Zebrafish klf4 plays a global role in primitive erythropoiesis

Mammalian Kruppel-like factors (KLFs) play essential roles during primitive and definitive erythropoiesis. In zebrafish myeloid and erythroid cell populations develop in spatially distinct regions of the developing embryo. Erythrocytes arise from the posterior LPM and express genes such as embryonic globins and gata1, whereas myeloid cells form in the anterior LPM and express genes such as spi1 and myeloperoxidase. Klf4 is the zebrafish homologue of neptune, a Xenopus KLF which acts early in the haematopoietic program [1,2]. Early expression of klf4 throughout the anterior and posterior LPM from the 1-somite stage of development suggested an early role in blood development. Knockdown of klf4 by two non-overlapping morpholinos (MOs) resulted in anaemia and pericardial oedema at 24 hpf. Expression profiling of klf4-MO versus control MO-injected embryos at 18 hpf using Compugen 16K oligo arrays suggested a role for klf4 in determining various cell fates which are derived from the LPM, particularly primitive erythropoiesis. Klf4 morphants showed a dramatic down-regulation of embryonic globin gene expression by both WISH and qRT–PCR (> 1000-fold) at 24 hpf, but no down-regulation of gata1 gene expression. There was a slight up-regulation (> 5-fold) in the level of definitive haematopoietic specific transcripts, runx1 and cmyb, at 24 hpf and adult globin (> 5-fold) at 36 hpf. This may reflect an expanded secondary/larval haematopoietic wave. These results suggest a restricted role for klf4 in primitive/ICM erythropoiesis in a manner analogous to the role of klf2 in mammalian primitive/yolk sac erythropoiesis. We reinvestigated organisation of the zebrafish globin locus and found a conserved extended CACC box element within the embryonic β-like globin gene promoters which bind Klf4 well in electromobility gel shift assays. On the other hand, Klf4 does not bind well to a divergent CACC box element in the adult β-like globin gene promoter