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

我们的长期目标是揭示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，我们正在使用遗传学、基因组学和蛋白质组学相结合的方法来研究肢体发育过程中的转录因子和调控级联，最终目的是阐明早期诱导前后模式（拇指到小指）和不同手指最终形态发生之间的调控层次。了解这种三维结构是如何形成的，对于理解器官发生是如何实现的，以及对生长和形态发生是如何安排的见解，将促进我们对如何治疗当这些调节成分突变或表达时出现的遗传疾病和癌症的理解异常 ...............................................................................................................................................................1) Shh的早期下游事件：我们对突变小鼠肢体芽中Shh信号的时间需求的分析表明，Shh在早期阶段通过间接信号中继指定数字，而不是作为经典形态因子，并且更有可能将肢体场划分为离散的区域，这些区域对次级下游信号的响应潜力不同，而不是指定“最终”不同的数字身份。为了确定在早期信号传导过程中建立的初始差异，我们将对Shh激活时和激活后不久的正常肢体芽进行单细胞转录组分析，以确定表达特征并表征早期反应区。这将为后续使用早期Shh活性改变的小鼠突变体的研究提供基础。此外，我们的遗传研究表明，有两类Shh应答靶基因具有非常不同的调控特征：一类是响应瞬时信号并稳定表达的基因，另一类是需要连续信号来维持其表达的基因。根据我们的分析，前一类将包括组织可以形成的肢体元件基本模式的关键目标，后一类将包括这些元件后期扩展和形态发生所必需的生长和生存调节因子。我们正在使用Shh突变肢体芽的转录组分析来开始表征这两种不同靶类的基因，并确定其差异调控的基础。Shh突变体与通过短暂Shh暴露（早期“触发”功能）和绕过Shh支持细胞存活的需要（Bax/Bak敲除，参见ZIA BC 011118项目）拯救的Shh突变体的转录组谱将被比较，以确定瞬时Shh信号阶段的表达特征，并表征即时早期反应基因。sh -null与对照胚胎的类似比较将确定所有靶标，包括那些依赖于在细胞存活中起重要作用的持续Shh信号的靶标。了解Shh在这些差异调控靶标类别中的增殖和抗凋亡作用，将为破译和拦截Shh在癌症和正常发育中的作用提供参考.....................................2) Shh和Fgf信号传导之间的反馈回路：肢体芽中表达Shh的中胚层和表达Fgf的外胚层信号传导中心之间的正负反馈回路相互作用，在调节手指形态和生长过程中维持和限制彼此的活性，并最终在肢体器官发生完成时终止活性。我们正在使用遗传策略来操纵不同肢体萌芽阶段的Shh和Fgf水平，开始揭示控制它们表达的正调控和负调控输入。这些结果将被纳入对肢体形态发生不同阶段的调控网络的分析，以得出一个更完整的模型，说明这些回路是如何整合的。3) Gli3-Hox相互作用和形态发生的调控：Gli3-Hox蛋白-蛋白相互作用控制肢体形态发生的多个过程，包括祖骨凝聚形成过程中的增殖速率和细胞粘附时间，以及邻近每个趾原基的指间组织（蹼）的晚期信号对不同的最终趾形态的控制。我们之前在Gli3中发现了一个高度保守的结构域，它与Hox因子和其他几个关键的发育调节因子（Smad1, β -catenin）相互作用。我们将使用质谱法（使用bioid -生物素标签接近标记来捕获和丰富相互作用伙伴）来阐明可以调节肢体中Gli3活性的伙伴的范围，并且也可能竞争Hox蛋白结合，这将使用其他生化和遗传策略进行验证。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，以确定潜在基因靶点的表达变化。我们的结果将与已知的Hoxd13和Gli3的直接转录靶点（来自现有的ChIPseq数据）进行比较，并在我们的实验室中补充ChIPseq用于后期肢体阶段。确定晚期Hoxd和Gli3靶点将有助于深入了解共调控基因和Gli3-Hoxd的作用，并阐明Hoxd基因在肢体形态发生中的晚期效应。肢体中由Hoxd和Gli3调控的转录网络也将被分析与sh通路靶标的关系，这些靶标形成两种不同的类别，需要短暂或持续的信号传导才能稳定激活. .........................3)最后，单细胞表达谱将用于表征手指祖区（指尖），该区域由Gli3-Hox平衡控制的指间信号网络指示形成指骨节和关节（另见Project ZIA BC 011119）。这个区域作为指骨的干细胞库，即使在哺乳动物中也有一些有限的再生潜力。使用这种方法的组合，我们希望揭示导致具有不同数量的节段和关节的定义趾形态形成的调控级联。激活和Hox基因也会异常发生在某些癌症和可能导致他们的发病机理,和这些研究也阐明其可能的角色在这些上下文 . ........................................................................................................................4)肢体适应性修饰对调控网络的启示：指导肢体骨骼形成的基本调控网络在脊椎动物中基本上是保守的。揭示进化适应背后的调控变化可以阐明关键的网络参数和鲁棒性的基础。先前在鸡和小鼠的实验表明，趾形态（身份）在后期由趾间信号调节。我们的遗传证据表明，5'Hoxd和Gli3是调控最终数字身份的数字间信号中心的一部分。阐明不同趾间信号通路的差异将为趾间身份在后期如何调节以及Hoxd和Gli3基因作用的潜在机制提供新的见解。

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 using transcriptome profiling of Shh mutant limb buds to begin to characterize the genes in these two distinct target classes and determine the basis of their differential regulation. Transcriptome profiles of Shh mutant vs. Shh mutant rescued by a transient Shh exposure (early "trigger" function) and bypassing the need for Shh to support cell survival (Bax/Bak knockout, see also Project ZIA BC 011118) will be compared to identify expression signatures in the transient Shh signaling phase and characterize immediate-early response genes. Similar comparisons of Shh-null with control embryos will identify all targets, including those dependent on sustained Shh signaling that play important roles in cell survival. 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 (with BioID-biotin tag proximity labeling to capture and enrich interaction partners) 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. 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. ......................... 3) 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 (see also Project ZIA BC 011119). 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.