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.

R03DE018159

2007-08-01

2010-07-31