Fonction et évolution de la régulation du gène Hoxa11 au cours du développement des membres chez les vertébrés

Limb development requires the proper function of genes belonging to the HoxA and HoxD gene clusters, which are differentially expressed along the proximal-distal in a collinear fashion corresponding to their position along the chromosome. This regulatory strategy is essential to specify the identify of each limb segment during limb development. We have previously shown that the expression of HoxA genes in the distal limb relie on several remote transcriptional enhancers which come into 3D contact with the Hoxa9 to Hoxa13 genes. However, Hoxa11 is not expressed in the distal limb and multiple lines of evidence suggest that a mechanism exists to specifically prevent Hoxa11 expression in distal limb  In the course of evolution, the fin to limb transition represents one of the most crucial morphological adaptations associated with the switch from an aquatic to terrestrial lifestyle. While most modern tetrapods are pentadactyl, fossil records indicate that the first tetrapods were polydactylous.   In chapter 2, we have shown that a change in Hoxa11 gene regulation over the course of vertebrate evolution has likely played a role in the acquisition/stabilization of the pentadactyl state in tetrapods. In zebrafish, hoxa11 and hoxa13 genes are expressed in overlapping domains in the fin bud. On the other hand, in the mouse, Hoxa11 and Hoxa13 have mutually exclusive expression domains in the limb bud. Hoxa11 is expressed in the proximal region of the limb bud (future zeugopod) whereas Hoxa13 is expressed in the most distal region (future autopod). We have shown that the absence of Hoxa11 expression in the distal limb bud is due to the presence of an enhancer located in the intron of Hoxa11. Under the control of HOXA13 and HOXD13 proteins, this enhancer induces antisense transcription which consequently prevents the transcription of Hoxa11 in the autopod. Moreover, we have shown that this enhancer is absent in zebrafish, suggesting that its emergence occurred during the fin to limb transition. We proceeded to delete this enhancer in the mouse and observed a distal expansion of the Hoxa11 expression domain in the limb bud. We have also shown in the mouse that the expression of Hoxa11 in the Hoxa13 expressing domain of the limb bud results in the formation of a polydactylous limb (Prx1Cre ; RosaHoxa11/Hoxa11 or A11KI mouse line). As such, our results indicate that the emergence of this enhancer in the intron of Hoxa11, throughout evolution, has permitted the restriction of the Hoxa11 expression domain to the proximal region of the limb by a mechanism of repression implicating HOX13 proteins and the antisense transcription. In addition, based on the polydactyly phenotype resulting from the expression of Hoxa11 in the distal region of the limb, our results suggest that the evolution of Hoxa11 regulation (which separated the expression domains of Hoxa11 and Hoxa13) has contributed to the transition from polydactyly in early tetrapods to pentadactyly in extant tetrapods.   In chapter 3, we have studied the molecular causes for the development of the polydactylous limb which results from the distal expression of Hoxa11 in the A11KI mouse line. Polydactyly is often caused by the ectopic expression of Shh in the anterior limb bud. To the contrary, our results suggest that ectopic Shh expression in limb buds of the A11KI mouse line is a consequence and not a cause of the polydactyly. Transcriptome analysis of A11KI limb buds has shown that the distal expression of Hoxa11 results in both the downregulation of genes involved in establishing the anterior identity of the limb bud as well as the anteriorization of genes posteriorly expressed. Moreover, we have shown that HOXA11 binds the regulatory region of Grem1, a gene whose expression is essential for limb bud growth and digit formation. Our results indicate that the distal expression of Hoxa11 reinforces the SHH-GREM1-FGF feedback loop which is probably the cause of polydactyly in the A11KI mouse line. Furthermore, studying the double mutant Xt/Xt ; A11KI allowed us to show that the polydactyly phenotype, resulting from distal Hoxa11 expression, is dependent on Gli3. Indeed, the double mutant Xt/Xt ; A11KI mice develop oligodactylous limbs (digit number less than 5). Molecular analysis of this double mutant allowed us to establish that expression of Grem1 is affected in limb buds and this strongly suggests that the observed oligodactyly is due to alteration of the SHH-GREM1-FGF feedback loop, which in turn causes a defect in expansion of the digit progenitor cells. Therefore, to conclude, our results demonstrate that the distal expression of Hoxa11 contributes to the regulation of the SHH-GREM1-FGF feedback loop. In the presence of Gli3, distal Hoxa11 expression positively regulates Grem1 expression, resulting in the development of a polydactylous limb. In the absence of Gli3, distal Hoxa11 represses Grem1 expression and causes the formation of an oligodactylous limb.   This work has provided a better understanding of the importance of changes in Hox genes regulation acquired over the course of evolution and for limb development, notably for the control of digit number and to establish the pentadactyl state in modern tetrapods.

Keyphrases: Hox genes, Enhancer, Regulation, Evolution, Limb, Fin, Pentadactyly, Polydactyly, Oligodactyly