Role of Shh in developmental patterning and growth of digit skeleton

Shh在正常组织更新的许多成人过程中和在许多类型的癌症中作为有丝分裂原和细胞生存因子，但在一些发育背景下作为形态原。Shh的有丝分裂作用是否以及如何在发育背景下与形态发生作用相结合仍然知之甚少。在肢体中，Shh同时调节数字和不同手指的身份(A-P、拇指-小指)。Shh被认为是一种沿着肢体AP轴形成梯度的形态生成物，浓度越高，指定的后部手指类型越多。我们已经确定了肢体Shh功能的时间要求(使用他莫昔芬调节的Cre在不同时间在小鼠体内清除Shh)。为了进行这一分析，我们构建并鉴定了一个在早期肢体中胚层、神经脊、肠道和尾芽中选择性表达的条件性Cre重组酶系。这条线为科学界提供了一个极好的工具，可以用来阐明关键发育调节因子在几个使用小鼠突变的重要发育模型中的不同时间角色，以及用于小鼠的遗传谱系追踪研究。作为对该模型的一种检验，我们发现不同类型的减少位数的突变体的Shh活性谱支持该模型。特别是，中央指头首先丢失的突变体Shh活性持续时间缩短，但指定后端指头的早期瞬时模式阶段保持不变。相反，选择性缺失数字5的突变体在早期构型阶段延迟了Shh表达的开始，并改变了Shh的活性。为了进一步测试Shh的功能，我们评估了后期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。为了挽救细胞的存活，已经引入了促凋亡的Bcl2家族成员Bax/Bak的复合突变(Bax/Bak在正常的指间凋亡中发挥作用)来灭活固有的死亡途径。MYC已被确定为Hedgehog信号的直接靶点，该信号在几个系统中调节增殖。为了试图挽救增殖，我们正在使用RosaMycER转基因(来自Gerard Evan博士)来提供一种增殖冲动，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，可以挽救正常的位数和模式(形态发生)。到目前为止，我们的结果挑战了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。此外，我们的结果表明，Shh反应靶基因有两类，一类是对瞬时信号做出反应并稳定表达的基因，另一类是需要持续信号来维持表达的基因。我们正在比较Shh突变和挽救的肢芽的转录本，以表征这两个不同调节的靶类中的基因类型。了解Shh在这些不同调控靶类背景下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招募如何与凝集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对非常早期的凝集进行成像，并从基因上追踪细胞谱系(与NCI的Stephen Lockett博士合作)。例如，缩合是通过交替序列中前体的空间受限增殖还是通过细胞黏附的交替焦点形成的？我们还在使用基因工具，包括在很早的时候就能改变手指模式的突变体，以了解手指形成的交替顺序是何时以及如何决定的。这些工具将有助于解开观察到的从前到后的交替凝聚顺序是如何调节的，以及这个序列是否在不同的脊椎动物中进化保守。数字形成的交替模式的可能性也可能导致对具有适应数字变化的物种进化中的数字丢失的机制的新的见解。

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. 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. 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. 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 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. 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.