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

Hoxd 基因在数字形态发生中的功能以及 Gli3-Hoxd 相互作用的作用

• 批准号: 8552994
• 负责人: Susan Mackem
• 金额: $ 43.4万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552994
• 关键词: Address; Adult; Binding; Biochemical; Birds; Cancer Biology; Cartilage; Cell Death; Cell physiology; Cells; Cessation of life; Chick Embryo; Chiroptera; Collaborations; Complex; Congenital Abnormality; DNA Microarray Chip; Data Analyses; Dependence; Development; Developmental Biology; Differentiation and Growth; Digit structure; Early identification; Elements; Employee Strikes; Ensure; Epithelium; Erinaceidae; Excision; Failure; Fibroblast Growth Factor; Fingers; Future; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomics; Goals; Growth; Hindlimb; Homeostasis; Human; Individual; Intercept; Joint by Site; Joints; Knock-out; Lead; Learning; Length; Limb Development; Limb structure; Link; Malignant Neoplasms; Mediator of activation protein; Memorial Sloan-Kettering Cancer Center; Mesenchyme; Mission; Modeling; Molecular; Molecular Profiling; Morphogenesis; Morphology; Mus; Mutation; Neoplasm Metastasis; Neoplasms; Nuclear; Organ; Organism; Outcome; Output; Pathology; Pattern; Peptides; Phalanx; Phenotype; Physical condensation; Physiological; Physiological Processes; Plastics; Play; Positioning Attribute; Process; Proteins; Proteomics; Regulation; Research; Role; Sampling; Shapes; Signal Pathway; Signal Transduction; Site; Skeletal system; Skin; Staging; Structure; Study models; Syndrome; System; Systems Biology; Tamoxifen; Testing; Thumb structure; Time; Tissues; Transfection; Tumor Biology; Vertebrates; beta catenin; bone; cell behavior; cell motility; cellular targeting; design; dosage; gastrointestinal epithelium; gene function; genome wide association study; insight; mutant; neoplastic; neoplastic cell; programs; promoter; research study; skeletal; skeletal disorder; smoothened signaling pathway; transcription factor; tumorigenesis

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 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 are summarized below. 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. 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 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 cartilage differentiation. 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 can restore 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. 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. We plan to extend our analysis to determine the molecular mechanism: 1) what are the target promoters regulated by Gli3-Hoxd interaction and 2) are there 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 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, 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 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. 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.

5'HOXD基因在肢体发育过程中起着许多作用，并且可以控制晚期形态发生的效应因子。 HOXD基因如何引导数字形态发生及其下游靶标仍然神秘。我们有遗传证据表明，HOXD基因已经形成了数字凝结后，在后期阶段调节了数字模式和形态发生，并且可以通过直接逆转特定部位的软骨分化来调节关节位置。在数字分割中的这种作用可能是HOXD基因调节数字形态的主要机制。 我们还发现了5HOXD和GLI3之间的遗传和物理相互作用，从而改变了GLI3R函数（以及SHH输出），将GLI3R转换为激活剂，目前正在研究GLI3-HOXD相互作用在发育中的肢体中的作用。我们和其他人以前表现出5'HOXD基因在激活声波刺猬（SHH）表达以及Hand2，HoxD和Gli3蛋白之间的相互作用中的早期作用，调节了SHH表达水平。 GLI3-HOX相互作用可能会调节GLI3阻遏活性，并在其他依赖性依赖性的情况下（例如皮肤和肠道上皮的正常或肿瘤更新）激活靶标。 GLI3-HOX相互作用也可能在软骨与关节形成的调节中起作用，这可能与骨骼系统和骨骼疾病的体内稳态以及骨骼的先天缺陷有关。 我们要解决的主要问题如下。 5'HOXD基因函数的时间要求是什么？：数字身份仍然存在塑性，即使在数字原始软骨凝结形成后，数字身份仍然受到塑性的要求，并且受互嵌套区域的调节，该区域也是5'HOXD和GLI3表达的晚期位点。我们与Denis Duboule（Univ。Geneva）合作，使用条件HOXD13-D11（5'HOXD）敲除和依赖他莫昔芬依赖性CRE分析了HOXD功能在肢体中的时间依赖性。我们发现，在跨阶段，HOXD功能的后期丧失会导致一种与早期HOXD基因去除非常相似的表型，其两次双臂数字（拇指样）与人毛rachydactylyly综合征的表型相似。这表明肢体中HOXD功能的迟到。在与Alex Joyner（纽约州MSKCC）合作的一项并行研究中，还检查了肢体中GLI3功能的时间要求，我们发现GLI3从肢体开发的早期到后期开始需要GLI3，扮演多个角色。 HOXD基因在软骨分化和关节形成中起什么作用？：除了间充质的间质外，HOXD表达在软骨模型的外围持续很晚，用于未来的数字骨骼，并且通常在区分软骨中关闭。 HOXD表达的关闭对于软骨分化的进行是必要的，并且在正常分割中可能起关键作用，这会导致数字关节形成，这是由于软骨分化程序的局部反转而发生的。 我们发现，遗传去除几个HOXD基因会导致关节形成异常，这可能是由于未能在关节分割位点反向软骨分化。 这与我们发现HOXD基因抑制SOX9的表达并提出HOXD基因在关节形成中的主要作用是一致的。 已知规范的Wnt信号通路在关节形成中起着至关重要的作用，也通过对抗Sox9功能并逆转软骨分化。 我们正在使用遗传和生化方法来分析5'HOXD基因与β-catenin之间在促进关节形成中的关系。 我们发现活化的β-catenin可以恢复5？HOXD突变体数字中的正常关节形成。 令人惊讶的是，需要选择性激活稳定的β-catenin在二阶间组织中（与在晚期阶段相关的指导数字身份有关）进行救援，这表明可能会通过互置发出信号来间接地发生β-catenin和5'HoxD功能的某些方面。 Gli3（与HOXD蛋白质上相互作用的声波刺猬信号传导的转录效应子）对软骨分化也具有非常引人注目的影响，并且可能在调节软骨形成和关节形成之间的细胞命运决策中起作用（见下文）。 Gli3-HoxD相互作用在数字模式中的作用是什么？：HOXD转录因子以增材方式合作以调节数字模式，并被认为是SHH信号的关键目标。我们先前发现，HOXD-GLI3相互作用可以通过将Gli3-抑制剂转换为目标启动子的激活因子和/或拮抗GLI3 Repressor函数来改变GLI3作为核声音刺猬介导的功能。 在数字前体的关节形成期间，GLI3突变体形成异常片段，过度，异常的关节形成延伸到软骨元件中。 一半将5？hoxd剂量降低，完全挽救了这种表型，允许形成正常的接头和数字，并具有正常的3个骨段。 我们计划扩展分析以确定分子机制：1）在肢体发育过程中GLI3-HOXD相互作用的gli3-HoxD相互作用调节的目标启动子是什么。虽然HOXD基因不再在成年人中表达，但其他相关的HOX基因表达，在GLI3结合结构域中具有高度保守，并且可以在其他情况下（例如皮肤和肠道）在这些上皮症或肿瘤增殖期间正常更新期间改变HH-GLI3靶标。我们已经确定了GLI3-HOXD蛋白相互作用的要求，并正在测试转染和雏鸡胚胎中GLI3（肽）的主要干扰形式的功能效应。取决于此类实验的结果，将进行将GLI3中HOX交互域突变引入小鼠的远程计划进行分析。 哪些信号通路与HOXD基因相互作用以调节最终数字形态发生？：数字形状和关节数量在晚期通过互化信号调节。由于HOXD基因同时发挥作用，因此它们很可能与互相中的某些信号通路相互作用并调节，如β-catenin救援实验所示，5？hoxd突变体表型（见上文）。 阐明不同互相之间的信号通路差异将提供有关如何在晚期调节数字身份以及HOXD基因在这些阶段起作用的潜在机制的新见解。我们正在评估具有数字形态进化适应的物种中的互化，以将形态发生变化与信号活性的变化相关，比较了三个脊椎动物：小鸡，小鼠和BAT（与J. Rasweiler，Suny，Suny）。蝙蝠和鸟类都进化出了惊人的数字改编以进行飞行，并具有高度适应的后肢。我们正在使用DNA微阵列和/或RNASEQ对基因表达进行全局分析，以筛选RNA表达水平下各个互嵌套样品之间各种信号通路的差异。表达数据分析是与DRS的协作工作。 NCBI的Ovcharenko和Agarwala。比较不同生物体中的基因表达和响应性的数字凝结将提供有关如何调节数字身份并发生进化适应性的新见解。全局表达分析分析也将应用于5'HOXD突变体，并在拯救后（通过β-catenin活性恢复联合形成），以进一步深入了解调节数字形态的关键信号通路并与软骨生长和关节段有关。