Isthmin 1 (ism1) is required for normal hematopoiesis in developing zebrafish

Abstract

Hematopoiesis is an essential and highly regulated biological process that begins with hematopoietic stem cells (HSCs). In healthy organisms, HSCs are responsible for generating a multitude of mature blood cells every day, yet the molecular pathways that instruct HSCs to self-renew and differentiate into post-mitotic blood cells are not fully known. To understand these molecular pathways, we investigated novel genes expressed in hematopoietic-supportive cell lines from the zebrafish (Danio rerio), a model system increasingly utilized to uncover molecular pathways important in the development of other vertebrate species. We performed RNA sequencing of the transcriptome of three stromal cell lines derived from different stages of embryonic and adult zebrafish and identified hundreds of highly expressed transcripts. For our studies, we focused on isthmin 1 (ism1) due to its shared synteny with its human gene ortholog and because it is a secreted protein. To characterize ism1, we performed loss-of-function experiments to identify if mature blood cell production was disrupted. Myeloid and erythroid lineages were visualized and scored with transgenic zebrafish expressing lineage-specific markers. ism1 knockdown led to reduced numbers of neutrophils, macrophages, and erythrocytes. Analysis of clonal methylcellulose assays from ism1 morphants also showed a reduction in total hematopoietic stem and progenitor cells (HSPCs). Overall, we demonstrate that ism1 is required for normal generation of HSPCs and their downstream progeny during zebrafish hematopoiesis. Further investigation into ism1 and its importance in hematopoiesis may elucidate evolutionarily conserved processes in blood formation that can be further investigated for potential clinical utility.

son is necessary for proper vertebrate blood development

“…These injections allowed us to then observe phenotypic changes caused by a reduction of son levels. After seeing no blood-specific phenotypic differences between embryos injected with the control MO and uninjected embryos (data not shown, [ 17 , 38 , 39 ], S3 and S4 Figs), we used uninjected embryos as the control for the remaining experiments. We first performed experiments in a transgenic zebrafish line that has the alpha globin locus control region (lcr) driving GFP expression; these lcr :GFP transgenic zebrafish only express GFP in red blood cells (RBCs) [ 29 ].…”

“…Briefly, five embryos were selected at random from uninjected animals, digested, and plated in supportive media. Five animals were also randomly selected from the son MO-injected group and plated at the same time to compare the colony growth seen in uninjected versus MO-injected samples [ 38 , 39 ]. The digested embryos were grown in methylcellulose media with carp serum, Gcsf, and Epo added to selectively stimulate myeloid and erythroid differentiation from HSPCs.…”

Hodgkin Lymphoma Monozygotic Triplets Reveal Divergences in DNA Methylation Signatures

“…Several genes related to HL, cancer or hematopoiesis are located in or near the differentially methylated regions in marginal zone-like B-cells. For example, ISM1 has been detected in lymphocytes, bone marrow, and embryonic blood islands and found to contribute to the hematopoiesis in zebrafish ( 10 ). KLHL22 is likely involved in tumorigenesis through the activation of the amino-acid-dependent mTOR pathway ( 11 ).…”

How HSCs Colonize and Expand in the Fetal Niche of the Vertebrate Embryo: An Evolutionary Perspective

“…The development of these zebrafish stromal cell lines is an important step and represents a valuable tool to study hematopoiesis in this model. Indeed, by comparing the transcriptome of CHEST, ZKS (zebrafish kidney stromal cells) and ZEST (zebrafish embryonic stromal trunk cells), Berrun and colleagues highlighted the hematopoietic role of isthmin 1 ( ism1 ), a secreted protein required for HSC development as well as erythro-myeloid differentiation ( Berrun et al, 2018 ).…”