Late stage signaling centers and Gli3-Hoxd interaction roles in Hedgehog regulated digit morphogenesis

Division Of Basic Sciences - Nci

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Abstract

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. Using genetic approaches in mice we find that, in addition to a role in initiating Sonic hedgehog (Shh) expression, 5'Hoxd genes determine the polarity of the primary limb axis early, and regulate digit pattern and morphogenesis at late stages, after digit condensations have already formed, including joint formation and positioning; a major mechanism by which Hoxd genes regulate digit identity. We previously discovered genetic and physical interactions between 5Hoxd and Gli3 that modify and antagonize Gli3 repressor (Gli3R) function. We find that Gli3-Hox interactions both modulate the polarity of limb axis formation and regulate the pacing of cartilage vs joint formation in digits, which may have relevance for skeletal homeostasis and disease, as well as skeletal birth defects, and for regenerative potential. Using genetic and CRISPR-based knockdown approaches to compare axis formation in a mammal (mouse) with urodele amphibian (axolotl,capable of complete limb regeneration) and we showed that 5'Hoxd-Gli3 interaction determines the polarity of primary limb axis formation. In axolotl, Gli3R dominates and the polarity is reversed relative to mammal. Likewise in the 5'Hoxd mutant, Gli3R also dominates and axis polarity is similar to axolotl. CRISPR knockdown of Gli3 in axolotl results in mammalian limb axis polarity. 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. We found that genetic removal of several Hoxd genes (d11-d13) results in abnormal joint formation, both loss of digit joints and/or abnormal joint position, as well as short, biphalangeal digits. The canonical Wnt pathway plays an essential role in joint formation and we find that activated beta-catenin restores normal joint formation in the 5'Hoxd mutant digits. But surprisingly, selective activation of stabilized beta-catenin in the interdigital tissues is required for rescue, indicating that at least some aspects of beta-catenin and 5'Hoxd function in joint formation occur indirectly, via interdigit signaling. Gli3 (the transcriptional effector of Shh and Hoxd protein interactor) also has striking effects on cartilage differentiation and joint formation in digits. During joint formation in digit precursors, Gli3 mutants form abnormal segments with excessive joint formation extending into the cartilage elements. Genetically, the balance between total 5'Hoxd and Gli3 gene dosage regulates the periodic formation of normal joints and the normal 3 bony segments typical of mammalian digits. Our genetic evidence indicates that the Hoxd-Gli3 balance acts indirectly, from interdigital mesenchyme, to modulate Bmp activity and thereby regulate the periodic appearance of digit elements (phalanges) and joints from a digit tip progenitor pool. We have extended our analysis to determine other signaling inputs that regulate the digit tip progenitor pool to determine phalanx number and size, including signals induced by beta-catenin activation in interdigits. Using a combination of transcriptome comparisons between WT control, 5'Hoxd mutant, and 5'Hoxd mutant with digit joint formation restored by interdigit beta-Catenin activation, and validation with genetic and pharmacologic analyses, we have found that Wnt antagonists, which are induced as direct targets by selective beta-catenin activation in interdigits, are responsible for joint restoration. This was unexpected because at later stages, Wnts are thought to play a key role in promoting synovial joint formation. We showed that in the digit tip, during early phalanx and joint progenitor formation, Wnts inhibit Gsk3 phosphorylation of pSmad effectors of Bmp signaling, leading to pSmad stabilization and promoting phalanx digit fate. By counteracting this effect, Wnt antagonists enable progenitors to also adopt a joint progenitor fate, so that both elements can form proportionately. This work has been submitted for publication and deposited at BioRxiv (https://doi.org/10.1101/2025.07.17.665381). To gain further insight into the mechanisms by which Gli3 and Hoxd proteins act antagonistically, we also plan to compare the normal WT limb transcriptome with 5'Hoxd, Gli3, and compound mutants, to identify expression changes in potential gene targets. Our results will be compared with the known direct transcriptional targets of Hoxd13 and of Gli3 (from available ChIPseq data) and ATACseq data from 5'Hoxd and Gli3 mutants. Identifying late stage Hoxd and Gli3 targets will provide insight into coregulated genes and Gli3/Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. We also intend to characterize the digit progenitor regions (digit tips) that are instructed to form periodic, alternating phalangeal segments and joints by the interdigit signaling network that is controlled by Gli3-Hox balance using single cell transcriptional profiling. This region behaves as a stem cell pool for the digit skeleton and has some limited regenerative potential even in mammals. Even in mammals, distal digit tips retain a limited capacity for regeneration and understanding the regulation of this distal digit progenitor pool and its maintenance will provide new and testable insights relevant to skeletal regeneration potential in adults. With this goal in mind, we are developing organ culture techniques to reproduce digit-like structures from limb mesenchyme in culture, to interrogate regulation by signaling factors active in vivo as well as methods to genetically mark embryonic digit tip progenitors and test their ability to contribute to post-natal digit regeneration, following digit tip amputation. The basic regulatory network instructing formation of the limb skeleton is largely conserved throughout vertebrates. Uncovering regulatory changes that underlie evolutionary adaptations can illuminate critical network parameters and basis for robustness. Previous work in chick, and in mouse from our lab, have shown that digit morphology (identity) is regulated at late stages by interdigit signals in both species. However, in chick the phalanx-forming potential of the progenitor pool is greatly enhanced leading to formation of different hindlimb digits with from 2 to 5 phalanges. Using a similar single cell profiling approach, we intend to compare the chick digit tip progenitor pools between digits that form fewer vs many phalanges at early stages (when both pools produce phalanges) and later stages when some have ceased phalanx induction while others have not. These comparisons together with characterization of the more limited progenitor pool in mouse will yield insights on the factors regulating progenitor pool maintenance and loss. In collaboration with Marian Ros (U. Cantabria) we will also compare terminal phalanx formation during development with regenerating terminal phalanx expression profiles to assess the similarity of programs regulating the morphogenesis of this structure, which can uniquely regenerate in mammals. Using this combination of approaches, we hope to uncover the regulatory cascade leading to formation of defined digit morphologies with distinct numbers of segments and joints. Gli3 and Hox genes are also aberrantly coexpressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts.