5'Hoxd genes play many roles during limb development and may control the effectors of morphogenesis at late stages. How Hoxd genes guide digit morphogenesis and their downstream targets remain enigmatic. We have genetic evidence that Hoxd genes regulate digit pattern and morphogenesis at late stages, after digit condensations have already formed, and may regulate joint position by directly reversing cartilage differentiation at particular sites. This role in segmentation of digits may be a major mechanism by which Hoxd genes regulate digit morphology. We have also discovered genetic and physical interactions between 5Hoxd and Gli3 that modify Gli3R function (and hence Shh output), converting Gli3R to an activator, and are currently investigating Gli3-Hoxd interaction roles in developing limb. We and others previously demonstrated a very early role for 5'Hoxd genes in activating Sonic hedgehog (Shh) expression and interactions between Hand2, Hoxd, and Gli3 proteins regulate the level of Shh expression. Gli3-Hox interactions may modulate Gli3 repressor activity and activate targets in other Shh-dependent contexts, such as normal or neoplastic renewal of skin and gut epithelia. Gli3-Hox interactions may also play a role in regulation of cartilage vs joint formation, which may have relevance for the homeostasis of the skeletal system and skeletal diseases, as well as skeletal birth defects. What are the time requirements for 5'Hoxd gene function? Digit identity remains plastic even after the formation of the digit primordial chondrogenic condensations and is regulated by interdigit zones, which are also late sites of 5'Hoxd and Gli3 expression. Collaborating with Denis Duboule (Univ. Geneva), we are analyzing the time dependence of Hoxd function in the limb using a conditional Hoxd13-d11 (5'Hoxd) knock-out and tamoxifen-dependent Cre. We find that late loss of Hoxd function at interdigit stages results in a phenotype very similar to early Hoxd gene removal, with short biphalangeal digits (thumb-like), similar to the phenotype in human brachydactyly syndromes, indicating a late requirement for Hoxd function in the limb. In a parallel collaboration with Dr. Joyner (SKI, NY), temporal requirements for Gli3 function in limb were also examined, and we found that Gli3 is required from early through late stages of limb development, playing several different roles. What role do Hoxd genes play in cartilage differentiation and joint formation?: In addition to interdigit mesenchyme, Hoxd expression continues very late at the periphery of the cartilage models for future digit bones, and normally shuts off within differentiating cartilage. Shut-off of Hoxd expression is necessary for chondrogenic differentiation to proceed and may play a key role in the normal segmentation that leads to digit joint formation, which occurs by local reversal of the cartilage differentiation program. We have found that genetic removal of several Hoxd genes results in abnormal joint formation, probably by failure to reverse cartilage differentiation at sites of joint segmentation. This is consistent with our finding that Hoxd genes repress Sox9 expression and suggests a major role for Hoxd genes in joint formation. The canonical Wnt signaling pathway is known to play an essential role in joint formation, also by antagonizing Sox9 function and reversing chondrogenesis. We are using genetic and biochemical approaches to analyze the relation between 5'Hoxd genes and beta-catenin in promoting joint formation. We find that activated beta-catenin restores normal joint formation in the 5'Hoxd mutant digits. Surprisingly, selective activation of stabilized beta-catenin in the interdigital tissues (which have been implicated in regulating digit identity at late stages) is required for rescue, suggesting that at least some aspects of beta-catenin and 5'Hoxd function in joint formation may occur indirectly, via signaling from interdigits. Gli3 (the transcriptional effector of sonic hedgehog signaling with which Hoxd proteins physically interact) also has very striking effects on cartilage differentiation and may play a role in conjunction with Hoxd genes in regulating the cell fate decision between cartilage and joint formation (see below). What is the role of Gli3-Hoxd interaction in digit pattern? Hoxd transcription factors cooperate in an additive fashion to regulate digit pattern and are thought to be key targets of Shh signals. We previously found that Hoxd-Gli3 interactions serve to modify the function of Gli3 as a nuclear Sonic hedgehog-mediator either by converting Gli3-repressor into an activator of its target promoters and/or antagonizing Gli3 repressor function. Collaborating with Chris Westlake (CCR,NCI), we are extending this finding by examining Hox-Gli3 interaction in vivo with fluorescent imaging to assess whether interaction plays a role only in the nucleus, or at other sites where Gli proteins are functionally processed in response to hedgehog signaling (specifically cilia). During joint formation in digit precursors, Gli3 mutants form abnormal segments with excessive, abnormal joint formation extending into the cartilage elements. Reducing the 5'Hoxd dosage by half completely rescues this phenotype, allowing formation of normal joints and digits with the normal 3 bony segments. Our genetic evidence suggests Hoxd-Gli3 interaction modulates Bmp pathway activity. We plan to extend our analysis to determine the molecular mechanism: 1) target promoters regulated by Gli3-Hoxd interaction and 2) other physiologic roles of Gli3-Hoxd interaction during limb development. While Hoxd genes are no longer expressed in the adult, other related Hox genes are expressed, have highly conserved in Gli3-binding domains and may modify Hh-Gli3 targets in other contexts, such as skin and gut, during normal renewal of these epithelia or during neoplastic proliferation. We have determined requirements for Gli3-HoxD protein interaction and are testing the functional effects of a dominant interfering form of Gli3 (peptide) in chick embryos. What signaling pathways interact with Hoxd genes to regulate final digit morphogenesis? Digit shape and numbers of joints are regulated at late stages by interdigit signals. Since Hoxd genes are functioning at the same time, it is likely that they interact with and regulated some of the signaling pathways active in interdigits, as suggested by beta-catenin rescue experiments of 5'Hoxd mutant phenotypes (see above). Elucidating signaling pathway differences between different interdigits will provide new insights on how digit identity is regulated at late stages and the potential mechanisms by which Hoxd genes may act at these stages. We are evaluating interdigits in species with evolutionary digit adaptations, to correlate morphogenetic changes with changes in signaling activity, comparing three vertebrates: chick, mouse, and bat (collaborating with J. Rasweiler, SUNY, and Marian Ros, Univ. Cantabria). Both bats and birds have evolved striking digit adaptations for flight and also have highly adapted hindlimbs. We are undertaking a global analysis of gene expression using DNA microarrays and/or RNAseq to screen for differences in various signaling pathways between individual interdigit samples at the RNA expression level (data analysis collaborative effort with Dr. Agarwala,NCBI). Comparing gene expression in the interdigits and responsive digit condensations of different organisms will provide new insights on how digit identity is regulated and evolutionary adaptation occurs. Global expression profiling analyses will also be applied to 5'Hoxd mutants and following rescue (joint formation restored by beta-catenin activity) to gain further insight into critical signaling pathways regulating digit morphology and implicated in cartilage growth and joint segmentation.
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