Functional analysis of a hematopoietic-specific gene identified using high-throughput tissue-specific cDNA screening in zebrafish

Abstract

Zebrafish hematopoiesis begins in an intra-embryonic tissue called the intermediate cell mass (ICM), which specifically expresses the erythroid-specific transcription factor GATA-1. Using a cDNA library constructed from embryonic zebrafish GATA-1- positive cells, we identified a prothymosin alpha-like gene, ProTaL-1. RNA whole mount in situ hybridization showed that ProT~-1 was specifically expressed in the ICM. Microinjection of a construct encoding ProTa.L-1 fused to the green fluorescent protein revealed nuclear localization of ProTa.L-1. Oye~-expression of ProTaL-1 mRNA increased gata-1 expression, whereas knockdown ofProTaL-1 using an antisense, morpholino oligonucleotide resulted in a specific decrease of gata-1 expression in whole embryos. Fluorescence activated cell sorting analy~is of green fluorescent proteinlabeled GATA-I-positive cells from transgenic zebrafish indicated that ProTa.L-1 acted primarily to increase the total number· of ~mbryonic hematopoietic cells. s·ince prothymosin alpha is conserved among species, ProTa.L-1 may play a role in promoting , ' proliferation ofhematopoietic-ptogenitor cells in,higher vertebrates.