Hoxd gene functions in digit morphogenesis and role of Gli3-Hoxd interaction

注：本项目是前项目“基因在胚胎发育过程中调节模式形成”（Z01 SC 009170）的一部分的延续。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。我们有遗传学证据表明，Hoxd基因在手指缩合形成后的后期调控手指形态和形态发生，并可能通过直接逆转特定部位的软骨分化来调节关节位置。这一作用可能是Hoxd基因调控趾形态的主要机制。我们还发现了5Hoxd和Gli3之间的遗传和物理相互作用，这些相互作用改变了Gli3R的功能（从而改变了Shh的输出），将Gli3R转化为激活剂，目前正在研究Gli3- hoxd相互作用在肢体发育中的作用。Gli3-Hox相互作用可能激活其他sh依赖环境中的靶标，如皮肤和肠道上皮的正常或肿瘤更新。Gli3-Hox的相互作用也可能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体内平衡以及骨骼出生缺陷有关。我们在研究中要解决的主要问题总结如下。Hoxd基因功能的时间要求是什么？趾间区是Hoxd和Gli3表达的后期位点，趾间区是指间区调控的。我们与Denis Duboule（日内瓦大学）合作，使用条件Hoxd13-d11敲除和他莫昔芬依赖性Cre分析了Hoxd功能在肢体中的时间依赖性。我们发现，指间期Hoxd功能的晚期缺失导致表型与早期Hoxd基因去除非常相似，具有短的双指指（拇指状），类似于人类短指综合征的表型。这表明肢体对Hoxd功能的要求较晚。在与Alex Joyner （MSKCC， NY）合作的一项平行研究中，Gli3在肢体中功能的时间要求也在研究中。Hoxd基因在软骨分化和关节形成中起什么作用？除了指间质外，Hoxd基因在未来指骨软骨模型的外周继续很晚地表达。Hoxd的表达通常在软骨分化过程中关闭，而在周围则保持正常。为了评估这种关闭对软骨分化是否重要，我们开发了一种可诱导的转基因模型来维持Hoxd基因在软骨形成过程中的表达。我们的初步结果表明Hoxd基因表达的关闭是软骨分化发生的早期步骤所必需的。我们发现软骨前体中活跃的Hoxd基因抑制Sox9的表达（软骨命运的主要调节剂）。这种抑制可能在导致手指关节形成的正常分割中起关键作用，这是通过软骨分化程序的局部逆转发生的。我们还发现，基因去除几个Hoxd基因会导致关节形成异常，这可能是由于软骨分化无法逆转，从而在分割部位形成成熟的关节。这与我们的发现一致，即Hoxd基因抑制Sox9和软骨形成，并表明Hoxd基因在关节形成中起主要作用。众所周知，典型的Wnt信号通路在关节形成中发挥重要作用，也可拮抗Sox9功能和逆转软骨分化。我们正在使用遗传和生化方法（用Hoxd小鼠突变体和β -连环蛋白获得和失去功能的小鼠突变体）来分析这两种调节因子在促进关节形成中的关系。有趣的是，Gli3（与Hoxd蛋白物理相互作用的超音刺猬信号的转录效应因子）也对软骨分化有非常显著的影响，并可能与Hoxd基因一起在调节软骨和关节形成之间的细胞命运决定中发挥作用（见下文）。Gli3-Hoxd相互作用在数字模式中的作用是什么？Hoxd转录因子以累加的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来修饰Gli3作为Shh的核介质的功能。我们正在扩展这一分析，以确定：1)Gli3-Hoxd相互作用调控的靶启动子；2)Gli3-Hoxd相互作用在肢体发育中的生理作用。虽然Hoxd基因在成人中不再表达，但其他相关的Hox基因在gli3结合域中表达，并在其他情况下，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，可能修饰Hh-Gli3靶点。我们已经确定了Gli3- hoxd蛋白相互作用的需求，并正在测试Gli3（肽）的主要干扰形式在转染和鸡胚胎中的功能影响。根据这些实验的结果，将进行长期计划，将Gli3中的hox相互作用结构域突变引入小鼠进行分析。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。由于Hoxd基因同时起作用，它们很可能与指间活动的一些信号通路相互作用并调节它们。阐明不同趾间间信号通路的差异将为趾间身份在晚期如何调节以及Hoxd基因在这些阶段可能起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（与纽约州立大学的J. Rasweiler合作），评估了物种中趾间指形态的进化适应性，以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。我们正在使用DNA微阵列进行基因表达的全球分析，以筛选个体指间样本在RNA表达水平上的各种信号通路的差异。我们通过对相邻指间组织通路激活的蛋白质组学分析来补充这一分析，以将激活信号磷酸化中间体的水平与相邻指间组织中差异活跃的信号联系起来。我们正在与L. Liotta（乔治梅森大学）合作，使用逆相蛋白质阵列（Bmp, Wnt, Fgf, Hh）分析这些对信号的反应，他开创了这种蛋白质组学方法。比较不同生物指间和反应性指间缩合的基因表达和信号磷酸化状态，将为研究指间身份是如何调节和进化适应的发生提供新的见解。[摘要截短为7800个字符]

Note: This project is a continuation of a part of the former project titled Genes regulating pattern formation during embryonic development, Z01 SC 009170. 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. Gli3-Hox interactions may 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. 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 to screen for differences in various signaling pathways between individual interdigit samples at the RNA expression level. We are complementing this analysis with a proteomic analysis of pathway activation in adjacent digit condensations to correlate levels of activated signaling phospho-intermediates with signals that are differentially active in adjacent interdigit tissues. We are assaying these responses to signaling using reverse phase protein arrays (Bmp, Wnt, Fgf, Hh), in collaboration with L. Liotta (George Mason Univ.), who pioneered this proteomic approach. Comparing both gene expression and signaling phosphorylation status in the interdigits and responsive digit condensations of different organisms will provide new insights on how digit identity is regulated and evolutionary adaptation occurs. [summary truncated at 7800 characters]