SoxE gene regulation of Type II collagen in the sea lamprey Petromyzon marinus

描述（由申请人提供）：所有脊椎动物，包括人类，都含有分泌软骨基质的软骨细胞，但脊椎动物中软骨形成机制的起源尚不清楚。Sox 9是一种在神经嵴中表达的HMG盒转录因子，其调节脊椎动物软骨中的主要基质蛋白II型胶原（Col 2a 1）的表达。Sox 9是Sox基因的SoxE亚家族的成员。最近的研究表明，SoxE基因在低等脊椎动物鳃弓软骨的正常发育和咽囊的形成中起重要作用。本研究将阐明SoxE基因SoxE 1、SoxE 2和SoxE 3在七鳃鳗Petromyzonmarinus鳃弓软骨形成过程中的功能。目前的项目有两个具体目标：（1）确定SoxE基因是否在发育中的咽中交叉调节其自身的表达;（2）确定SoxE基因是否调节七鳃鳗中Col 2a 1的表达。将使用分子技术的组合，包括过表达、吗啉代敲低和原位杂交。这项研究将有助于实现两个目标。主要目的是确定Col 2a 1是否受多个SoxE基因调控。患有Sox 9单倍不足的人的特征是肢端发育不良，一种以骨骼异常为特征的半致死性骨软骨发育不良，Sox 9单倍不足的小鼠在围产期死亡前发生腭裂，这表明哺乳动物（包括人类）的正常颅面和骨骼发育需要Sox 9的两个功能拷贝。这项研究将形成背景的理解，如果需要在软骨形成过程中的单个Sox 9基因的多个拷贝的表达可能已经出现在早期脊椎动物的软骨形成神经嵴中表达的多个SoxE因子。第二个目标是确定SoxE基因是否调节七鳃鳗中其他SoxE转录因子的表达。在迄今为止研究的脊椎动物模型中，Sox 9和Sox 10具有部分重叠的表达结构域，Sox 10的过表达或功能丧失可以影响Sox 9的表达，这表明这些相关基因之间的调控相互作用。这项研究将确定是否需要多个拷贝的SoxE基因适当的软骨基因可能已经出现在脊椎动物的历史早期复制后，一个单一的祖先SoxE基因，并将形成进一步调查的基础，以揭示软骨形成和咽部图案机制的控制SoxE转录因子的起源。该项目旨在确定在原始脊椎动物中，SoxE转录因子的复制对于所有脊椎动物中软骨发育的适当控制可能是重要的。这将是有价值的，以了解如何调节胶原蛋白表达的专性多功能拷贝的SoxE基因（Sox 9）出现，因为其他作用的Sox 9不依赖于多个拷贝。由于更好地理解Sox 9对软骨形成的控制取决于理解它可能在早期脊椎动物中出现的这种作用，因此这项研究服务于更大的公共利益，因为它将揭示人类软骨形成病理学的起源，例如Campomelic发育不良，这取决于多个功能Sox 9等位基因，以适当表达II型胶原蛋白和软骨形成。

DESCRIPTION (provided by applicant): All vertebrates, including humans, contain chondrocytes that secrete a cartilaginous matrix, but the origin of chondrogenic mechanisms within the vertebrates is not well understood. Sox9 is an HMG-box transcription factor expressed in the neural crest that regulates expression of Type II collagen (Col2a1), the major matrix protein in vertebrate cartilage. Sox9 is a member of the SoxE subfamily of Sox genes. Recent research indicates SoxE genes play an essential role in proper development of branchial arch cartilage and formation of pharyngeal pouches in lower vertebrates. The research described in this proposal will elucidate the function of SoxE genes, SoxE1, SoxE2 and SoxE3, during chondrogenesis of the branchial arches in the sea lamprey Petromyzon marinus. The current project has two Specific Aims: (1) to determine if SoxE genes cross-regulate their own expression in the developing pharynx; (2) to determine if SoxE genes regulate expression of Col2a1 in the sea lamprey. A combination of molecular techniques, including overexpression, morpholino knock-down, and in situ hybridization will be used. This research will contribute to two goals. The primary goal is to determine if Col2a1 is regulated by multiple SoxE genes. People who suffer from haploinsufficiency of Sox9 are characterized by campomelic dysplasia, a semilethal osteochondrodysplasia characterized by skeletal abnormalities, and mice haploinsufficient for Sox9 develop cleft palate prior to perinatal death, suggesting two functioning copies of Sox9 are required for normal craniofacial and skeletal development in mammals, including humans. This research will form the background for understanding if the need for expression of multiple copies of the single Sox9 gene during chondrogenesis may have arisen from multiple SoxE factors expressed in the chondrogenic neural crest in early vertebrates. The secondary goal is to determine if SoxE genes regulate the expression of other SoxE transcription factors in the lamprey. In vertebrate models studied to date, Sox9 and Sox10 have partially overlapping expression domains and overexpression or loss of function of Sox10 can affect the expression of Sox9, suggesting regulatory interaction between these related genes. This research will determine if the requirement of multiple copies of SoxE genes for proper chondrogenes may have arisen early in vertebrate history following duplication of a single ancestral SoxE gene, and will form the basis for further investigation to uncover the origin of the control of chondrogenesis and pharyngeal patterning mechanisms by SoxE transcription factors. This project seeks to determine in a primitive vertebrate, how duplication of SoxE transcription factors may be important for proper control of cartilage development in all vertebrates. It will be valuable to understand how the regulation of collagen expression by obligate multiple functioning copies of a SoxE gene (Sox9) arose since other roles of Sox9 do not depend on multiple copies. Since a greater understanding of the control of chondrogenesis by Sox9 depends on understanding this role where it may have arisen in early vertebrates, this study serves the greater public good because it will shed light on the origin of chondrogenic pathologies in humans, such as campomelic dysplasia, that depend on multiple functioning Sox9 alleles for proper expression of type II collagen, and chondrogenesis.