Determining the role of klf4 in zebrafish development

The Krüppel-like factor (KLF) family of genes encode transcriptional regulatory proteins that play key roles in differentiation. Gene knockout studies of KLFs have shown essential roles in organogenesis. Although much is known about the biochemistry of interaction between the three C-terminal C2H2 zinc fingers and CACC or GC rich DNA sequences, very little is known about the genetic pathways in which KLFs act. The founding member of the KLF family, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. To date the KLF responsible for primitive erythropoiesis remains elusive. A screen for KLF family members expressed in zebrafish kidney (the site of adult haematopoiesis), identified a number of family members, many of which are expressed in blood and vasculature suggesting possible roles in haematopoiesis and blood vessel function. One which is of particular interest, klf4/blood island krüppel like factor (biklf), is expressed in the intermediate cell mass of Oellacher (ICM), the site of zebrafish primitive haematopoiesis. Additionally it is expressed in the hatching gland suggesting a key role for klf4 in primitive haematopoiesis and hatching gland development. The role of klf4 in zebrafish development was analysed using morpholino-based antisense oligonucleotides and microarray analysis. The results presented in this thesis, identify an essential role for klf4 in hatching gland gene expression and morphogenetic movement of the hatching gland. Although a critical role for klf4 in primitive erythropoiesis has been described, klf4 does not play a role in myeloid or endothelial cell development. Finally, the studies presented provide strong evidence that klf4 preferentially binds CACCC box elements in the primitive zebrafish 􀀁-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions