Critical role of connexin43 in zebrafish late primitive and definitive hematopoiesis.

In vitro studies have suggested that connexin43 (cx43) expression is of particular importance during establishment and regeneration of the mammalian hematopoietic system. However, little is known about its in vivo functions during hematopoiesis due to the embryonic lethality of mammalian knockout models. In this study, we observed that zebrafish cx43 is not only expressed in the eyes, cerebellum, heart, and vasculature, but also expressed, albeit at low levels, in intermediate cell mass (ICM, the primitive hematopoietic site). Knockdown of cx43 leads to vacuolization in the wedge of the ICM and an apparent reduction in the number of circulating blood cells, but does not affect their cellular morphology. Whole-mount in situ hybridization analysis revealed that the hemangioblastic marker flk-1 and the primitive hematopoietic markers lmo2 and scl are basically maintained at normal levels in cx43 morphant embryos at 12-13 h postfertilization (hpf) compared with the con-MO injected embryos. However, subsequent expression of the definitive hematopoietic stem cell (HSC) marker c-myb was severely downregulated in the ventral wall of the dorsal aorta of cx43-depleted embryos at 36 hpf. Furthermore, we confirmed this phenotype by injection of cx43-MO into Tg(gata1:EGFP) embryos. Together, our results show that cx43 contributes to late primitive and definitive hematopoiesis in zebrafish embryos.