Nfe2 Is Dispensable for Early, but Required for Adult Thrombocyte Formation and Function in Zebrafish
Authors: Rost Megan S , Shestopalov Ilya , Liu Yang , Vo Andy H , Barrett Francesca , Stachura David L , Zon Leonard I. , Shavit Jordan A. 
Published: October 2019

Abstract
The NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 completely lack circulating platelets, causing early lethality due to neonatal hemorrhage. Recent data in mice suggest some differences in embryonic and adult thrombopoiesis, and overexpression of NFE2 in murine bone marrow cells increases megakaryocyte maturation and platelet release, suggesting a role for NFE2 in both early and late megakaryocyte development. Zebrafish have emerged as an excellent model for studying hematopoiesis and thrombopoiesis due to their external development, optical transparency, high fecundity, and conservation of nearly the entire hemostatic system. Rather than platelets, zebrafish possess thrombocytes - nucleated cells believed to be the functional equivalent in mammals.
We designed TALENs to target exon 4 of zebrafish nfe2, producing two mutant strains containing either an 8 or 10 base pair deletion, both resulting in a frameshift and null allele. We tracked survival for over one year and found that unlike mammals, zebrafish survive into adulthood in the absence of Nfe2 function with no signs of overt bleeding or lethality. We bred the nfe2 mutation into a transgenic background in which thrombocytes and hematopoietic progenitor cells express green fluorescent protein (Tg(cd41:GFP)) and are characterized by GFPhigh and GFPlow expression, respectively. We performed flow cytometry analysis and found that the percentage of GFPhigh cells (circulating thrombocytes) in the peripheral blood was significantly decreased from 0.67% to 0.2% in homozygous mutants (p < 0.001). In contrast, the percentage of GFPlow cells in the kidney marrow, the site of hematopoiesis in adult zebrafish, was increased from 0.47% to 1.17% in nfe2-/- mutants (p < 0.001). Surprisingly, quantification of circulating thrombocytes in 6 day old nfe2 null zebrafish larvae showed no significant differences from wild type siblings. Finally, we performed colony forming assays on whole kidney marrow lysates to measure the ability of hematopoietic progenitors to differentiate into thrombocytes. Both mutant and wild type adults are capable of producing thrombocytic colonies in the presence of thrombopoietin and erythropoietin.
We and others have shown that thrombocytes participate in the formation of induced thrombi upon laser-mediated endothelial injury in zebrafish embryos and larvae. We tested the functionality of nfe2-/- thrombocytes and were surprised to find that wild type and nfe2 null zebrafish larvae form fibrin- and thrombocyte-rich clots in response to endothelial injury at day of life 3 (venous circulation) and 6 (arterial circulation), respectively. Measurement of both the time to occlusion as well as the total number of thrombocytes adhering to the site of injury revealed no significant differences between wild type and nfe2-/- larvae.
These data suggest that loss of Nfe2 results in a late block in thrombopoiesis with secondary expansion of thrombocytic precursors, both features that are consistent with mammals. Surprisingly, Nfe2 appears to be dispensable for early embryonic thrombocyte production and function. These results suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development. This includes the potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. The long term homozygous mutant survival will also facilitate more in depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production in vivo or ex vivo.

Disclosures
Zon: Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder.