Dominant-interfering C/EBPα stimulates primitive erythropoiesis in zebrafish

Abstract

Objective-We investigated the role of CCAAT enhancer-binding protein-α (C/EBPα) during zebrafish embryonic blood development.Methods-Whole-mount mRNA in situ hybridization was performed to determine the spatiotemporal expression pattern of zebrafish cebpa in developing hematopoietic progenitors. A deletion mutation of cebpa (zD420), which mimics the human dominant-negative mutations of C/EBPα, was transfected into CV1 cell line to evaluate its transcriptional activity in vitro and injected into zebrafish embryos at the one-to two-cell stage to examine its effects on primitive hematopoiesis during early zebrafish development.Results-Zebrafish cebpa is expressed in the anterior and posterior lateral plate mesoderm at 12 hours postfertilization, along with scl, pu.1 and gata1 in developing hematopoietic progenitors. In vitro, the deletion mutation of cebpa (zD420) prevents expression of the full-length protein, allowing the expression of truncated isoforms from internal translational initiation sites. As in the human, the truncated zebrafish C/ebpα proteins did not activate the expression of known target granulocytic genes, and in fact, suppressed transactivation that was induced in vitro by the full-length protein.Forced expression of the zD420 mRNA in zebrafish embryos led to an expansion of primitive erythropoiesis, without a discernible effect on granulopoiesis.Conclusion-Expression of the truncated isoforms of cebpa alters the developmental pattern of hematopoietic progenitor cells during embryogenesis.

Human C/EBP-ϵ activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

“…The ability of the C/EBP-⑀ 14 repressor isoform to drive progenitors to the erythroid lineage is consistent with results in zebrafish in which enforced ectopic expression of C/EBP-␣ dominant-negative isoforms, through deletion mutations of C/EBP-␣ (zD420), which mimic human dominant-negative mutations, induced primitive erythropoiesis with no discernible effect on granulopoiesis. 33 C/EBP-⑀ 14 may increase erythroid progenitor proliferation and differentiation by increasing the expansion of GATA-1-expressing committed erythroid progenitors, and because GATA-1 expression is autoregulated, may serve to expand this cell population.…”

Sumoylation of CCAAT/enhancer–binding protein α promotes the biased primitive hematopoiesis of zebrafish

“…This was not unexpected because several different C/ebp␣ MOs had been tested without success previously. 9 We also tried the most recently developed approach, custom-made zinc-finger nucleases made by OPEN (Oligomerized Pool ENgineering)-mediated gene knockout, 42 but failed to get any working zinc-finger nucleases (T.X.L., unpublished data). Such a technical obstacle may have been due to the fact that C/ebp␣ is a single-exon gene that is very rich in GC.…”

“…6,7 The zebrafish CCAAT/enhancer-binding protein ␣ (C/ebp␣), a master regulator of granulopoiesis, 8 is also expressed, along with pu.1 and gata1, in the hematopoietic progenitors of P-LPM during primitive hematopoiesis. 9 C/ebp␣ is the founding member of a family of transcription factors containing a basic leucine zipper domain. 10 Loss of C/ebp␣ in mice results in the complete absence of mature neutrophils and eosinophils and increased numbers of erythroid progenitors and erythroid cells in the fetal liver.…”

eena Promotes Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation in Zebrafish

“…It was therefore interesting to explore the possible function of een on hematopoietic cells. We first microinjected synthetic zebrafish een mRNA into wild-type embryos at the one-cell stage and examined the hematopoietic marker expression, including scl (hematopoietic progenitors), gata1 (primitive erythroid lineage), and pu.1 (primitive myeloid precursors) (29,30).…”