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节和表达的早期模式与结构的最终实现联系起来的步骤，以作为信号网络如何策划复杂组织的形成的范式。为此，我们使用遗传，基因组和蛋白质组学方法的联合研究在肢体发育过程中运行的转录因子和调节级联反应，其最终目的是阐明早期诱导前后形态的调节层次结构（拇指对小指）与最终的不同数字的形态学。了解这种三维结构形式通常与理解如何实现器官发生有关以及对生长和形态发生的方式的见解将促进我们对如何治疗如何治疗这种调节成分突变或表达这种调节成分时出现的遗传疾病和癌症的理解。 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.为了确定早期信号传导期间建立的初始差异，我们将从正常的肢体芽AT和SHH激活后不久进行单细胞转录组分析，以识别表达特征并表征即时响应区域。这将为随后的研究提供基础，使用小鼠突变体改变了早期SHH活性。此外，我们的遗传研究表明，有两类的SHH响应靶基因具有截然不同的调节特征：对瞬态信号响应并稳定表达的响应特征，以及需要连续信号传导以维持其表达的。从我们的分析中，前类将包括对可以形成的肢体元素的基本模式至关重要的目标，后者将包括生长和生存的调节因子，这些元素的膨胀和形态发生所需。我们正在比较对照，SHH突变体和救出的SHH突变肢体的转录组（强制性细胞存活替代后期功能），以开始表征这两个Dstinct目标类别中的基因，并确定其差异调节的基础。在这些差异调节的目标类别的背景下，了解SHH的增生和抗凋亡作用将为解密和拦截SHH在癌症以及正常发育中的作用提供参考。 2）SHH和FGF信号之间的反馈电路：中胚层表达SHH和外胚层成纤维成纤维细胞生长因子（FGF）表达肢体芽的表达信号传导中心之间的相互正面和负反馈回路，以保持数字模式和外观的活性，以保持和限制彼此的活性，以使彼此的活性在跨度和外观中限制在限制和外观中，以终止生物的生动限制。我们正在使用遗传策略在不同的肢体芽阶段操纵SHH和FGF水平，以开始揭示控制其表达的正面和负调控输入。这些结果将纳入对肢体形态发生不同阶段运行的调节网络的分析中，以更完整地了解这些电路的整合方式。 3）GLI3-HOX相互作用和形态发生的调节：Gli3-Hox蛋白 - 蛋白质相互作用控制肢体形态发生的多个过程，包括祖细胞骨骼凝结的形成过程中的增殖速率和细胞粘附的时间，并通过不同的信号来控制原位的最终信号。我们先前鉴定出GLI3中高度保守的结构域，该结构域与HOX因子以及其他几个关键的发育调节剂（SMAD1，β-catenin）相互作用。我们将使用质谱法来阐明可以调节肢体中Gli3活性的伴侣范围，并且还可以竞争HOX蛋白结合，该结合将使用其他生化和遗传策略进行验证。同时，为了深入了解GLI3和HOXD蛋白在拮抗作用的机制，我们将正常的肢体转录组与5'HoxD，Gli3和复合突变体进行比较，以识别潜在基因靶标的表达变化。我们的结果将与已知的HOXD13和GLI3的已知直接转录靶标（来自可用的Chipseq数据）进行比较，并在我们的实验室中补充Chipseq，以便在需要时进行以后的肢体阶段。我们已经与史蒂夫·沃克斯（Steve Vokes）（UT Austin）合作设计了一个表位标记的HOXD13条件转基因等位基因，后者生成了用于全基因组芯片的类似表位标记的GLI3小鼠系。识别晚期HOXD和GLI3靶标将提供对共同调节的基因和GLI3-HOXD角色的洞察力，以及在肢体形态发生中照亮HOXD基因的后期效应子。肢体中由HOXD和GLI3调节的转录网络也将与SHH-Pathway目标相关，这些目标形成了两个不同的类别，这些目标需要瞬时或持续的信号传导才能进行稳定激活。最后，将使用单细胞表达分析来表征数字祖细胞区域（数字尖端），这些区域被指示由由Gli3-Hox平衡控制的InterDigit信号网络形成指向段和关节。该区域作为数字骨骼的干细胞库，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，我们希望揭开法规级联反应，从而形成具有不同数量的片段和关节的定义数字形态。 GLI3和HOX基因在某些癌症中也异常共表达，并可能导致其发病机理，这些研究还将阐明它们在这些情况下的可能作用。 4）从自适应肢体修改中对调节网络的见解：基本的调节网络指导肢体骨骼的形成在整个脊椎动物中都在很大程度上保存。揭示构成进化适应的监管变化可以照亮关键网络参数和鲁棒性的基础。先前在小鸡和我们实验室的小鼠中的工作表明，数字形态（身份）受到跨阶段的阶段进行调节。我们的遗传证据表明，5'HOXD和GLI3是调节最终数字身份的互化信号中心的一部分。阐明不同互相之间的信号通路差异将提供有关在晚期如何调节数字身份以及HOXD和GLI3基因作用的潜在机制的新见解。我们正在比较具有数字适应性的物种中的互化表达谱，以将形态发生变化与信号活性的变化相关，并比较了三个脊椎动物：小鸡，小鼠和蝙蝠（合作者J. Rasweiler，Suny； M. Ros，M。Ros，U.Cantabria）。蝙蝠和鸟类都进化出了惊人的飞行数字适应性，并且还具有高度适应的后肢，包括变化的变化和联合形成。比较转录组分析（与R. agarwala，NCBI的合作）和具有截然不同的数字形态的不同生物体的响应性数字冷凝，将为数字身份的调节和进化适应性提供新的见解。总之，这些研究也将与先天性畸形和再生医学高度相关。