Recapitulating the Expression Pattern of Jak2 in Transgenic Zebrafish Using Jak2 Promoters and a GFP Reporter Gene.

The non-receptor tyrosine kinase Jak2 plays an important role in regulating erythro and thrombopoiesis. There has been intense interest in Jak2 since the observation that an activating mutation V617F is present in almost all patients with Polycythemia vera and many patients with Essential Thrombocytosis and Myelofibrosis. The analysis of Jak2 function in vivo has been limited as the murine jak2 knockout is lethal at day 10.5 of embryogenesis. Our laboratory has taken advantage of an ancestral partial duplication of the zebrafish genome, which has yielded two jak2 alleles --- jak2a and jak2b to study jak2 expression and function. Whole mount in situ hybridization studies confirm that the jak2a gene is only expressed in hematopoietic tissues, while jak2b is expressed in the developing lens and nephritic ducts. We have cloned and characterized the full-length jak2a and jak2b cDNAs and characterized the jak2a and 2b genomic loci. The jak2b gene has 24 exons spanning 79kb of genomic DNA. We amplified a 4kb zebrafish genomic fragment upstream of the first exon of the jak2b gene and linked it to the enhanced green fluorescent protein (EGFP) cDNA reporter and then microinjected the construct into single-cell zebrafish embryos. At 24 hours post fertilization (hpf), we observed fluorescence in the lens and nephritic ducts of developing embryos, with some expression in skin, muscle and notochord. The jak2a gene locus is complex as the jak2a gene is linked to a gene of unknown function, STARD4, in a head-to-tail manner with a small intergenic region of 1kb. As observed with jak2b, the first exon contains the jak2b 5′-UTR and the second exon contains the translation initiation site. We cloned a 1.9kb DNA fragment that included exons 1 and 2 the intervening first intron and an additional 800bp upstream of exon 1. This 1.9 kb promoter fragment was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that recapitulated the native expression pattern of jak2a. In injected embryos 24hpf, GFP⁺ cells were present in the anterior intermediate cell mass (ICM) and the lens. Fluorescent circulating blood cells, largely erythrocytes, were detected in 12 of 124 microinjected embryos 48 hours after fertilization. The jak2-EGFP transgenic zebrafish strains should be useful in the study of normal and pathologic hematopoiesis and in future studies of the pathogenesis of the MPDs.