Role of Shh in developmental patterning and proliferation of digit skeleton

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的促有丝分裂作用是否以及如何与发育背景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，我们产生并表征了在早期肢中胚层、神经嵴、肠和尾芽中选择性表达的条件性Cre重组酶系。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。作为该模型的一个测试，我们表明，Shh活性曲线在不同类别的突变体与减少的数字支持该模型。 特别是，突变体中，中央数字首先失去了嘘活动的持续时间减少，但早期短暂的图案化阶段，其中后数字指定，保持完整。相反，突变体与数字5的选择性损失有延迟的发病Shh的表达和改变Shh的活动在早期图案化阶段。为了进一步测试该模型的Shh功能，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。Myc已被鉴定为Hedgehog信号转导的直接靶点，其在几个系统中调节增殖。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，其时机，持续时间和作用水平可以通过他莫昔芬密切调节。我们的初步研究结果表明，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。 在补充实验中，我们将使用由Allan布拉德利（Nature，2011）开发的Shh的敲除第一靶向等位基因来检查在肢体发育期间延迟正常Shh表达的开始的后果（数字形成，并且身份改变）。到目前为止，我们的研究结果挑战了Shh作为经典形态发生因子的观点，并表明Shh在器官形态发生和肿瘤发生过程中的作用可能非常相似。在发现肢体中的Shh缺失导致G1期阻滞后，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凝聚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凝聚，并在遗传上追踪细胞谱系（与Stephen Lockett博士合作，NCI）。例如，凝聚形成通过空间限制增殖的前体在一个交替的序列，或通过交替的焦点细胞粘附？ 我们还使用遗传工具，包括突变体，这些突变体在很早的时候就改变了数字模式，以了解数字形成的交替序列何时以及如何确定。这些工具将有助于揭示所观察到的交替的前到后的凝聚顺序是如何调节的，以及这种序列在不同的脊椎动物中是否进化保守。

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.