Morpholino Knockdown of Jak2a Inhibits Primitive and Definitive Hematopoiesis in Zebrafish

In zebrafish, the primitive wave of hematopoiesis is specified at 3 somites and is marked by two lateral stripes, which express early hematopoietic genes. The coalescence of these stripes forms the intermediate cell mass (ICM). <internal deletion due to database size limit - Gene Tools> We and others have observed that Jak2 is duplicated in zebrafish with one of the duplicated genes, jak2a, expressed exclusively in hematopoietic tissues. We have knocked down jak2a with anti-sense morpholinos to assess its role in hematopoiesis. O-dianisidine staining of embryos 2dpf shows a reduction in the primitive wave of erythropoiesis in the jak2a morphants. Using the CD41-GFP transgenic fish line we observed markedly reduced and, in some cases, the complete absence of circulating thrombocytes in jak2a morphants. To help understand how jak2a knockdown impairs hematopoiesis we examined the expression of a panel of early hematopoietic genes in the ICM of 20 to 24 hpf fish by whole mount in situ hybridization (WISH). There was an overall reduction in the expression of lmo2, scl, gata-1 and gata-2. Upon closer observation it was apparent that the reduction was due to the loss of certain cells from the ICM with normal staining intensity in the remaining cells. We also observed reduced cyclin D1 expression and elevated caspase 3 activity, which strongly suggested programmed cell death was activated in the jak2a morphant. We believe that there is a selective loss due to cell cycle arrest of the more mature hematopoietic cells, which require the expression of jak2a for their survival with retention of early hematopoietic progenitors and hematopoietic stem cells in the ICM. These observations in developing zebrafish embryos establish the important role of jak2a in hematopoiesis and provide new insights into the stage of hematopoiesis where jak2a is critically important.