The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish

Karen Stanic,Miguel L Concha,Pedro A Retamal,Juan C Opazo,Alejandro H Corvalán,Gareth I Owen,Ignacio A Wichmann,Ricardo J Figueroa,Julio D Amigo,German Reig

doi:10.1038/s41598-019-43436-8

Abstract

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Highlights

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood
By loss-of-function assays either using generated knockdown CRISPR mutants (G0) or morphant embryos, we demonstrate that rprml is required during both waves of definitive hematopoiesis for the specification of both erythroid-myeloid progenitors (EMP) and hematopoietic stem/progenitor cells (HSPCs)
A potential role of RPRML in human hematopoiesis has not been yet assessed, the recent association between the loss of RPRM and highly aggressive acute myeloid leukemia (AML)[15] raises the question of whether products from the RPRM gene family play a relevant role during hematopoiesis

Summary

We previously reported distinctive

RNA expression pattern profiles for RPRM gene family members[10,14]. We showed that rprml embryonic expression domains include vascular and mesodermal-derived tissues. The number of neutrophils was drastically reduced in the CHT of transgenic Tg(mpx:GFP) embryos between 32 and 48 hpf (Fig. 2I–J′), consistent with the decreased mpx expression visualized by WISH These results indicate that rprml is required for EMP formation during the transient wave of definitive hematopoiesis. At 28 hpf, the cmyb+ HSPC population was greatly reduced in rprmlkd when compared with control embryos (Fig. 1I,J). The significant difference in apoptosis between knockdown embryos and wild type/control siblings by 48 hpf, indicates that rprml is required for cell survival during the formation of the CHT. This finding partly explains the reduction in the HSPC niche by rprml loss-of-function. These mechanisms need to be further explored in the future

Discussion

Materials and Methods

Author Contributions

Findings

Additional Information