3173 – AP2A2 KO MICE LINK FETAL LIVER HAEMATOPOIESIS EXHAUSTION TO LOSS OF HSC QUIESCENCE, PERTURBED ASYMMETRICAL FATE AND ALTERED LIPID METABOLISM

Abstract

We identified Ap2a2 as an enhancer of mouse long-term (LT-) HSC function via a bone marrow (BM) transplantation screen, and a HSC cell fate determinant during live cell HSC videomicroscopy. We hypothesised Ap2a2-transduced HSCs acquired in-vitro quiescence. Using the Tet-On H2B-GFP mouse line - We transduced vector versus AP2A2 into H2B-GFP HSCs for an in-vivo pulse-chase transplantation assay. This showed a 3-fold increase in the dormant GFP-high (CD150+48-LSK) LT-HSC subpopulation of Ap2a2-transduced-HSC recipients. We present two new mouse transgenic lines: an Ap2a2-LacZ reporter where X-Gal/FDG staining of BM cells confirmed higher endogenous Ap2a2 expression in LT- versus short-term (ST- CD150-48-LSKs) HSCs and a constitutive Ap2a2 knock-out (KO) line. Analyses of heterozygote Ap2a2 KO matings showed less than expected 25% Ap2a2-/- Mendelian inheritance at weaning consistent with fetal and peri-natal lethality. E14. 5 Ap2a2-/- embryos had smaller livers but twice as many LT-HSCs, confirmed by limit dilution assay. Ki67/DAPI cell cycle analyses showed loss of G0 in E14.5 LT- versus ST-HSCs. At E16.5-E18.5, there were two embryonic phenotypes: Ap2a2-/- survivors defined by restoration of FL LT-HSC G0 and differentiation as opposed to Ap2a2-/- non-survivors with continued loss of G0 and failed differentiation. Numb staining of Ap2a2-/- E14.5 LT-HSCs showed perturbed asymmetrical to symmetrical divisions. RNAseq analyses of Ap2a2-/- compared to Ap2a2+/+ E14.5 LT-HSCs showed differential upregulation of PPAR and lipid signaling pathways. Functionally, E14.5 Ap2a2-/- FL cells have maintained splenic colony formation but significantly impaired competitive transplant reconstitution. We conclude constitutive Ap2a2 deletion results in FL HSC exhaustion via loss of LT-HSC G0 quiescence and differentiation, which mechanistically involves the PPAR-lipid metabolism pathways.