Genome-wide target analysis of Shh-activated transcription network in limb bud

肢芽中Shh激活转录网络的全基因组目标分析

• 批准号: 10014541
• 负责人: Susan Mackem
• 金额: $ 17.13万
• 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/10014541
• 关键词: Adult; Alleles; Apoptotic; Binding Proteins; Biochemical Genetics; Birds; Cell Adhesion; Cell Death; Cell Survival; Cell physiology; Cells; Cessation of life; Chiroptera; Collaborations; Complex; Congenital Abnormality; Data; Development; Developmental Biology; Digit structure; Elements; Engineering; Ensure; Epitopes; Equilibrium; Erinaceidae; Event; Expression Profiling; Feedback; Fibroblast Growth Factor; Fingers; Foundations; GLI gene; GLI3 gene; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genetic study; Genomics; Goals; Growth; HOX protein; Hindlimb; Homeobox Genes; Homeostasis; Human; Intercept; Internet; Joints; Lead; Learning; Length; Light; Limb Bud; Limb Development; Limb structure; Link; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mission; Modeling; Modification; Molecular; Molecular Genetics; Morphogenesis; Morphology; Mus; Mutant Strains Mice; Mutate; Neoplasm Metastasis; Neoplasms; Organ; Organism; Organogenesis; Pathogenesis; Pathologic; Pathology; Pathway interactions; Pattern; Phalanx; Physical condensation; Physiological Processes; Process; Proteins; Proteomics; Regenerative Medicine; Regulation; Research; Role; SHH gene; Secondary to; Shapes; Signal Pathway; Signal Transduction; Skeleton; Skin; Sonic Hedgehog Pathway; Stem cells; Structure; Study models; System; Systems Biology; Thumb structure; Tissues; To specify; Transgenes; Tumor Biology; Vertebrates; Work; austin; beta catenin; cell behavior; cell motility; cellular targeting; chromatin immunoprecipitation; comparative; design; genome-wide; insight; joint formation; morphogens; mutant; neoplastic cell; progenitor; programs; protein protein interaction; regenerative; response; self-renewal; skeletal; smoothened signaling pathway; three dimensional structure; transcription factor; transcriptome; transcriptome sequencing; tumorigenesis

Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are using combined genetic, genomic, and proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior pattern (thumb to pinky) and the final morphogenesis of distinct digits. Learning how this 3-dimensional structure forms will be generally relevant for understanding how organogenesis is achieved and insights on how growth and morphogenesis are orchestrated will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. 1) Early events downstream of Shh: Our analyses of temporal requirements for Shh signals in mutant mouse limb buds suggests that Shh acts at early stages to specify digits through an indirect signal relay rather than acting as a classical morphogen, and more likely acts to divide the limb field into discrete domains with differing potential to respond to secondary downstream signals, than to specify 'final' distinct digit identities. To determine the initial differences established during early signaling, we will perform single cell transcriptome analysis from normal limb buds at, and shortly after Shh activation to identify expression signatures and characterize immediate-early response zones. This will provide a foundation for subsequent studies using mouse mutants in which early Shh activity is altered. Furthermore, our genetic studies indicate that there are 2 classes of Shh responsive target genes with very different regulatory features: those that respond to a transient signal and become stably expressed, and those that require continuous signaling to maintain their expression. From our analysis, the former class would include targets critical for organizing a basic pattern of limb elements that can form, and the latter would include regulators of growth and survival necessary for the later expansion and morphogenesis of these elements. We are comparing the transcriptomes of control, Shh mutant, and rescued Shh mutant limb buds (enforced cell survival substituting for late function), to begin to characterize the genes in these two dstinct target classes and determine the basis of their differential regulation. Understanding the proliferative and anti-apoptotic roles of Shh in the context of these differentially regulated target classes will provide a reference for deciphering and intercepting Shh roles in cancer as well as normal development. 2) Feedback circuits between Shh and Fgf signaling: Reciprocal positive and negative feedback loops between the mesodermal Shh-expressing and ectodermal Fibroblast growth factor (Fgf)-expressing signaling centers in the limb bud act to both maintain and restrict each other's activity in regulating digit pattern and outgrowth and eventually to terminate activity when limb organogenesis is complete. We are using genetic strategies to manipulate Shh and Fgf levels at different limb bud stages, to begin to unravel the positive and negative regulatory inputs controlling their expression. These results will be incorporated into the analysis of the regulatory networks operating at different stages of limb morphogenesis to arrive at a more complete model of how these circuits are integrated. 3) Gli3-Hox interactions and regulation of morphogenesis: Gli3-Hox protein-protein interactions govern multiple processes in limb morphogenesis, including the rate of proliferation and timing of cell adhesion during formation of progenitor skeletal condensations, and the control of distinct final digit morphologies by late signals from interdigital tissues (webbing) adjacent to each of the digit primordia. We previously identified a highly conserved domain in Gli3 that interacts with Hox factors and also several other key developmental regulators (Smad1, beta-catenin). We will use mass spectrometry to elucidate the range of partners that can modulate Gli3 activity in the limb, and may also compete Hox protein binding, which will be validated using other biochemical and genetic strategies. In parallel, to gain insight into the mechanisms by which Gli3 and Hoxd proteins act antagonistically, we are comparing the normal limb transcriptome with 5'Hoxd, Gli3, and compound mutants, to identify expression changes in potential gene targets. Our results will be compared with known direct transcriptional targets of Hoxd13 and of Gli3 (from available ChIPseq data) and supplemented with ChIPseq in our lab for later limb stages if needed. We have engineered an epitope-tagged Hoxd13 conditional transgene allele for ChIP in collaboration with Steve Vokes (UT Austin), who generated a similarly epitope-tagged Gli3 mouse line used for genomewide ChIP. Identifying late-stage Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. The transcriptional network regulated by Hoxd and Gli3 in the limb will also be analyzed in relation to Shh-pathway targets that form two distinct classes, requiring either transient or sustained signaling for their stable activation. Finally, single cell expression profiling will be used to characterize the digit progenitor regions (digit tips) that are instructed to form phalangeal segments and joints by the interdigit signaling network that is controlled by Gli3-Hox balance. This region behaves as a stem cell pool for the digit skeleton and has some limited regenerative potential even 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 co-expressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts. 4) Insights on regulatory network from adaptive limb modifications: 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. Our genetic evidence indicates that 5'Hoxd and Gli3 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 potential mechanisms by which Hoxd and Gli3 genes act. We are comparing interdigit expression profiles in species with digit adaptations, to correlate morphogenetic changes with changes in signaling activity, comparing three vertebrates: chick, mouse, and bat (collaborators J. Rasweiler, SUNY; M. 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. Comparative transcriptome analysis (collaboration with R. Agarwala, NCBI) of 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. Together, these studies will also be highly relevant to congenital malformations and regenerative medicine.

我们的长期目标是阐明将基因调控和表达的早期模式与结构的最终实现联系起来的步骤，作为信号网络如何协调复杂组织形成的范例。为了实现这一目标，我们正在使用组合的遗传、基因组和蛋白质组学方法来研究肢体发育过程中的转录因子和调控级联，最终目的是阐明前后模式（拇指到小指）的早期诱导和不同数字的最终形态发生。了解这种 3 维结构的形成方式通常有助于理解器官发生是如何实现的，以及对生长和形态发生如何协调的见解将促进我们对如何治疗当此类调节成分突变或表达时出现的遗传疾病和癌症的理解不正常地。 1）Shh下游的早期事件：我们对突变小鼠肢芽中Shh信号的时间要求的分析表明，Shh在早期阶段通过间接信号中继来指定数字，而不是充当经典的形态发生素，并且更有可能起到分裂的作用将肢体场划分为具有不同潜力的离散域，以响应次级下游信号，而不是指定“最终”不同的数字身份。为了确定早期信号传导过程中建立的初始差异，我们将对正常肢芽在Shh激活时和之后不久进行单细胞转录组分析，以识别表达特征并表征立即早期反应区。这将为后续使用小鼠突变体进行研究奠定基础，其中早期Shh活性发生改变。此外，我们的遗传学研究表明，有两类 Shh 响应性靶基因具有非常不同的调控特征：一类对瞬时信号做出响应并稳定表达，另一类需要持续信号传导以维持其表达。根据我们的分析，前一类将包括对组织可形成的肢体元素的基本模式至关重要的目标，而后者将包括这些元素后来的扩展和形态发生所必需的生长和生存的调节因子。我们正在比较对照、Shh 突变体和获救的 Shh 突变体肢芽（强制细胞存活替代晚期功能）的转录组，以开始表征这两个不同目标类别中的基因并确定其差异调节的基础。在这些差异调节靶标类别的背景下了解Shh的增殖和抗凋亡作用将为破译和拦截Shh在癌症以及正常发育中的作用提供参考。 2）Shh和Fgf信号之间的反馈回路：肢芽中表达Shh的中胚层和表达外胚层成纤维细胞生长因子（Fgf）的信号中心之间的相互正反馈回路和负反馈回路起到维持和限制彼此调节手指的活动的作用模式和生长，并最终在肢体器官发生完成时终止活动。我们正在使用遗传策略来操纵不同肢芽阶段的Shh和Fgf水平，以开始解开控制其表达的正向和负向调节输入。这些结果将被纳入对肢体形态发生不同阶段运行的调节网络的分析中，以获得这些回路如何集成的更完整的模型。 3）Gli3-Hox相互作用和形态发生的调节：Gli3-Hox蛋白-蛋白相互作用控制肢体形态发生的多个过程，包括祖骨骼凝结形成过程中的增殖速率和细胞粘附时间，以及不同最终手指形态的控制通过来自邻近每个指原基的指间组织（蹼）的晚期信号。我们之前在 Gli3 中发现了一个高度保守的结构域，它与 Hox 因子以及其他几个关键的发育调节因子（Smad1、β-连环蛋白）相互作用。我们将使用质谱来阐明可以调节肢体中 Gli3 活性的合作伙伴范围，并且还可能竞争 Hox 蛋白结合，这将使用其他生化和遗传策略进行验证。与此同时，为了深入了解 Gli3 和 Hoxd 蛋白拮抗作用的机制，我们将正常肢体转录组与 5'Hoxd、Gli3 和复合突变体进行比较，以确定潜在基因靶标的表达变化。我们的结果将与 Hoxd13 和 Gli3 的已知直接转录靶点（来自可用的 ChIPseq 数据）进行比较，并在需要时在我们的实验室中为后续肢体阶段补充 ChIPseq。我们与 Steve Vokes (UT Austin) 合作，设计了用于 ChIP 的表位标记的 Hoxd13 条件转基因等位基因，后者生成了用于全基因组 ChIP 的类似表位标记的 Gli3 小鼠品系。识别晚期 Hoxd 和 Gli3 靶标将有助于深入了解共同调控的基因和 Gli3-Hoxd 的作用，并阐明 Hoxd 基因在肢体形态发生中的晚期效应子。肢体中由 Hoxd 和 Gli3 调节的转录网络也将与形成两个不同类别的 Shh 通路目标相关进行分析，需要瞬时或持续信号传导才能稳定激活。最后，单细胞表达谱将用于表征数字祖细胞区域（数字尖端），这些区域被 Gli3-Hox 平衡控制的指间信号网络指示形成指骨节段和关节。该区域充当手指骨骼的干细胞库，即使在哺乳动物中也具有有限的再生潜力。使用这种方法的组合，我们希望揭示导致形成具有不同数量的节段和关节的定义的数字形态的调控级联。 Gli3 和 Hox 基因也在一些癌症中异常共表达，可能有助于其发病机制，这些研究也将阐明它们在这些情况下可能发挥的作用。 4）来自适应性肢体修饰的调节网络的见解：指导肢体骨骼形成的基本调节网络在整个脊椎动物中很大程度上是保守的。揭示进化适应背后的监管变化可以阐明关键的网络参数和鲁棒性的基础。之前对小鸡和我们实验室的小鼠的研究表明，手指形态（身份）在后期受到指间信号的调节。我们的遗传证据表明 5'Hoxd 和 Gli3 是调节最终数字身份的指间信号中心的一部分。阐明不同指间信号通路之间的差异将为了解数字身份在后期如何调节以及 Hoxd 和 Gli3 基因发挥作用的潜在机制提供新的见解。我们正在比较具有数字适应的物种中的指间表达谱，以将形态发生变化与信号活动的变化联系起来，比较三种脊椎动物：小鸡、小鼠和蝙蝠（合作者 J. Rasweiler，纽约州立大学；M. Ros，U. Cantabria）。蝙蝠和鸟类都进化出了惊人的飞行适应能力，并且还具有高度适应的后肢，包括指骨数量和关节结构的变化。对具有截然不同的数字形态的不同生物体的指间和响应性数字浓缩进行比较转录组分析（与 NCBI 的 R. Agarwala 合作），将为数字身份的调节和进化适应的发生提供新的见解。总之，这些研究也将与先天畸形和再生医学高度相关。