Ying Chen

Date of Award

8-2013

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Advisor

Thomas Gridley

Second Committee Member

Mary Ann Handel

Third Committee Member

Greg Cox

Abstract

Endochondral bone formation is a multistep process during which a cartilage primordium is replaced by mineralized bone. Several genes involved in cartilage and bone development have been identified as target genes for the Snail family of zinc finger transcriptional repressors, and a gain-of-function study has demonstrated that upregulation of Snail activity in mouse long bones caused a reduction in bone length. However, no in vivo loss-of-function studies have been performed to establish whether Snail family genes have an essential, physiological role during normal bone development. I demonstrate here that the Snail and Snai2 genes function redundantly during embryonic long bone development in mice. Deletion of the Snai2 gene, or limb bud-specific conditional deletion of the Snail gene, did not result in obvious defects in the skeleton. However, limb bud-specific Snail deletion on a Snai2 null genetic background resulted in substantial defects in the long bones of the limbs. Long bones of the Snai1ISnai2 double mutants exhibited defects in chondrocyte morphology and organization, inhibited trabecular bone formation and delayed ossification. Snai2 transcript levels were increased in Snail mutant femurs, while Snail transcript levels were increased in Snai2 mutant femurs. In addition, in the mutant femurs the Snail and Snai2 genes compensated for each other’s loss not only quantitatively, but also by expanding their expression into the other genes’ normal expression domains. These results demonstrate that the Snail and Snai2 genes transcriptionally compensate temporally, spatially, and quantitatively for each other’s loss.

In order to understand the genetic redundancy observed in the mouse loss of function models at the molecular level, I employed the mouse ATDC-5 chondrogenic cell line and performed ChIP assays to examine the binding patterns of the SNAI1 and SNAI2 proteins to their own and each other's promoter during chondrogenic differentiation of the ATDC5 cells. Consistent with the in vivo data, my ATDC-5 cell results support the model that expression of the Snail and Snai2 genes is negatively regulated by their protein products occupying each other’s promoter during chondrogenesis, which helps provide an explanation for the genetic redundancy observed in the mouse loss of function models.

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