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 have previously demonstrated a very early role for 5'Hoxd genes in activating Sonic hedgehog (Shh) expression. In a collaborative project with Dr. Rolf Zeller (Univ. of Basel), we have now found that interactions between Hand2, Hoxd, and Gli3 proteins regulate the level of Shh expression, highlighting the complex regulatory feedback circuits involved in the control of Shh. 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 versus joint formation, which may have relevance for the homeostasis of the skeletal system and skeletal diseases, as well as skeletal birth defects. The major questions we are addressing in our research are summarized below. What are the time requirements for 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 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 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. This indicates a late requirement for Hoxd function in the limb. In a parallel study collaborating with Alex Joyner (MSKCC, NY), temporal requirements for Gli3 function in limb are also being examined, and we have 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 genes continue to be expressed very late at the periphery of the cartilage models for future digit bones. Hoxd expression normally shuts off as cartilage differentiation proceeds, while remaining on at the periphery. To assess whether this shut-off is important to allow chondrogenic differentiation to proceed, we developed an inducible transgenic model to sustain expression of Hoxd gene expressing in forming cartilage. Our preliminary results indicate that shut off of Hoxd gene expression is necessary for early steps in cartilage differentiation to occur. We find that active Hoxd genes in cartilage precursors repress Sox9 expression (a master-regulator of cartilage fate). This repression 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 also found that genetic removal of several Hoxd genes results in abnormal joint formation, probably by failure to reverse cartilage differentiation in order to form a mature joint at sites of segmentation. This is consistent with our finding that Hoxd genes repress Sox9 and cartilage formation 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 cartilage differentiation. We are using genetic and biochemical approaches (with Hoxd mouse mutants and beta-catenin gain- and loss-of-function mouse mutants) to analyze the relation of these two regulators in promoting joint formation. Interestingly, 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 mediator of Shh by converting Gli3-repressor into an activator of its target promoters. We are extending this analysis to determine: 1) target promoters regulated by Gli3-Hoxd interaction and 2) physiologic role 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 transfections and in chick embryos. Dependent on the outcome of such experiments, long-range plans to introduce Hox-interaction domain mutations in Gli3 into mice for analysis will be undertaken. 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. 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 adaptations of digit morphology, to correlate morphogenetic changes with changes in signaling activity, comparing three vertebrates: chick, mouse, and bat (collaboration with J. Rasweiler, SUNY). 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. The expression data analysis is a collaborative effort with Drs. Ovcharenko and Agarwala in 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. In the mouse, well-characterized mutants are available that have digit alterations such as brachydactyly (short, biphalangeal digits), as seen in Hoxd13-11 mutants. Global genomic and proteomic analyses will also be applied to these mutants to gain further insight into critical signaling pathways regulating digit morphology and implicated in cartilage growth and joint segmentation.
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