The type I TGF-beta receptor Alk8 participates in neural crest cell (NCC) formation and dorsoventral patterning of the zebrafish embryo. Preliminary studies demonstrated that decreased Alk8 signaling, through injection of dominant negative (DN)-
alk8 mRNA, resulted in a dorsalized phenotype, and disrupted pharyngeal arch development. Complete inhibition of Alk8 signaling prevented the formation of NCCs altogether (Payne-Ferreira and Yelick, 2003). While the early influence of Alk8 on embryonic patterning was demonstrated to occur through modulation of BMP signaling (Bauer et al, 2001, Mintzer et al, 2001, Payne et al, 2001), we have also demonstrated that Alk8 can specifically bind TGF-beta ligand (deCaestekker et al., 2002). We hypothesize that Alk8 mediates multiple signaling pathways during early zebrafish development. Objective: The objective of this study is to determine the components of the Alk8 signaling pathway. Methods: Microarray analysis revealed genes affected by injection of DN-
alk8 mRNA. The top 100 most significantly up- or down-regulated ESTs have been determined, and those involved in cell signaling have been selected for further characterization. Real-time PCR analyses were performed to determine gene expression levels and verify the change indicated in the array analysis. Temporospatial changes in gene expression between wild type (WT) and DN-
alk8 mRNA injected embryos were examined by Whole Mount In Situ Hybridization (WISH). Results: The helix-loop-helix protein, Id2, has been found to be down-regulated in DN-
alk8 mRNA injected embryos. Id proteins have been described as key targets of normal neural crest cell development. We are characterizing the role of Id2 in Alk8 signaling pathways and believe that Id2 may act down-stream of Alk8 regulating zebrafish neural induction and patterning. Conclusion: We conclude that Alk8 is an integral component of embryonic signaling pathways and may direct a variety of cellular fate decisions in part through modulation of Id2. Supported by NIDCR grant K22 DE 14683-2.