How Critical is the Hedgehog Pathway During Early Skeletogenesis?

One major feature of the vertebrate body plan is the skeletal apparatus and its development is regulated in part by the Hedgehog (Hh) family of signaling proteins. Skeletal defects are known to arise in Hh deficient vertebrates, however, Hh genes are required throughout ontogeny, therefore elucidating its bone regulatory function in this context remains a challenge. Using transgenic zebrafish whereby the Hh pathway may be up- or downregulated via heat shock, we were able to regulate global expression of the Hh pathway in a time specific manner. Transgenic fish were outcrossed with a wild-type strain to produce transgenic and non-transgenic offspring which could be distinguished by the presence or lack of green fluorescence protein expression. Zebrafish were heatshocked starting after the completion of the hatching stage for one week then cleared and stained using Alizarin Red and Alcian Blue followed by imaging with light and fluorescent microscopy. Differences in bone development were observed by measuring length, area, and number of several prominent bone structures including the cleithrum, opercle, branchiostegal rays and vertebral central. Downregulating the Hh pathway reduced the lengths and sizes of early forming bones, and delayed the formation of later forming ones. While measurement data for Hh upregulatory transgenic fish are less clear, the appearance of fusion events between branchiostegal rays in multiple fish suggest an undiscovered pathological role for this pathway.