The role of inka1b in zebrafish primitive hematopoiesis

Abstract

Inka1b gene was shown up-regulated in the intermediate cell mass (ICM) of the zebrafish chordin morphant but its role in embryonic hematopoiesis is unknown. In this study, we characterized the spatio-temporal expression pattern and hematopoietic functions of for inka1b in zebrafish embryos. Spatio-temporal expression of zebrafish inka1b gene was examined by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). Inka1b was knocked-down by anti-sense morpholino (MO) (herein inka1bMO embryos) and the hematopoietic phenotype was analyzed by WISH, real-time RT-PCR, O-dianisidine, Sudan Black staining and quantitatively by flow cytometry in Tg(mpo:EGFP) embryos. INKA is present in vertebrates and relatively conserved from human to zebrafish. In zebrafish embryos, inka1b is expressed since the zygotic stage, indicating the presence of maternal transcript. At 18 and 24 hpf, inka1b can be detected in the ICM in wild-type and chordin morhants. Erythropoiesis was reduced in inka1bMO embryos, as shown by reduction of gata1 and alpha- and beta-embryonic hemoglobin expression as well as O-dianisidine staining at 48 hpf and the hematopoietic defects could be rescued by co-injection with wild-type inka1b mRNA. Differentiation was not affected and cytological examination of erythrocytes in inka1bMO embryos showed no difference from those in un-injected control. Inka1b knock-down also resulted in modest up-regulated expression of myelomonocytic markers pu.1 and l-plastin but reduced granulopoiesis as confirmed by RT-PCR, WISH and flow cytometric analysis in Tg(mpo:EGFP) embryos. Definitive hematopoiesis and angiogenesis were not affected. Zebrafish inka1b is involved in the maintenance of erythropoiesis and macrophage in primitive hematopoiesis. Its roles in murine embryonic stem cells (ESC) are being evaluated.