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。我们和其他人之前证明了5'Hoxd基因在激活Sonic hedgehog （Shh）表达中的早期作用，以及Hand2、Hoxd和Gli3蛋白之间的相互作用调节Shh表达水平。Gli3- hox的相互作用可能调节Gli3抑制因子活性，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。我们正在处理的主要问题总结如下。5'Hoxd基因功能的时间要求是什么？趾间区是5'Hoxd和Gli3表达的后期位点，趾间区是指间区调控的。我们与日内瓦大学的Denis Duboule合作，使用条件Hoxd13-d11 （5'Hoxd）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被检查，我们发现Gli3在肢体发育的早期到晚期都是必需的，扮演着几个不同的角色。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在未来指骨软骨模型周围的表达持续到很晚，通常在分化软骨内关闭。Hoxd表达的关闭是软骨分化进行的必要条件，并可能在正常分割中发挥关键作用，从而导致手指关节形成，这是通过软骨分化程序的局部逆转发生的。我们发现几个Hoxd基因的遗传去除会导致关节形成异常，可能是由于关节分割部位的软骨分化无法逆转。这与我们的发现一致，即Hoxd基因抑制Sox9的表达，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。我们正利用遗传学和生物化学方法分析5'Hoxd基因与β -连环蛋白在促进关节形成中的关系。我们发现活化的-连环蛋白可以恢复5？Hoxd突变数字。令人惊讶的是，在指间组织中选择性激活稳定的β -catenin（在后期涉及调节手指身份）是挽救所必需的，这表明β -catenin和5'Hoxd在关节形成中的功能至少在某些方面可能通过指间的信号间接发生。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和/或拮抗Gli3抑制因子的功能来修饰Gli3作为核Sonic hedgehog介质的功能。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、异常的关节形成延伸到软骨元件。减少5？一半的Hoxd剂量完全恢复了这种表型，允许形成正常的关节和手指，正常的3个骨节。我们计划扩展我们的分析，以确定分子机制：1)Gli3-Hoxd相互作用调节的目标启动子是什么？ 2)Gli3-Hoxd相互作用在肢体发育过程中是否有其他生理作用。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性的一些信号通路相互作用并调节，正如5？Hoxd突变表型（见上文）。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微阵列和/或RNAseq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。表情数据分析是与dr。奥夫查连科和阿加瓦拉在NCBI。比较不同生物指间和反应性缩合的基因表达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。全球表达谱分析也将应用于5'Hoxd突变体和随后的修复（通过β -连环蛋白活性恢复关节形成），以进一步了解调节手指形态和涉及软骨生长和关节分割的关键信号通路。