Role of Shh in developmental patterning and growth of digit skeleton

Sonic hedgehog （Shh）在正常组织更新和许多类型的癌症的许多成体过程中作为有丝分裂原和细胞存活因子，但在一些发育环境中作为形态因子。在发育背景下，Shh的有丝分裂作用是如何与形态发生作用结合在一起的，人们对其了解甚少。在肢体中，Shh调节手指数量和不同手指的身份（从a到p，拇指到小指）。Shh被认为是一种形态因子，沿着肢体AP轴形成梯度，浓度越高，后趾类型越多。我们已经确定了肢体Shh功能的时间要求（在小鼠中使用三苯氧胺调节的Cre在不同时间去除Shh）。为了进行这一分析，我们产生了一种新的条件Cre重组酶系，并对其进行了表征，该重组酶系在早期肢体中胚层、神经嵴、肠道和尾芽中选择性表达。这条细胞系为科学界提供了一个很好的工具，用于阐明关键发育调节因子在几种重要的小鼠突变发育模型中的不同时间作用，以及小鼠遗传谱系追踪研究。我们在不同时间点删除Shh功能的结果与一个模型最一致，在这个模型中，Shh活性只需要非常短暂的（几个小时）来指定5种不同手指类型的完整补体，但需要很长时间（大约2天）来维持细胞存活和增殖，使5个正常手指形成。为了进一步测试这个Shh功能模型，我们评估了在Shh基因缺失导致短暂的Shh活性终止后，恢复细胞存活是否可以恢复突变胚胎中正常的手指形成。为了挽救细胞存活，研究人员引入了促凋亡Bcl2家族成员Bax/Bak（在正常指间细胞凋亡中起作用）的复合突变体来灭活内在死亡途径。我们的研究结果表明，通过简单地恢复Shh突变胚胎的细胞存活和增殖，可以挽救正常的数字和模式（形态发生）。此外，对不同时间点对Shh信号有反应的肢体细胞的遗传谱系追踪表明，当所有5趾祖细胞都被指定时，Shh仅直接向肢体芽的最后部分发出信号。这一结果表明，Shh通过一个中继信号系统间接地在早期肢体芽中起作用，以指定数字。我们开发了一种遗传分析方法，通过在缺乏所有Shh功能（Shh KO）的胚胎后肢芽中人工增强Shh反应来测试中继信号。这在一定程度上挽救了前指的形成，并强烈表明存在Shh下游的中继信号。我们的研究结果与Shh在肢体中作为一种经典形态原的作用不相容，并表明Shh作为激活传递机制的触发器。此外，我们的研究结果表明，有两类Shh应答靶基因，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表达的基因。我们比较嘘突变体的转录组和拯救肢芽”来形容这两个不同类型的基因在监管目标类(参见项目:全基因组目标分析Shh-activated转录网络肢芽,齐亚公元前0111120 ). .........................................................................................................................................................................我们的研究结果还表明，sh的表达和反应都是高度动态的，我们正在使用几种方法来表征这些特征。我们对Shh产生和反应的谱系追踪结果表明，Shh产生细胞来自近端肢体芽缘更新的祖细胞库，为肢体中的Shh信号提供了高水平的稳健性。同时，我们对正常肢体芽进行了单细胞转录组分析，以确定瞬时Shh信号传导阶段的表达特征，并表征了即时早期反应基因。这些研究通过轨迹分析和确定的祖特异性标记揭示了一个潜在的更新祖群体。我们正在使用由标记调控区域驱动的条件Cre来跟踪这些观察结果，以进行遗传标记并进一步表征祖谱系。理解Shh-expressing祖细胞的鲁棒性和更新能力的基础可能是宝贵的在设计Shh-driven癌症的治疗干预 . .........................................................................................................................................................................sh产生和应答的谱系分析还表明，sh产生细胞完全不受自分泌信号反应的影响，这是人类许多sh驱动肿瘤的共同特征（信号通常是旁分泌的，调节支持性肿瘤生态位）。我们的遗传和转录组学研究表明，对Shh信号的无反应性与Shh配体的产生紧密相关，但在产生Shh的细胞中以细胞自主的方式发生。我们正在剖析组织培养模型中对配体产生的要求，使用内源性Shh位点与仅编码外显子序列的Shh cDNA的比较来评估这种自主无反应性的基础。了解这种严格抑制自主反应的潜在机制也将有助于了解在一些sh驱动的癌症中如何绕过这种抑制。

Sonic hedgehog (Shh) acts as a mitogen and cell survival factor in many adult processes during normal tissue renewal and in many types of cancer, but acts as a morphogen in several developmental contexts. How the mitogenic role of Shh is integrated with the morphogenetic role in developmental contexts is still poorly understood. In the limb, Shh regulates both digit number and identity of different digits (A-to-P,thumb to pinky). Shh is thought to act as a morphogen forming a gradient along the limb AP axis, with higher concentrations specifying more posterior digit types. We have determined the time-requirements for Shh function in limb (using a tamoxifen-regulated Cre to remove Shh at different times in mice). To perform this analysis, we generated and characterized a novel conditional Cre recombinase line selectively expressed in early limb mesoderm, neural crest, gut and tailbud. This line provides an excellent tool available to the scientific community to illuminate different temporal roles of key developmental regulators in several important developmental models using mouse mutants, as well as for genetic lineage tracing studies in mice. Our results, deleting Shh function at different time points, are most consistent with a model in which Shh activity is required only very transiently (several hours) to specify the complete complement of 5 different digit types, but is required for a prolonged time (about 2 days) to maintain cell survival and proliferation, enabling 5 normal digits to form. To further test this model for Shh function, we have assessed whether restoring cell survival can rescue normal digit formation in mutant embryos after a transient period of Shh activity that is terminated by Shh gene deletion. To rescue cell survival, the compound mutant for the pro-apoptotic Bcl2 family members Bax/Bak (which play roles in normal interdigital apoptosis) has been introduced to inactivate the intrinsic death pathway. Our results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. In addition, genetic lineage tracing of cells in the limb that have responded to Shh signals at different time points indicates that Shh only signals directly to the very posterior part of the limb bud at the time when all 5 digit progenitors have been specified. This result indicates that Shh acts indirectly in the early limb bud, via a system of relay signals, to specify digits. We developed a genetic assay to test for relay signaling by artificially enforcing Shh-response in the posterior limb bud in embryos lacking all Shh function (Shh KO). This partly rescues anterior digit formation, and strongly suggests the presence of relay signals acting downstream of Shh. Our results are incompatible with Shh acting as a classic morphogen in the limb and suggest that Shh acts as a trigger to activate a relay mechanism. Furthermore, our results indicate that there are 2 classes of Shh responsive target genes, those that respond to a transient signal and become stably expressed, and those that require continuous signaling to maintain expression. We are comparing the transcriptomes of Shh mutant and rescued limb buds to characterize the types of genes in these two differentially regulated target classes (see also Project : Genome-wide target analysis of Shh-activated transcription network in limb bud; ZIA BC 0111120). ......................................................................................................................................................................... Our results also indicate that both Shh-expression and response are highly dynamic and we are characterizing these features using several approaches. Our lineage tracing results of Shh production and response indicate that Shh producing cells arise from a renewing progenitor pool in the proximal limb bud margin, providing a high level of robustness to Shh signaling in the limb. In parallel, we performed single cell transcriptome profiling from normal limb buds to identify expression signatures in the transient Shh signaling phase and characterize immediate-early response genes. These studies have revealed a potential renewing progenitor population by trajectory analysis and identified progenitor-specific markers. We are following up on these observations using a conditional Cre driven by marker regulatory regions to genetically mark and further characterize the progenitor lineage. Understanding the basis for robustness and renewal capacity in Shh-expressing progenitors may be valuable in devising therapeutic intervention for Shh-driven cancers. ......................................................................................................................................................................... The lineage analysis of Shh-production and -response also suggest that Shh-producing cells are completely refractory to autocrine signaling response, which is a feature shared by many Shh-driven tumors in humans (signaling is often paracrine and regulates a supportive tumor niche). Our genetic and transcriptomic studies suggest that non-responsiveness to Shh signaling is tightly coupled to functional Shh-ligand production, but occurs in a cell autonomous manner in the producing cells. We are dissecting the requirements for ligand production in a tissue culture model using comparison of the endogenous Shh locus vs Shh cDNA with only coding exon sequences to assess the basis for this autonomous non-responsiveness. Understanding the underlying mechanisms for this strict inhibition of autonomous response will also provide insights on how this inhibition is bypassed in some Shh-driven cancers.