2008 – ADULT HEMATOPOIETIC STEM CELL CLONALITY IS DETERMINED BY EMBRYONIC MACROPHAGE SENSING OF CALRETICULIN

Abstract

In development local signals induce hematopoietic stem cell (HSC) emergence, trafficking, and division. Using a brainbow color barcoding system, we previously found that zebrafish produce 20-30 HSCs in the embryonic aorta. Using spinning disk confocal microscopy, we imaged runx1+23:mCherry+ HSPCs in the fetal niche and found surprising interactions with mpeg1:GFP+ primitive macrophages. At any one time, 20-30% of HSPCs contacted macrophages and were either fully engulfed or had a piece of cytoplasm removed. Using the transgenic cell cycle marker FUCCI, we found 65% of HSPCs in G2M phase engaged with macrophages. To evaluate HSC clonality, we depleted macrophages in brainbow embryos and raised fish to adulthood. Embryonic macrophage depletion via the irf8 morpholino or clodronate liposomes significantly reduced the number of HSC clones in adulthood with an average of 14 clones vs 24.6 in controls (p = 0.0002). To better characterize the interaction between macrophages and HSPCs we performed few-cell proteomics. This identified 166 peptides enriched in macrophages which had recently engaged HSPCs, including three isoforms of calreticulin (calr). Normally ER bound, calr may become surface bound during stress, where it acts as a ‘come-eat-me' signal. Antibody staining confirms surface calr on HSPCs and scRNA-seq identifies expression of lrp1ab, the canonical calr receptor, specifically in recently-engaged macrophages. Morpholino knockdown of calr isoforms reduces macrophage-HSPC interactions by up to 2-fold (p = 0.0008), and HSPCs overexpressing a non-ER bound form of calr are 4-fold more likely to engage macrophages (p < 0.0001). Together, our data support a model in which the environmental stimuli that induce HSCs also result in variable surface display of calr, leading to either macrophage grooming or phagocytosis, which ultimately regulates HSC clonality.