Hoxd and Gli3-Hoxd interaction roles in Hedgehog regulated digit morphogenesis

5'Hoxd基因在肢体发育过程中发挥了许多作用，并可能在后期控制形态发生的效应。Hoxd基因如何引导手指形态发生及其下游目标仍然是一个谜。我们发现，除了启动Sonic hedgehog （Shh）表达外，5'Hoxd基因还在早期决定初级肢轴的极性，并在手指凝聚形成后的后期调节手指模式和形态发生，包括关节的形成和定位；这是Hoxd基因调控数字身份的主要机制。我们还发现了5Hoxd和Gli3之间的遗传和物理相互作用，这些相互作用可以改变Gli3R的功能（从而改变Shh的输出），拮抗Gli3R并可能将其转化为激活剂。Gli3- hox相互作用可能调节Gli3抑制因子（Gli3R）活性，并激活其他sh依赖环境中的靶标，如正常或肿瘤皮肤和肠道上皮的更新。Gli3-Hox相互作用还在软骨与关节形成的调节中发挥作用，这可能与骨骼系统和骨骼疾病的稳态以及骨骼出生缺陷有关。Gli3-Hoxd相互作用的作用是什么？Hoxd转录因子以累加方式合作调节数字模式，被认为是Shh信号的关键靶点。我们之前发现Hoxd-Gli3相互作用通过将Gli3抑制因子转化为其靶启动子的激活因子和/或拮抗Gli3抑制因子的功能来修饰Gli3作为核sh介质的功能。与Chris Westlake （CCR，NCI）合作，我们正在扩展这一发现，通过荧光成像检查Hox-Gli3在体内的相互作用，以评估相互作用是否在细胞核中起作用，或者在Gli蛋白响应Shh信号（纤毛）的其他部位起作用。虽然Hoxd基因在成人中不广泛表达，但其他相关的Hox基因在gli3结合域高度保守，可能在其他情况下，如皮肤和肠道，在这些上皮细胞的正常更新或肿瘤增殖过程中修饰sh - gli3靶点。我们还确定了Gli3- hoxd蛋白相互作用的需求，并正在测试Gli3（肽）的主要干扰形式在鸡胚胎中的功能影响。Hoxd-Gli3相互作用在决定主肢轴极性中起什么作用？在大多数脊椎动物中，主肢轴穿过后肢，尺骨/指（d4）首先收缩。在蝾螈等尾纲两栖动物中，它们在成年后仍具有肢体再生的能力，前肢轴占主导地位（首先出现半径/d2）。基于表达模式的改变，有人提出尾犬的轴移是由于末端肢体中5'Hoxd基因表达扩展失败造成的。我们分析了5'Hoxd突变体（Hoxd11-13缺失）的肢轴形成，发现与尾犬一样，前肢轴首先形成。此外，我们发现化合物5'Hoxd；Gli3突变体，后轴优势恢复。5'Hoxd同源盒转录因子在复制许可和细胞粘附中起作用。Gli3抑制因子，表达在前面，也调节增殖和凝结，并拮抗5'Hoxd功能。我们正在分析在这些突变体中，肢体芽不同区域的增殖和细胞聚集/凝结的相对时间和速率的变化是如何改变的，以及它们是否与前轴优势和后轴优势相关。我们提出拮抗5'Hoxd-Gli3功能之间的平衡决定了主肢轴形成的极性，并正在研究轴极性改变与再生能力之间的潜在关系。与Marian Ros（坎塔布里亚大学）合作，我们也在研究Hoxa13在手指形成中的作用。Hoxa13也与Gli3相互作用，诱导Hoxd13的晚期表达，并可能在调节正常拇指的形成中发挥独特的作用。Hoxd基因在软骨分化和关节形成中起什么作用？趾间区是5'Hoxd和Gli3表达的后期位点，即使在趾原始软骨凝聚形成后，趾间区仍然具有可塑性，并受趾间区调控。我们发现几个Hoxd基因（d11-d13）的遗传去除会导致关节形成异常，手指关节丢失和/或关节位置异常，以及短的双指指。典型的Wnt通路在关节形成中起着至关重要的作用，我们发现激活的β -连环蛋白可以恢复5'Hoxd突变趾的正常关节形成。但令人惊讶的是，在指间组织中，稳定的β -catenin的选择性激活是修复所必需的，这表明β -catenin和5'Hoxd在关节形成中的作用至少在某些方面是通过指间信号间接发生的。Gli3 （Shh和Hoxd蛋白相互作用因子的转录效应因子）在趾软骨分化和关节形成中也有显著的影响。在手指前体的关节形成过程中，Gli3突变体形成异常节段，过度的关节形成延伸到软骨元件。从遗传学上讲，5'Hoxd和Gli3基因总剂量之间的平衡调节着正常关节和哺乳动物指骨典型的正常3骨节的周期性形成。我们的遗传证据表明，Hoxd-Gli3平衡间接地通过指间间质调节Bmp活性，从而调节指尖祖细胞池中指元素（指骨）和关节的周期性出现。我们正在扩展我们的分析，以确定：1)Gli3-Hoxd相互作用调节的靶标和2)调节指尖祖细胞池的其他信号输入，以确定指骨阵的数量和大小。与Steve Vokes （UT-Austin）合作，我们将研究在指尖祖细胞中选择性表达的染色质修饰子Prmt5在维持这个祖细胞库中的作用。哪些信号通路与Hoxd基因相互作用以调控最终趾形态发生？手指形状和关节数量在后期由指间信号调节。我们的遗传证据表明，5'Hoxd和Gli3基因是调控最终数字身份的指间信号中心的一部分。阐明不同趾间信号通路的差异将为趾间身份在后期如何调节以及Hoxd和Gli3基因作用的潜在机制提供新的见解。我们通过比较三种脊椎动物：小鸡、老鼠和蝙蝠（合作者J. Rasweiler， SUNY; Marian Ros， U. Cantabria），评估了物种中趾间的进化适应，将形态发生变化与信号活动的变化联系起来。蝙蝠和鸟类都进化出了适应飞行的惊人手指，也有高度适应的后肢，包括指骨数量和关节形成的变化。我们正在进行转录组分析（RNAseq），以筛选个体指间样本之间各种信号通路的差异（与NCBI的Agarwala博士合作）。比较具有不同趾形态的不同生物趾间和反应性趾缩合中的基因表达将为趾身份如何调节和进化适应的发生提供新的见解。全球表达谱分析也将应用于5'Hoxd突变体和后续修复（通过β -catenin活性或Gli3缺失恢复关节形成），以进一步了解通过软骨生长和关节分割调节手指形态的关键信号通路。这些发现也将与先天性畸形和骨骼再生问题高度相关。

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 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 have also discovered genetic and physical interactions between 5Hoxd and Gli3 that modify Gli3R function (and hence Shh output), antagonizing Gli3R and potentially converting it to an activator. Gli3-Hox interactions may modulate Gli3 repressor (Gli3R) activity and activate targets in other Shh-dependent contexts, such as normal or neoplastic renewal of skin and gut epithelia. Gli3-Hox interactions 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 is the role of Gli3-Hoxd interaction? 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 Shh-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 in the nucleus, or at other sites where Gli proteins are functionally processed in response to Shh signaling (cilia). While Hoxd genes are not broadly expressed in the adult, other related Hox genes are and, having highly conserved in Gli3-binding domains, may modify Shh-Gli3 targets in other contexts, such as skin and gut, during normal renewal of these epithelia or during neoplastic proliferation. We have also 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 role does Hoxd-Gli3 interaction play in determining polarity of the primary limb axis? In most vertebrates, the primary limb axis runs through the posterior limb with the ulna/digit4 (d4) condensing first. In urodele amphibians such as axolotl, which retain the ability to regenerate limbs as adults, the anterior limb axis is dominant (radius/d2 appear first). Based on altered expression patterns, it has been proposed that the axis shift in Urodeles results from a failure to expand 5'Hoxd gene expression in the late distal limb. We have analyzed limb axis formation in the 5'Hoxd mutant (Hoxd11-13 deleted) and found that the anterior axis forms first as in urodeles. Furthermore, we find that in compound 5'Hoxd;Gli3 mutants, posterior axial dominance is restored. The 5'Hoxd homeobox transcription factors play roles in replication licensing and cell adhesion. Gli3 repressor, expressed anteriorly, also regulates proliferation and condensation, and antagonizes 5'Hoxd function. We are analyzing how changes in the relative timing and rate of proliferation and of cell aggregation/condensation in different zones of the limb bud are altered in these mutants, and if they correlate with anterior vs posterior axial dominance. We propose that the balance between antagonistic 5'Hoxd-Gli3 functions governs the polarity of primary limb axis formation and are investigating the potential relation between altered axis polarity and regenerative capacity. Collaborating with Marian Ros ((Univ. Cantabria) we are also examining the role of Hoxa13 in digit formation. Hoxa13 also interacts with Gli3 and induces the late phase of Hoxd13 expression and may have a distinct role in regulating the formation of a normal thumb. What role do Hoxd genes play in cartilage differentiation and joint formation? 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, suggesting 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 are extending our analysis to determine: 1) targets regulated by Gli3-Hoxd interaction and 2) other signaling inputs that regulate the digit tip progenitor pool to determine phalanx number and size. Collaborating with Steve Vokes (UT-Austin) we will examine the role of the chromatin modifier Prmt5, which is selectively expressed in the digit tip progenitors, in maintaining this progenitor pool. 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. Our genetic evidence indicates that 5'Hoxd and Gli3 genes are part of an interdigit signaling center that regulates final digit identity. 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 and Gli3 genes act. 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 (collaborators J. Rasweiler, SUNY; Marian Ros, U. Cantabria). Both bats and birds have evolved striking digit adaptations for flight and also have highly adapted hindlimbs including changes in phalanx number and joint formation. We are undertaking transcriptome analysis (RNAseq) to screen for differences in various signaling pathways between individual interdigit samples (collaboration with Dr. Agarwala, NCBI). Comparing gene expression in the interdigits and responsive digit condensations of different organisms with very different digit morphologies 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 or Gli3 loss) to gain further insight into critical signaling pathways regulating digit morphology via cartilage growth and joint segmentation. These findings will also be highly relevant to the problems of congenital malformations and skeletal regeneration.