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Spatial transcriptomic profiling reveals local and domain-establishing regulatory signaling networks within the bone marrow

ABSTRACT: The application of single-cell RNA sequencing (scRNAseq) to the bone field has led to significant advancements in our understanding of skeletal stem cell (SSC) heterogeneity, yet disparity remains in the characterization of these cells in the context of their native environment. To resolve these limitations, we combined scRNAseq and spatial transcriptomics in adult femurs to provide endogenous, in vivo context. First, predictive modeling was used to determine the spatial location of SSCs within the bone marrow. These results localized CXCL12-abundant reticular cells to the bone marrow, while PDGFRα+SCA1+ were found to enrich within the outer periosteum. Correlative analyses were next used to define the cellular niche composition, identifying smooth muscle cells, as well as endothelial cell and macrophage subtypes overrepresented within the SSC niche. Using cell-cell communication prediction and spatial transcriptomics, we defined ligand-receptor pairs specifically expressed within the niche and mapped their expression back to niche-resident cells. Finally, these SSC niches were placed in the context of bone microdomains. Signaling gradients derived from the vasculature and bone surfaces were unbiasedly assessed using SpatialTime. This data indicated a striking, spatially-restricted activation of metabolic and major morphogenetic pathways. This project demonstrates the ability of this technique to spatially localize SSC subpopulations, define the cellular components of the stem cell niche, unravel cell-cell communication and place this niche in the context of its bone microdomain to gain a comprehensive understanding of local and global SSC regulatory networks within the bone.

ORGANISM(S): Mus musculus

PROVIDER: GSE228534 | GEO | 2023/11/10

REPOSITORIES: GEO

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Project description:Single cell transcriptomic analyses (scRNAseq) of hepatocytes and liver endothelial cells (L-EC) have revolutionized the understanding of the spatial architecture of liver structure and function. The spatial alignment of L-EC and hepatocytes is pivotal for liver function in health and disease given that L-EC act as instructive gatekeeper of nearby hepatocytes including the maintenance of liver metabolic zonation in a Wnt-dependent manner. Advancing liver biology beyond the ’transcript-centric’ view of scRNAseq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyse post-translational modifications. Here, by combining spatial cell sorting methodology with transcriptomic and quantitative proteomic/phospho-proteomic analyses, we established the first functionally and spatially-resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signalling mechanisms. Phosphorylation of receptor tyrosine kinases (RTK) was detected preferentially in the central vein area resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation of the identified strong phosphorylation gradient of the vascular RTK Tie1 by blockade resulted in the rapid peri-central dysregulation of the L-EC transcriptome. Notably, the expression of Wnt9b in L-EC was discovered as Tie receptor controlled with reciprocal regulation by FoxO1 and STAT3 transcription factors. Genetic inactivation of Tie1 in L-EC or antibody blockade resulted in reduced liver regeneration following partial hepatectomy with reduced Wnt ligand and Wnt target gene expression, including Axin2, Sox9, Tbx3 and Lgr5. Taken together, the study has yielded unparalleled insight into the spatial organization of L EC signalling and discovered a vascular Tie-Wnt signalling axis as regulator of liver function. The employed spatial sorting technique followed by phospho-proteomic analysis may be employed as a universally adaptable strategy for the spatial phosphoproteomic analysis of scRNAseq data-defined relevant cellular (sub)-populations.

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Spatial transcriptomic profiling reveals local and domain-establishing regulatory signaling networks within the bone marrow

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