Differential Regulation of Primitive Myelopoiesis in the Zebrafish by Spi-1/Pu.1 and C/ebp1

Abstract

The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.

c-Myb acts in parallel and cooperatively with Cebp1 to regulate neutrophil maturation in zebrafish

“…51,54,55 These findings prompted us to speculate that, in zebrafish, Cebp1 and c-Myb may form a genetic network to regulate neutrophil development.…”

Common genetic control of haemangioblast and cardiac development in zebrafish

“…pu1 expression is lost in gata5 and gata6 morphants but, although this could explain the absence of myelopoiesis, a role for pu1 in haemangioblast formation or endothelial development has not been established (Rhodes et al, 2005;Su et al, 2007). However, analysis of pu1 morphants showed that pu1 is required only for myeloid development and not at all for the endothelial programme (see Fig.…”

Ncor1 and Ncor2 play essential but distinct roles in zebrafish primitive myelopoiesis

“…However we could not preclude the possibility that Ncor2 may function to activate the transcription of spi1b required for primitive myelopoiesis directly by remodeling their chromatin, because such mechanisms have been reported in a few cases for the other co-repressors' function (O'Malley and McKenna, 2008). mfap4 1 macrophages and mpx 1 neutrophils are the mature myeloid cells developing from spi1b 1 myeloid cells (Su et al, 2007;Xu et al, 2012). The regulation mechanism of fate determination of macrophages and neutrophils has been extensively studied.…”

“…mRNAs containing full-length of coding sequences of tal1, lmo2, and spi1b were in vitro transcribed as we reported previously (Liang et al, 2012). The full-length coding sequence of spi1b (Su et al, 2007) was cloned by RT-PCR using primers of ATGCTGCATCCGTACAG (forward) and TTACATGTAATGCTTTC (reverse).…”