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。这条线为科学界提供了一个很好的工具，可以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。我们发现Shh在肢体中具有可分离的双重功能：早期瞬时作用控制手指模式，以及持续作用调节细胞质量（存活和增殖），决定手指总数。我们发现数字损失的不变顺序与当前形态梯度模型的预测不一致。趾丢失的先后顺序与趾原基形成的先后顺序成反比，不同时期的表型严重程度与细胞凋亡程度和有丝分裂指数的降低相关。我们的研究结果表明，Shh仅在非常早期和短暂的时间内调节趾型，但在很长一段时间内主要需要确保细胞存活和/或增殖。如果肢芽的细胞较少，能形成的指原基就较少，但能形成的指原基是正常的。Gli3转录因子是细胞核中Shh信号的主要下游效应因子。在与Dr. Alex Joyner （Sloan Kettering institute .）的合作研究中，也分析了Gli3功能的时间要求，本研究的结果支持我们的模型，也表明Shh在生长过程中扩展数字所需的时间比模式所需的时间要长得多。为了测试这个Shh功能模型，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。为了挽救细胞存活，将引入促凋亡Bcl2家族成员Bax/Bak（在正常指间凋亡中起作用）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缺失导致g1阻滞之后，我们将同时分析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析物的交替形成相关，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成像非常早期的凝析物，并从遗传学上追踪细胞谱系（与Yosuke Mukoyama博士合作）。这些工具将有助于揭示所观察到的交替的A-P缩合顺序是如何被调节的，以及这个序列在不同的脊椎动物中是否具有进化保守性。我们还分析了另一种在软骨开始形成后产生的刺猬信号（印度刺猬，Ihh）是否与Shh一起在决定每个趾的最终形态或身份方面发挥任何作用。我们将通过使用条件突变等位基因在不同时间移除信号受体相互作用下游的一个重要通路成分（smoothened, smo），来评估早期肢体发育过程中所有刺猬配体的时间需求。在一项互补的合作研究中，我们也分析了相反的，功能获得型构成活性smo（与Sohyun Ahn博士，NICHD）。

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).