Zebrafish KLF4 Is Essential for Primitive Haematopoiesis.

Gene knockout studies of Krüppel-like factors (KLFs) in mice have demonstrated key roles in organogenesis. EKLF (Klf1) plays an essential role in definitive erythropoiesis and b-globin gene expression, but primitive (yolk sac) erythropoiesis and embryonic globin gene expression are essentially normal. Since expression of embryonic globin genes is dependent upon proximal CACC box elements, additional KLFs must function during the embryonic wave of erythropoiesis. A number of KLFs have been described in zebrafish. One of these, zKLF4, the homologue of neptune, a Xenopus Laevis KLF expressed in the ventral blood island, cement and hatching glands, is an early marker of lateral plate mesoderm (LPM). From 1 somite and prior to expression of scl and flk1, there is a continuous rim of expression of zKLF4 2–3 cells wide within the LPM extending from the postero-lateral boundary of the polster to the tail. Expression in the posterior lateral plate persists as it migrates medially in a zipper-like movement to generate the intermediate cell mass (ICM), the site of embryonic haematopoiesis. zKLF4 is also expressed in a patch of dorsal mesendodermal cells from 70% epiboly which are fated to become the polster. Morpholino knockdown of zKLF4 results in moderate anaemia which resolves as definitive erythropoiesis replaces primitive erythropoiesis at 48–72 hpf. zKLF4 morphants show a down-regulation in the expression of GATA-1 (>100 fold) and embryonic globin (>10 fold) by real time RT-PCR. Both are expressed during the primitive wave of haematopoiesis. In addition there is a dramatic up-regulation (>100 fold) in the level of cmyb expression in zKLF4 morphant compared to control embryos at 72 hpf, which is consistent with expansion of the secondary definitive wave of erythropoiesis, possibly in response to anemia. These results suggest mammalian KLF orthologs of zKLF4, either KLF4 or KLF2, may play roles in primitive erythropoiesis and embryonic globin gene expression in mice or man.