A Durable Anatomy with Local Plasticity Enables Normal and Stress Hematopoiesis
Authors: Wu Qingqing , Zhang Jizhou , Johnson Courtney , Weinhaus Benjamin , Slaughter Anastasiya , Valladares-Nuñez Andre , Sherman Andre , Filippi Marie-Dominique , Vellai Badradoss Surya Prasath , Way Sing Sing , Koffron J. Mathew , Lucas-Alcaraz Daniel 
Published: November 2021

Abstract
Knowledge of the anatomy of each tissue and the relationships between progenitors and daughter cells is necessary to understand physiology and pathology. The anatomy of hematopoiesis in the marrow remains largely unknown. Here we identify strategies to image all steps of blood cell production in the mouse sternum using confocal microscopy. We show that long-distance migration of multipotent progenitors, lineage-committed progenitor recruitment to vessels, and generation of lineage-specific oligoclonal structures that are the main production sites for immature cells are key features of the anatomy of blood production. This structural organization is extremely durable and resilient to insults as it was maintained after hemorrhage, Listeria monocytogenes infection, and aging (80-weeks). Importantly, this anatomy is also enabled with local plasticity as production sites for each blood lineage selectively expand/contract in respond to insults followed by a return to homeostasis.
We used immunophenotyping to identify fifty-six surface markers that can be combined to image any populations of interest. For example, we found that ESAM is selectively expressed in 100% of LT-HSC, 90% of ST-HSC, 70% of MPP2 and MPP3, 30% of MPP4, 10% of CMP, and 90% of MkP and megakaryocytes but absent in more mature cells. Transplantation experiments revealed that all functional LT- and ST-HSC, MPP2, MPP3 and CMP were contained -exclusively- in the ESAM positive fraction (p&lt;0.05 when compared with ESAM- cells n= 7 mice per group). ESAM + MPP4 displayed 5-fold more engraftment than ESAM - MPP4 (p&lt;0.05). Combining ESAM with classical HSPC markers allowed imaging of all LT-HSC, ST-HSC, MkP, Pre-MegE, MPP2 and a mixed population of MPP3, CMP and MPP4. We developed similar strategies to map erythropoiesis (Pre-MegE → Pre-CFU-E → CFU-E → early erythroblast → late erythroblast → reticulocyte → erythrocyte) and lymphopoiesis (CLP→ PreProB → ProB →PreB→ Immature B). All strategies allowed clonal fate-mapping using Ubc-cre ERT2:confetti mice. In this model tamoxifen treatment leads to irreversible expression of one out of four fluorescent proteins. We found that multipotent and oligopotent progenitors are found as single cells, evenly distributed through the marrow (e.g. mean LT-HSC distance to closest ST-HSC, MPP2, MPP3, MkP, Pre-MegE &gt;100 μm, no different from random simulations, n=41 LT-HSC from 5 sternum sections of 4 wild-type mice) and are clonally unrelated between them. Multipotent and oligopotent progenitors reside near sinusoids (mean distance =9.7 μm) but this association is not different from that observed for random cells. In contrast, as progenitors become lineage-restricted, they localize to arterioles (for lymphoid progenitors) or sinusoids (all other progenitors) where they enter oligoclonal structures that are the main production sites for immature cells in each lineage. Each production site has distinct architectures: lymphoid sites are characterized by tight clusters of PreProB cells surrounding CLP; erythroid sites are characterized by strings of 4-21 CFU-E decorating the surface of sinusoids with early erythroblasts differentiating orthogonally to the vessel surface; in megakaryocyte sites one or two megakaryocyte progenitors produce megakaryocytes that decorate blood vessels over large (&gt;200μm 3) marrow regions. We previously showed that production sites for neutrophils contain 1 or 2 granulocyte progenitors tightly clustered with preneutrophils and that sites for monocytes/dendritic cells contain loose clusters of dendritic cells surrounding monocyte dendritic cell progenitors (Zhang Nature 2021).
This spatial architecture is durable and resilient and is maintained after acute challenge via phlebotomy, L. monocytogenes infection, or physiological aging (80-week-old mice). However, we also observed plasticity of production sites. Two days after phlebotomy we found increases in erythroid site numbers (368 vs 945 per mm 3, p&lt;0.05). These expansions were reversed by day 8 after phlebotomy. Similarly, infection led to increases in the size of neutrophil and dendritic cell production sites (~2-fold by day 6 post-infection) but these changes are reverted by day 20 post-infection.
In summary, we have developed strategies that allow imaging of differentiation in situ and defined a complex - but durable and plastic- anatomy for the hematopoietic tissue.

Disclosures
No relevant conflicts of interest to declare.