1). The positive cross-regulation between Shh and the HoxA and HoxD clustered genes identified an indirect effect of Hox genes on the maintenance of AER-FGFs but the respective function of Shh and Hox genes in this process remains unknown. Here, by uncoupling Hox and Shh function, we show that HoxA and HoxD genes are required for proper AER-FGFs expression, independently of their function in controlling Shh expression. In addition, we provide evidence that the Hoxdependent control of AER-FGF expression is achieved through the regulation of key mesenchymal signals, namely Grem1 and Fgf10, ensuring proper epithelial-mesenchymal interactions. Notably, HoxA and HoxD genes contribute to both the initial activation of Grem1 and the subsequent anterior expansion of its expression domain. We propose that the intricate interactions between Hox genes and the FGF and Shh signaling pathways act as a molecular network that ensures proper limb bud growth and patterning, probably contributing to the coordination of these two processes.

Background: Precise temporal and spatial expression of the clustered Hox genes is essential for patterning the developing embryo. Temporal activation of Hox genes was shown to be cluster-autonomous. However, gene clustering appears dispensable for spatial colinear expression. Generally, a set of Hox genes expressed in a group of cells instructs these cells about their fate such that the differential expression of Hox genes results in morphological diversity. The spatial colinearity is considered to rely both on local and long-range cis regulation. Results: Here, we report on the global deregulation of HoxA and HoxD expression patterns upon inactivation of a subset of HOXA and HOXD proteins. Conclusions: Our data suggest the existence of a "self-regulation" mechanism, a process by which HOX proteins establish and/or maintain the spatial domains of the Hox gene family and we propose that the functionally dominant HOX proteins could contribute to generating the spatial parameters of Hox expression in a given tissue, i.e., HOX controlling the establishment of the ultimate HOX code. Developmental Dynamics 00:000-000,

Removal of the posterior wing bud leads to massive apoptosis of the remaining anterior wing bud mesoderm. We show here that this finding correlates with an increase in the level of the repressor form of the Gli3 protein, due to the absence of the Sonic hedgehog (Shh) protein signaling. Therefore, we used the anterior wing bud mesoderm as a model system to analyze the relationship between the repressor form of Gli3 and apoptosis in the developing limb. With increased Gli3R levels, we demonstrate a concomitant increase in Bmp4 expression and signaling in the anterior mesoderm deprived of Shh signaling. Several experimental approaches show that the apoptosis can be prevented by exogenous Noggin, indicating that Bmp signaling mediates it. The analysis of Bmp4 expression in several mouse and chick mutations with defects in either expression or processing of Gli3 indicates a correlation between the level of the repressor form of Gli3 and Bmp4 expression in the distal mesoderm. Our analysis adds new insights into the way Shh differentially controls the processing of Gli3 and how, subsequently, BMP4 expression may mediate cell survival or cell death in the developing limb bud in a position-dependent manner.

In the tetrapod limb, the digits (fingers or toes) are the elements most subject to morphological diversification in response to functional adaptations. However, despite their functional importance, the mechanisms controlling digit morphology remain poorly understood. Here we have focused on understanding the special morphology of the thumb (digit 1), the acquisition of which was an important adaptation of the human hand. To this end, we have studied the limbs of the Hoxa13 mouse mutant that specifically fail to form digit 1. We show that, consistent with the role of Hoxa13 in Hoxd transcriptional regulation, the expression of Hoxd13 in Hoxa13 mutant limbs does not extend into the presumptive digit 1 territory, which is therefore devoid of distal Hox transcripts, a circumstance that can explain its agenesis. The loss of Hoxd13 expression, exclusively in digit 1 territory, correlates with increased Gli3 repressor activity, a Hoxd negative regulator, resulting from increased Gli3 transcription that, in turn, is due to the release from the negative modulation exerted by Hox13 paralogs on Gli3 regulatory sequences. Our results indicate that Hoxa13 acts hierarchically to initiate the formation of digit 1 by reducing Gli3 transcription and by enabling expansion of the 5′Hoxd second expression phase, thereby establishing anterior−posterior asymmetry in the handplate. Our work uncovers a mutual antagonism between Gli3 and Hox13 paralogs that has important implications for Hox and Gli3 gene regulation in the context of development and evolution.

The original article to which this Erratum refers was published in Developmental Dynamics 231:148–160