Role of Shh in developmental patterning and proliferation of digit skeleton

Shh在正常组织更新的许多成体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寿命追踪研究。我们发现Shh在肢体中具有可分离的双重功能：一个是控制手指模式的早期瞬时角色，另一个是调节细胞质量(生存和增殖)的持续角色，它决定了总的位数。我们发现了一个不变的数字丢失顺序，这与当前形态梯度模型的预测不一致。断指顺序与断指原基形成顺序呈负相关，不同时间的表型严重程度与细胞凋亡程度和核分裂指数下降呈负相关。我们的结果表明，Shh只在非常早期和短暂地调节手指模式，但在较长时间内需要，主要是为了确保细胞存活和/或增殖。如果肢芽的细胞较少，可以形成的指状原基也较少，但形成的指状原基是正常的。Gli3转录因子是Shh信号在细胞核中的主要下游效应因子。在与Alex Joyner(Sloan Kettering Inst.)博士的合作研究中，也分析了Gli3功能的时间要求，这项研究的结果支持了我们的模型，也表明生长中Shh扩大数字的需要比构图需要更长的时间。为了测试Shh功能的这一模型，我们将确定在后期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。为了挽救细胞的存活，将引入促凋亡的Bcl2家族成员Bax/Bak的复合突变体(它们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，我们将使用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。在发现肢体中Shh的丢失导致G1期停滞后，我们将同时分析Shh是如何调节细胞周期的。了解Shh的增殖和抗凋亡作用可能为破译Shh在肿瘤中的作用提供参考。为了了解间充质细胞的增殖和募集如何与凝集的交替形成相关，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，并追踪细胞的遗传谱系(与Yosuke Mukyama博士合作)。这些工具将有助于解开观察到的A-P凝聚交替顺序是如何调节的，以及这种顺序是否在不同的脊椎动物中进化上保守。我们还在分析后来软骨开始形成时产生的另一种刺猬信号(印度刺猬，IHH)是否在决定每个手指和Shh的最终形态或身份方面发挥作用。我们将使用条件突变等位基因在不同的时间通过移除信号-受体相互作用下游的一个重要通路组件(平滑的，SMO)来评估所有刺猬配体在肢体发育早期的时间需求。在一项互补性的合作研究中，我们还分析了结构性活跃的SMO的反向功能增益表型(与NICHD的Sohyun Ahn博士一起)。

Shh acts as a mitogen and cell survival factor in many adult processes during normal tissure 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 lieage tracing studies in mice. We found that Shh has separable, dual functions in limb: an early transient role controlling digit pattern, and an ongoing role regulating cell mass (survival and proliferation) that determines the total digit number. We find an invariant order of digit loss that is not consistent with predictions from current morphogen gradient models. The order of digit loss correlates inversely with the order in which digit primordia first form, and the phenotype severity at different times correlates with degree of apoptosis and decrease in mitotic index. Our results suggest that Shh regulates digit pattern only very early and transiently but is required over an extended time mainly to ensure cell survival and/or proliferation. If the limb bud has fewer cells, fewer digit primordia can form but those that do form are normal. The Gli3 transcription factor is the major downstream effector of Shh signaling in the nucleus. In collaborative studies with Dr. Alex Joyner (Sloan Kettering Inst.), the time-requirements for Gli3 function have also been analyzed and the results of this study support our model, also suggesting that the requirement for Shh in growth to expand digit numbers requires a much more prolonged time than the requirement for patterning. To test this model for Shh function, we will determine if 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) will be 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 will use 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. Having found that Shh loss in limb causes G1-arrest, in parallel we will analyze 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 formation of condensations, 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. Yosuke Mukoyama). These tools will help unravel how the observed alternating A-P order of condensation is regulated, and whether this sequence is evolutionarily conserved among different vertebrates. We are also analyzing whether another hedgehog signal (Indian Hedgehog, Ihh), produced later when cartilage begins to form, plays any role in determining the final morphology or identity of each digit together with Shh. We will assess the time requirements for all hedgehog ligands during early limb development by removing an essential pathway component (smoothened, smo) downstream of signal-receptor interaction at different times using a conditional mutant allele. In a complementary collaborative study, we are also analyzing the converse, gain-of-function phenotype of constitutively active smo (with Dr. Sohyun Ahn, NICHD).