Sonic Hedgehog Signaling in Zebrafish Branchial Arch Development

描述（由申请人提供）：拟定的研究旨在确定颅面骨骼中Shh信号传导的细胞和分子机制。这可能为在前脑无裂畸形患者中观察到的广泛表型提供新的解释。为了检查Shh在鳃弓发育中的功能，我们分析了斑马鱼变色龙（con）突变体，其具有缺陷的Displ蛋白，con突变体胚胎缺乏所有的鳃弓软骨，这一缺陷可以部分地由鳃弓前体维持必需的软骨形成转录因子Sox9a的失败来解释。拟议的研究将专门寻求描绘信号机制，从而Shh途径促进软骨形成的鳃弓。我们推测，Displ行为内咽内胚层非细胞自主释放Shh，指导鳃弓软骨前体向软骨分化。利用细胞移植研究，我们将产生嵌合胚胎，以证明Displ和Smo功能的细胞自主性。这将决定颅面区域中的哪些细胞必须释放（Displ）和响应（Smo）Shh配体。此外，我们的工作已经划定了多个时间要求的Shh信号在鳃弓的发展。利用一个良好的特征GN1活性报告构建体，我们将产生一种新的转基因斑马鱼，以精确地确定哪些细胞接受Shh配体，当这发生在软骨发育过程中。最后，人类颅面疾病是由SHH通路和SOX 9的误调节引起的，因此进一步研究这种潜在的调节关系具有相当大的意义。将进行体内过表达研究以评估鳃弓中的异位SoxQa是否可以挽救Shh途径的失活。Shh信号转导通路在大脑和面部的生长和图案化中起着核心作用。在胚胎发育过程中，这一途径的中断会导致相当大的疾病负担，最常被归类为谱系障碍性前脑无裂症。这项研究的总体目标是确定控制颅面骨骼发育的Shh通路的细胞和分子机制。

DESCRIPTION (provided by applicant): The proposed studies seek to determine the cellular and molecular mechanisms of Shh signaling in the craniofacial skeleton. This may provide new explanations for the wide range of phenotypes observed in Holoprosencephaly patients. To examine the function of Shh in branchial arch development we analyzed the zebrafish chameleon (con) mutant, harboring a defective Displ protein, con mutant embryos lack all branchial arch cartilage, a defect that can be partially explained by a failure of the branchial arch precursors to maintain the essential chondrogenic transcription factor Sox9a. The proposed research will specifically seek to delineate the signaling mechanism whereby the Shh pathway promotes chondrogenesis in the branchial arches. We hypothesize that Displ acts within pharyngeal endoderm non cell autonomously to release Shh which directs branchial arch cartilage precursors toward chondrogenic differentiation. Utilizing cell transplantation studies, we will generate chimeric embryos to demonstrate the cell autonomy of Displ and Smo function. This will determine which cells in the craniofacial region must release (Displ) and respond (Smo) to the Shh ligand. Furthermore, our work has delineated multiple temporal requirements for Shh signaling in branchial arch development. Utilizing a well-characterized GN1 activity reporter construct, we will generate a novel transgenic zebrafish to precisely determine which cells recieve the Shh ligand and when this occurs during cartilage development. Lastly, human craniofacial diseases arise from the misregulation of the SHH pathway and SOX9, therefore further investigation of this potential regulatory relationship is of considerable interest. In vivo overexpression studies will be performed to assess whether ectopic SoxQa in the branchial arches can rescue inactivation of the Shh pathway. The Shh signal transduction pathway plays a central role in growth and patterning of the brain and face. Disruptions in this pathway during embryonic development lead to a considerable disease burden, most often classified as the spectral disorder Holoprosencephaly. The overall goal of this proposed research is to determine the cellular and molecular mechanism of the Shh pathway that controls craniofacial skeleton development.