Functional Analysis of Pegasus: The ‘Atypical’ Member of the Ikaros Gene Family.

Abstract 3645

Poster Board III-581

The Ikaros family of zinc finger transcription factors are an important class of hematopoietic regulators involved in the development of the immune system. However, the in vivo role of its most divergent ‘atypical’ member, Pegasus, remains unknown. Therefore, we have utilised zebrafish to explore the function of Pegasus. Bioinformatics analysis identified a single pegasus orthologue in zebrafish. Wholemount in situ hybridisation revealed an initial broad distribution of maternally-derived pegasus transcripts during early zebrafish embryogenesis, which was replaced by specific expression in the brain, eye, thymus, gut and pancreas during late embryogenesis. Morpholino-mediated gene knockdown of zebrafish pegasus resulted in a reduction in cells expressing markers for hematopoietic stem cells (lmo2), granulocytes (mmp9), macrophages (fms), as well as early (ikaros) and late (rag1) lymphoid cells while, conversely, those expressing early (spi1) and late (l-plastin) myeloid markers were increased. No difference was noted in the early erythroid (gata1) compartment, although mature red blood cells appeared reduced, as judged by o-dianisidine staining. During emergency hematopoiesis (elicited by LPS injection), numbers of granulocytes (mpo+) and macrophages (lyz+) were both significantly further reduced. In addition, morphants also showed anterior defects, including reduced forebrain, smaller eyes and otoliths, which corresponded with a reduction of expression of the brain markers pax2.1, krox20 and dlx1. Gut expression of ifabp also appeared affected. Collectively, these experiments suggest a clear role for pegasus in hematopoiesis, as well as novel functions in brain and gut development.