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The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions(1,2). It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow(3-5). We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

A dual genetic strategy enables the labelling and in vivo imaging of native long-term haematopoietic stem cells in the mouse calvarial bone marrow.

Single-cell RNA sequencing and spatial transcriptomics reveal that the somitogenesis clock is active in mouse gastruloids, which can be induced to generate somites with the correct rostral-caudal patterning.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization(1-3). To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.