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基因突变与人类发现的许多临床疾病有关，包括全前脑畸形和各种形式的癌症。HH配体最显著的特征之一是能够在发育中的组织中充当经典的形态生成物。这在发育中的脊椎动物肢体中尤为明显，Shh通常表达在极化活动区(ZPA)的后肢边缘，能够以剂量依赖的方式逐渐指定具有更多后端身份的手指数量的增加。以前，我们的研究确定了Shh在肢体骨骼元素的生长和模式形成中的需求。此外，我们还证明了这一要求必须通过调节Gli3的加工来调节。在这种背景下，ZPA分泌的Shh抑制Gli3的加工进入其阻遏形式(GN3R)，从而促进全长GN3蛋白(Gli3-FL或GH3-190)的积累，而GN3蛋白可以在Shh信号范围内作为转录激活因子(GN3A)发挥作用，因此，GN3A和GN3R之间的相对平衡可能在介导Shh的肢体模式功能中发挥关键作用。在理解Shh活动梯度如何被调节以在肢体中产生明确的模式方面仍然存在主要差距。解决这个问题需要更好地了解影响Shh运动的因素、配体-受体相互作用、旁分泌活性的图案化效应以及反应组织中效应器的转录活性。因此，我们建议的研究主要是为了更好地了解Shh信号在肢体发育过程中是如何被调控和解释的，通过解决(1)胆固醇部分在调节哺乳动物组织环境中Shh运动/空间范围中的作用，(2)局部和旁分泌信号在产生复杂的数字模式中的作用，(3)不同形式的Gli3在调节组织图案中的生物功能。我们相信这一知识将与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.