Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow (BM) remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant tridimensional associations among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal BM. These arterioles are ensheathed exclusively by rare NG2+ pericytes, distinct from sinusoid-associated LepR+ cells. Pharmacological or genetic activation of HSC cell cycle alters the distribution of HSCs from NG2+ peri-arteriolar niches to LepR+ peri-sinusoidal niches. Conditional depletion of NG2+ cells induces HSC cycling and reduces functional long-term repopulating HSCs in BM. These results thus indicate that arteriolar niches are indispensable to maintain HSC quiescence.

Most species maintain abundant genetic variation and experience a range of environmental conditions, yet phenotypic variation is low. That is, development is robust to changes in genotype and environment. It has been claimed that this robustness, termed canalization, evolves because of long-term natural selection for optimal phenotypes. We show that the developmental process, here modeled as a network of interacting transcriptional regulators, constrains the genetic system to produce canalization, even without selection toward an optimum. The extent of canalization, measured as the insensitivity to mutation of a network's equilibrium state, depends on the complexity of the network, such that more highly connected networks evolve to be more canalized. We argue that canalization may be an inevitable consequence of complex developmental-genetic processes and thus requires no explanation in terms of evolution to suppress phenotypic variation.

Cell types are the basic building blocks of multicellular organisms and are extensively diversified in animals. Despite recent advances in characterizing cell types, classification schemes remain ambiguous. We propose an evolutionary definition of a cell type that allows cell types to be delineated and compared within and between species. Key to cell type identity are evolutionary changes in the 'core regulatory complex' (CoRC) of transcription factors, that make emergent sister cell types distinct, enable their independent evolution and regulate cell type-specific traits termed apomeres. We discuss the distinction between developmental and evolutionary lineages, and present a roadmap for future research.

In the bone marrow (BM), hematopoietic stem cells (HSCs) lodge in specialized microenvironments that tightly control their proliferative state to adapt to the varying needs for replenishment of blood cells while also preventing exhaustion1. All putative niche cells suggested thus far have a non-hematopoietic origin2-8. Thus, it remains unclear how feedback from mature cells is conveyed to HSCs to adjust proliferation. Here we show that megakaryocytes (Mk) can directly regulate HSC pool size. Three-dimensional whole-mount imaging revealed that endogenous HSCs are frequently located adjacent to Mk in a non-random fashion. Selective in vivo depletion of Mk resulted in specific loss of HSC quiescence and led to a marked expansion of functional HSCs. Gene expression analyses revealed that Mk were the source of chemokine C-X-C motif ligand 4 (Cxcl4, also named platelet factor 4, Pf4) in the BM and Cxcl4 injection reduced HSC numbers via increased quiescence. By contrast, Cxcl4−/− mice exhibited increased HSC numbers and proliferation. Combined use of whole-mount imaging and computational modelling was highly suggestive of a megakaryocytic niche capable of influencing independently HSC maintenance by regulating quiescence. Thus, these results indicate that a terminally differentiated HSC progeny contributes to niche activity by directly regulating HSC behavior.