Role of Shh in developmental patterning and proliferation 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。Myc已被确定为在几个系统中调节增殖的Hedgehog信号传导的直接靶标。为了尝试挽救增殖，我们正在使用RosaMycER转基因（来自Gerard Evan博士）来提供增殖冲动，其时间，持续时间和作用水平可以由他莫昔芬密切调节。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）。在补充实验中，我们将使用Allan Bradley （Nature, 2011）开发的首个靶向Shh等位基因敲除，以检查肢体发育过程中延迟正常Shh表达的后果（是否形成手指，身份是否改变）。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。在发现肢体中Shh缺失导致g1阻滞后，我们同时也在分析Shh如何调节细胞周期。了解Shh的增殖和抗凋亡作用可能为破译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？我们也在使用基因工具，包括在很早的时候就改变数字模式的突变体，以了解数字形成的交替序列是何时以及如何确定的。这些工具将有助于揭示所观察到的前后交替凝结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。

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. Myc has been identified as a direct target of Hedgehog signaling that regulates proliferation in several systems. To attempt rescue of proliferation, we are employing a RosaMycER transgene (from Dr. Gerard Evan) to provide a proliferative impulse whose timing, duration and level of action can be closely regulated by tamoxifen. 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. In complementary experiments we will use a knock-out first targeted allele of Shh developed by Allan Bradley (Nature, 2011) to examine the consequences of delaying the onset of normal Shh expression during limb development (do digits form, and are identities altered). 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. Having found that Shh loss in limb causes G1-arrest, in parallel we are also analyzing how Shh regulates the cell cycle. Understanding the proliferative and anti-apoptotic actions of Shh may provide a reference for deciphering 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 to alter digit patterns at very 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-to-posterior order of condensation is regulated, and whether this sequence is evolutionarily conserved among different vertebrates.