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.
VertebrateHoxgenes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that theHoxCgene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of theHoxCcluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function ofHoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of theHoxCgene cluster, which together regulateHoxcgene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation ofHoxcgenes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.
The distal part of the tetrapod limb, the autopod, is characterized by the presence of digits. The digits display a wide diversity of shapes and number reflecting selection pressure for functional adaptation. Despite extensive study, the different aspects of digit patterning, as well as the factors and mechanisms involved are not completely understood. Here, we review the evidence implicating Hox proteins in digit patterning and the interaction between Hox genes and the Sonic hedgehog/Gli3 pathway, the other major regulator of digit number and identity. Currently, it is well accepted that a self-organizing Turing-type mechanism underlies digit patterning, this being understood as the establishment of an iterative arrangement of digit/interdigit in the hand plate. We also discuss the involvement of 5’ Hox genes in regulating digit spacing in the digital plate and therefore the number of digits formed in this self-organizing system.
The developing vertebrate limb has long proved as an excellent system for studying the mechanisms involved in pattern formation and morphogenesis and more recently in transcriptional regulation and morphological evolution. To elucidate the stage-specific expression profiles of the components of the developing limb, we have generated the temporal transcriptome of the limb progenitors and of the overlying ectoderm separately. Our study has uncovered a collinear activation of Hoxc genes in the limb ectoderm that we have validated by in situ hybridization. However, while members of the HoxA and HoxD clusters show complex and dynamic patterns of expression during limb development that correlate with the morphology of the different limb segments, no specific function for the HoxC or HoxB clusters has been identified ( 1 - 3 ). To investigate the function of Hoxc genes in the limb ectoderm, we have reexamined the HoxC cluster null mice. Remarkably, and despite exhibiting normal terminal phalanges, these mice didn't form claws (anonychia). Morphological and immunohistochemical analysis identified a failure in the differentiation of the main components of the nail/claw organ. To unravel the transcriptional regulation of Hoxc genes in the limb ectoderm, we used the ATACseq technique. Using this approach, we identified two putative regulatory regions which activity was tested in mouse transgenic enhancer assays. It is currently considered that Hox genes have played a key role in the evolution of morphological traits, probably associated with changes in their regulatory landscapes ( 4 ). Given that the form and size of the distal limb integumentary organ (nail, claw or hoof) correlates with that of the distal phalanx and that the development of hooves was a major innovation in the evolution of a cursorial lifestyle, we are also exploring the possible implication of Hoxc genes in the nail/claw/hoof transition. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sp8 and Sp6 are two closely related Sp genes expressed in the limb ectoderm where they regulate proximo-distal and dorso-ventral patterning. Mouse genetics revealed that they act together in a dose-dependent manner but with Sp8 making a much greater contribution. Here, we combine ChIP-seq and RNA-seq genome-wide analyses to investigate the Sp8 regulatory network and mechanism of action. We find that Sp8 predominantly binds to putative distal enhancers to activate crucial limb patterning genes, including Fgf8, En1, Sp6 and Rspo2. Sp8 exerts its regulatory function by directly binding DNA at Sp consensus sequences or indirectly through Dlx5 interaction. Overall, our work underscores Sp8 master regulatory functions and supports a model in which it cooperates with other Dlx and Sp cofactors to regulate target genes. We believe that this model could help to properly understand the molecular basis of congenital malformations. Impact SentenceIn the limb ectoderm, Sp8 regulates master genes through a dual mechanism: directly binding DNA at Sp consensus sequences and indirectly engaging through Dlx5 interaction.
Vertebrate Hox genes are key players in the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was globally co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws.Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia) and also hairs (alopecia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two ectodermal, mammalian-specific enhancers located upstream of the HoxC gene cluster, which act synergistically to regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with mammals to provide the level of HOXC proteins necessary for the full development of hairs and nails. Significance StatementIn this study, we report a unique and necessary function for the HoxC gene cluster in the development of some ectodermal organs, as illustrated both by the hair and nail phenotype displayed by mice lacking the Hoxc13 function and by the congenital anonychia (absence of nails) in full HoxC cluster mutants. We show that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. We identify two mammalian-specific enhancers located upstream of the HoxC cluster with and exclusive ectodermal specificity. Individual or combined enhancer deletions suggest that they act
In the present study we have investigated the molecular causes of the absence of digit 1 in the Hoxa13 mutant and why the absence of Hoxa13 protein, whose expression spans the entire autopod, specifically impacts the anterior-most digit. We show that in the absence of Hoxa13, the expression of Hoxd13 does not extend into the anterior mesoderm consequently leaving the presumptive territory of digit1 devoid of distal Hox expression and providing an explanation for the agenesis of digit 1. We provide compelling evidence that the lack of Hoxd13 transcription in the anterior mesoderm is due to increased Gli3R activity, in turn resulting from the loss of transcriptional repression exerted by Hoxa13 on Gli3. Our results are compatible with a mutual transcriptional repression between Gli3 and Hox13 genes that determines the anteriorposterior asymmetry of the autopod.
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