A novel role of Inka1 in regulating zebrafish hematopoiesis

Abstract

Background: Inka1 (Induced in Neural Crest by AP2 1) is first discovered among up-regulated genes in Xenopus embryos stimulated by the transcriptional activator protein Tfap2a through microarray analysis, which shares no significant similarity to any known protein. While it is suggested that inka1 is related to cytoskeleton organization, the precise cellular function of Inka1 remain unclear. Our previous study demonstrated that inka1b is up-regulated in the intermediate cell mass (ICM) of the zebrafish chordin morphants, which is characterized by expanded hematopoiesis. However, its role in vertebrate hematopoiesis remains unknown. Methods: Spatio-temporal expression pattern of zebrafish inka1b was examined by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). Inka1b was knockdown by anti-sense morpholino (MO) and the hematopoietic phenotype was analyzed by WISH, real-time RT-PCR, O-dianisidine. Results: Inka1 is conserved in vertebrates and duplicated into inka1a and inka1b in Zebrafish. During embryonic development, inka1b, but not inka1a expressed along the hematopoietic compartment, ICM at 18 and 24 hpf, suggested a potential role of inka1b in embryonic hematopoiesis. In inka1b morphant (inka1bMO) embryos, erythropoiesis was significantly reduced, as shown by the reduced gata1 and embryonic hemoglobin expression as well as O-dianisidine staining. Cytological examination of erythrocytes in inka1bMO embryos showed no difference from control embryos. In contrary, Inka1b knockdown resulted in up-regulation of myeloid progenitor marker spi1 and pan-leukocyte marker l-plastin. Definitive hematopoietic stem cell (HSC), as shown by the expression of c-myb, was not affected. Upon co-injection of inka1b mRNA, hematopoietic defects in inka1bMO embryos could be rescued. Conclusion: Inka1b knock-down by MO significantly suppressed erythropoiesis but promoted myelopoiesis in Zebrafish embryos. These results supported the proposition that inka1b is important in embryonic hematopoiesis and may be involved in the regulation of erythroid/myeloid lineage commitment.