The role of vasculature during the development of intramembranous bones of the chick, Gallus gallus

Abstract

Intramembranous bone development is a complex multi-step process which relies on extensive interactions between bone cells and surrounding tissue types. The embryonic vasculature, composed primarily of vascular endothelial cells, is well known to play a key role in regulating bone development via endochondral ossification; however, its role during intramembranous ossification remains poorly understood, and in vivo studies are lacking. Here I use the scleral ossicles of the domestic chick (Gallus gallus), a ring of intramembranous bones located in the sclera of the eye, to investigate the role of vasculature in intramembranous bone development in vivo. My results indicate that vasculature begins to fill the posterior sclera at HH 35 and forms distinct avascular zones around the conjunctival papillae, structures involved in the induction of the scleral ossicles. Posteriorly, these avascular zones are obliterated by HH 36.5, concomitant with the end of ossicle induction. In situ hybridization and bead implantation experiments suggest a key role for vascular endothelial growth factor (Vegf) in coordinating vascular and scleral ossicle development. Vegfa is expressed in the conjunctival papillae and the surrounding mesenchyme during ossicle induction and vascular growth, and is downregulated thereafter. Localized knockdown of Vegf signaling via bead implantation results in both an expansion of the avascular zone and mispatterning of the scleral ossicles, suggesting a key role for Vegf in regulating vascular growth and ossicle development. This work provides important insights into the complex relationship between bone and vasculature during development of chick scleral ossicles and intramembranous bones in general.