Role of Shh in developmental patterning and growth of digit skeleton

Shh在正常组织更新和许多类型的癌症的许多成人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的有丝分裂作用是否以及如何与形态发生作用相结合仍然知之甚少。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）。为了进行这项分析，我们产生了一个条件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。作为该模型的一项测试，我们发现不同类别的数字减少突变体的Shh活性谱支持该模型。特别是，首先失去中央趾的突变体，Shh活动的持续时间缩短，但早期的瞬时模式阶段，即指定的后趾，保持完整。相反，选择性丢失5趾的突变体在早期模式形成阶段会延迟Shh的表达和改变Shh的活性。为了进一步测试这个Shh功能模型，我们评估了在后期Shh缺失后恢复突变胚胎的存活和/或增殖是否可以挽救手指的形成。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。我们的初步结果表明，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）。到目前为止，我们的研究结果挑战了Shh作为一种经典形态因子的观点，并表明Shh在器官形态发生和肿瘤发生中的作用可能非常相似。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。我们正在比较Shh突变体和获救肢体芽的转录组，以表征这两种不同调控靶类的基因类型。同时，将对正常肢体芽进行单细胞转录组分析，以确定瞬时Shh信号阶段的表达特征，并表征即时早期反应区。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在癌症中的作用提供参考。为了了解间充质细胞的增殖和募集如何与冷凝形成的交替顺序相关，并将我们的结果与Shh功能的其他工作相结合，我们正在开发工具来成像非常早期的冷凝，并从基因上追踪细胞谱系（与NCI的Stephen Lockett博士合作）。例如，冷凝是通过前体在交替序列中的空间限制增殖形成的，还是通过细胞粘附的交替焦点形成的？我们还使用基因工具，包括在hedgehog信号下游的突变体，在早期改变手指模式，以了解何时以及如何确定手指形成的交替序列。这些工具将有助于揭示所观察到的手指缩合前后交替顺序是如何被调节的，以及这一序列在不同脊椎动物中是否具有进化保守性。趾数形成交替模式的可能性也可能导致对趾数发生适应性变化的物种进化中趾数丢失机制的新见解。

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. Whether and 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 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. As one test of this model, we showed that the Shh activity profiles in different classes of mutants with reduced digit number support the model. In particular, mutants in which central digits are lost first have a reduced duration of Shh activity, but the early transient patterning phase, in which posterior digits are specified, remains intact. Conversely, mutants with selective loss of digit 5 have delayed onset of Shh expression and altered Shh activity during the early patterning phase. To further test this model for Shh function, we have assessed whether restoring survival and/or proliferation in mutant embryos after later-stage Shh deletion can rescue digit formation. 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 preliminary results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. Our results thus far challenge the view that Shh behaves as a classic morphogen and suggest that the roles of Shh during organ morphogenesis and during tumorigenesis may be very similar. 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. In parallel, single cell transcriptome analysis from normal limb buds will be performed to identify expression signatures in the transient Shh signaling phase and characterize immediate-early response zones. Understanding the proliferative and anti-apoptotic actions of Shh in the context of these differentially regulated target classes, will provide a reference for deciphering and intercepting Shh roles in cancer. To learn how proliferation and recruitment of mesenchymal cells relate to the alternating order in which condensations form and integrate our results with other work on Shh function, we are developing tools to image very early condensations, and to trace cell lineage genetically (collaboratively with Dr. Stephen Lockett, NCI). For example, do condensations form via spatially restricted proliferation of precursors in an alternating sequence, or by alternating foci of cell adhesion? We are also using genetic tools including mutants that act downstream of hedgehog signaling to alter digit patterns at early times to learn when and how the alternating sequence of digit formation is determined. These tools will help unravel how the observed alternating anterior-posterior order of digit condensations is regulated, and whether this sequence is evolutionarily conserved among different vertebrates. The possibility of alternating patterns of digit formation may also lead to new insights on the mechanisms of evolutionary digit loss in species with adaptive changes in digit number.