Role of Shh in developmental patterning and growth of digit skeleton

Sonic hedgehog（Shh）在正常组织更新期间的许多成人过程和许多类型的癌症中充当有丝分裂原和细胞存活因子，但在几种发育环境中充当形态发生剂。Shh的促有丝分裂作用如何与发育环境中的形态发生作用相结合仍然知之甚少。在肢体中，Shh调节不同手指的数字和身份（A到P，拇指到小指）。Shh被认为是一种形态发生素，沿肢体AP轴沿着形成梯度，浓度越高，说明更多的后趾类型。我们已经确定了肢体中Shh功能的时间要求（使用他莫昔芬调节的Cre在小鼠中的不同时间去除Shh）。为了进行这种分析，我们产生并表征了一种新的条件性Cre重组酶系，该重组酶系选择性地在早期肢体中胚层、神经嵴、肠和尾芽中表达。这条线提供了一个很好的工具，可供科学界阐明不同的时间作用的关键发育调节因子在几个重要的发育模型，使用小鼠突变体，以及遗传谱系跟踪研究小鼠。我们的研究结果，删除Shh功能在不同的时间点，是最一致的模型，其中Shh活动只需要非常短暂的（几个小时），以指定5个不同的数字类型的完整的补充，但需要一个延长的时间（约2天），以维持细胞的存活和增殖，使5个正常的数字形成。为了进一步测试该模型的Shh功能，我们评估了恢复细胞存活是否可以在Shh基因缺失终止的Shh活性短暂期后挽救突变胚胎中的正常指形成。为了挽救细胞存活，已经引入促凋亡Bcl 2家族成员Bax/巴克（其在正常的趾间凋亡中起作用）的复合突变体以阻断内在死亡途径。我们的研究结果表明，正常的数字和模式（形态发生）可以通过简单地恢复细胞存活和增殖Shh突变胚胎获救。此外，在不同时间点对Shh信号作出反应的肢体细胞的遗传谱系追踪表明，当所有5个指祖细胞都已被指定时，Shh仅直接向肢芽的非常后部发出信号。这一结果表明，Shh在早期肢芽中通过中继信号系统间接作用，以指定数字。我们开发了一种遗传分析，通过在缺乏所有Shh功能的胚胎（Shh KO）中人工强制Shh反应来测试中继信号。这在一定程度上挽救了前趾的形成，并强烈表明存在中继信号作用于Shh的下游。我们的研究结果是不兼容的Shh作为一个经典的形态发生在肢体，并建议，Shh作为触发激活中继机制。此外，我们的研究结果表明，有2类Shh响应靶基因，那些响应于瞬时信号，并成为稳定表达，和那些需要持续的信号传导，以维持表达。我们正在比较Shh突变体和获救肢芽的转录组，以表征这两种差异调节靶类中的基因类型（另见项目：肢芽中Shh激活转录网络的全基因组靶分析; ZIA BC 0111120）。.........................................................................................................................................................................我们的研究结果还表明，Shh-表达和反应是高度动态的，我们正在使用几种方法来表征这些功能。我们的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芽边缘的更新祖细胞库，为肢体中的Shh信号传导提供了高水平的鲁棒性。同时，我们进行了正常肢芽的单细胞转录组分析，以确定瞬时Shh信号传导阶段的表达特征，并表征立即早期反应基因。这些研究通过轨迹分析揭示了一个潜在的更新祖细胞群体，并确定了祖细胞特异性标记。我们正在跟踪这些观察结果，使用由标记调控区驱动的条件Cre来遗传标记并进一步表征祖细胞谱系。了解Shh表达祖细胞的稳健性和更新能力的基础可能对设计Shh驱动的癌症的治疗干预有价值。......................................................................................................................................................................... Shh产生和反应的谱系分析还表明，产生Shh的细胞完全不受自分泌信号反应的影响，这是人类许多Shh驱动的肿瘤所共有的特征（信号通常是旁分泌的，并调节支持性肿瘤生态位）。我们的遗传学和转录组学研究表明，对Shh信号传导的无响应性与功能性Shh配体产生紧密相关，但在产生细胞中以细胞自主方式发生。我们正在解剖的组织培养模型中的配体生产的要求，使用比较内源性的Shh基因座与Shh cDNA仅编码外显子序列，以评估这种自主的非响应性的基础。理解这种严格抑制自主反应的潜在机制也将提供关于这种抑制在一些Shh驱动的癌症中如何被绕过的见解。

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.