2009 – BNIP3LB REGULATED MITOPHAGY MAINTAINS THE EMBRYONIC POOL OF HEMATOPOIETIC STEM CELLS BY PROTECTING THEM FROM ROS INDUCED APOPTOSIS

Abstract

In contrast to the established detrimental effect that elevated Reactive Oxygen Species (ROS) has on adult hematopoietic stem and progenitor cells (HSPCs), our previous work has shown that ROS promotes HSPC formation in the dorsal aorta. In order to elucidate the mechanism and developmental timing of this shift in the role of ROS in HSC biology we sought to examine the initiation and regulation of mitophagy in these cells, this being a key process by which adult HSCs regulate ROS by removing and recycling damaged mitochondria. Our recent data shows that oxidative stress begins to limit HSPC numbers immediately upon their colonization of the secondary hematopoietic niche, the caudal hematopoietic tissue (CHT, the zebrafish analog of the fetal liver), and live imaging with Tg(ubi:mitoGR), a fluorescent transgenic reporter for mitophagy, shows that this coincides with the onset of mitophagy in HSPCs. Mitophagy can be induced via multiple pathways but our scRNAseq and in situ-hybridization analyses suggest that it is induced in these cells via the bnip3lb receptor. We validated the role of bnip3lb via morpholino directed knockdown and found that mitophagy was reduced alongside HSPC marker expression in the CHT, whereas induction of mitophagy by small molecules or hsp70:ΔOTC mutants elevated detection of HSPC markers runx1 and cmyb by WISH, and CD41:GFP by flow cytometry. Mechanistically, we found that bnip3lb knockdown increases ROS levels, and that the reduction in HSPC numbers can be rescued by chemically reducing ROS. Finally, we demonstrate that the enhancement in ROS levels caused by reduced mitophagy increases apoptosis in the CHT region and alters HSPC fate. We therefore propose that bnip3lb directed developmentally programmed mitophagy is responsible for protecting embryonic HSCs from the harmful effects of oxidative stress while the HSC pool expands. In contrast to the established detrimental effect that elevated Reactive Oxygen Species (ROS) has on adult hematopoietic stem and progenitor cells (HSPCs), our previous work has shown that ROS promotes HSPC formation in the dorsal aorta. In order to elucidate the mechanism and developmental timing of this shift in the role of ROS in HSC biology we sought to examine the initiation and regulation of mitophagy in these cells, this being a key process by which adult HSCs regulate ROS by removing and recycling damaged mitochondria. Our recent data shows that oxidative stress begins to limit HSPC numbers immediately upon their colonization of the secondary hematopoietic niche, the caudal hematopoietic tissue (CHT, the zebrafish analog of the fetal liver), and live imaging with Tg(ubi:mitoGR), a fluorescent transgenic reporter for mitophagy, shows that this coincides with the onset of mitophagy in HSPCs. Mitophagy can be induced via multiple pathways but our scRNAseq and in situ-hybridization analyses suggest that it is induced in these cells via the bnip3lb receptor. We validated the role of bnip3lb via morpholino directed knockdown and found that mitophagy was reduced alongside HSPC marker expression in the CHT, whereas induction of mitophagy by small molecules or hsp70:ΔOTC mutants elevated detection of HSPC markers runx1 and cmyb by WISH, and CD41:GFP by flow cytometry. Mechanistically, we found that bnip3lb knockdown increases ROS levels, and that the reduction in HSPC numbers can be rescued by chemically reducing ROS. Finally, we demonstrate that the enhancement in ROS levels caused by reduced mitophagy increases apoptosis in the CHT region and alters HSPC fate. We therefore propose that bnip3lb directed developmentally programmed mitophagy is responsible for protecting embryonic HSCs from the harmful effects of oxidative stress while the HSC pool expands.