Regulation of Shh Signaling Activity in Limb Patterning

描述（申请人提供）：由Hedgehog （Hh）基因家族编码的分泌分子已被认为是调节无脊椎动物和脊椎动物胚胎生长和模式的关键信号。编码Shh信号成分的Shh基因突变与许多人类临床疾病有关，包括前脑畸形和各种形式的癌症。Hh配体最显著的特征之一是在发育组织中充当经典形态形成因子的能力。这在发育中的脊椎动物肢体中尤为明显，Shh通常在极化活动区（ZPA）的后肢边缘表达，能够以剂量依赖的方式逐渐指定具有更多后肢身份的手指数量的增加。在此之前，我们的研究确定了Shh在肢体骨骼元素生长和模式形成中的需求。此外，我们发现这种需求是由Gli3加工的调节所介导的。在这种情况下，从ZPA分泌的Shh抑制Gli3加工成其抑制因子形式（GN3R），从而促进全长GN3蛋白（Gli3- FL或GH3-190）的积累，该蛋白在Shh信号传导范围内可作为转录激活因子（GN3A），因此，GN3A和GN3R之间的相对平衡可能在介导Shh的肢体模式功能中起关键作用。在了解Shh活性梯度如何被调节以在肢体中产生确定的模式方面，仍然存在主要的空白。解决这个问题需要更好地理解影响Shh运动的因素，配体-受体相互作用，旁分泌活性的模式效应，以及反应组织中效应物的转录活性。因此，我们提出的研究主要旨在通过解决(1)胆固醇片段在哺乳动物组织环境中调节Shh运动/空间范围的作用，(2)局部和旁分泌信号在产生复杂的手指模式中的贡献，(3)不同形式的Gli3在调节组织模式中的生物学功能，更好地了解肢体发育过程中Shh信号是如何被调节和解释的。我们相信这些知识将与Shh在人类器官发生、疾病和癌症中的作用直接相关。

DESCRIPTION (provided by applicant): Secreted molecules encoded by the Hedgehog (Hh) gene family have been recognized as key signals in regulating the growth and patterning of invertebrate and vertebrate embryos. Mutations in Shh genes encoding Shh signaling components have been associated with many clinical disorders found in humans including holoprosencephaly and various forms of cancer. One of the most salient features of Hh ligands is the ability to act as a classical morphogen in developing tissues. This is particularly evident in the developing vertebrate limbs, where Shh, normally expressed in the posterior limb margin of the zone of polarizing activity (ZPA), has ability to progressively specify increase in number of digits with more posterior identities in a dose-dependent manner. Previously, our studies identified the requirements of Shh in both growth and patterning of the skeletal elements of the limb. Additionally, we showed that this requirement is necessarily mediated by regulation of Gli3 processing. In this context, Shh secreted from the ZPA inhibits Gli3 processing into its repressor form (GN3R), hence promoting the accumulation of full-length GN3 protein (Gli3- FL or GH3-190), which can function as a transcriptional activator (GN3A) within the range of Shh signaling, thus, the relative balance between GN3A and GN3R likely plays a critical role in mediating the limb patterning function of Shh. Major gaps remain in understanding how the Shh activity gradient is regulated to generate defined patterns in the limb. Addressing this question requires a better understanding of factors affecting Shh movement, ligand-receptor interaction, patterning effects of paracrine activity, and transcriptional activity of effectors in the responsive tissue. Therefore, our proposed studies are aimed primarily at providing a better understanding of how Shh signaling is regulated and interpreted during limb development by addressing (1) the role of cholesterol moiety in regulating Shh movement/spatial range in a mammalian tissue environment, (2) the contribution of local and paracrine signaling in generating a complex pattern of digits, (3) the biological function of different forms of Gli3 in regulating tissue patterning. We believe this knowledge will be directly relevant to the role of Shh in human organogenesis, disease and cancer.