Characterization of Niche Cells and Signals Regulating Stepwise Embryonic Hematopoiesis Using Slide-Seq and Merfish Spatial Transcriptomics

Stagewise HSC development initiates in the aorta-gonad-mesonephros (AGM) region during mouse embryonic day (E)9.5-E12.5 and progresses through stages including hemogenic endothelial cell (HEC) specification, the endothelial to hematopoietic transition (EHT), and maturation. The nascent HSCs (pre-HSCs) form in the AGM region and subsequently migrate to the fetal liver where they undergo further maturation and expansion. Recent advances in single cell technologies have significantly enhanced our understanding of intrinsic cellular programs involved in HSC development. However, our knowledge about niche cells and the signals they emanate in the AGM region, as well as their contribution to HSC development, remain limited.

Here we've conducted spatial transcriptomics analysis using Slide-seq on mouse trunk tissues, including both the AGM region and the fetal liver, at E10.5, E11.5 and E12.5. In addition, we performed scRNA-seq on the cells collected from the AGM and trunk at E10.5, E11.5 and E12.5, as well as the fetal liver at E12.5. To integrate the Slide-seq and scRNA-seq data, we developed an algorithm that leverages capabilities from both spatial and in-depth transcriptional sequencing. As a result, we were able to successfully reconstitute the structures of the trunk tissues and obtain gene expression profiles of individual cells at 10 µm resolution. From the analysis, we identified 7 cell clusters surrounding the aorta, with one cluster comprised of Nfgr ⁺ cells predominantly occupying the dorsal aorta. The ventral part of aorta displayed a complex and multilayered cellular architecture, featuring pre-HSCs, endothelial cells, mesonephric cells, and macrophages. Notably, we identified a unique mesenchymal stromal cell (MSC) population that co-express Cdh2 (encodes N-cadherin or N-cad) and Pdgfra; thus, we call these cells N-cad ⁺ MSCs. These N-cad ⁺ MSCs were particularly enriched at the ventral part of the aorta, forming a single cell layer positioned between an inner layer of aortic endothelial cells and an outer layer of mesonephric cells. Next, we utilized several bioinformatics tools (CellphoneDB, CellChat, NicheChat, and StringDB) in effort to identify potential signals that could impact embryonic hematopoiesis. The results revealed intricate interactions between N-cad ⁺ MSCs and pre-HSCs, for example, involving JAG1-NOTCH1 and SDF-1 (encoded by Cxcl12)-CXCR4 ligand-receptor pairs.

To validate the findings obtained by the Slide-seq data, we selected and designed customized RNA probes for 140 genes of interest, including cell markers and potential signals. We then conducted MERFISH (Multiplexed Error-Robust Fluorescence in situ Hybridization) on 18 E11.5 trunk sections. The MERFISH data enabled us to characterize the spatially resolved niche cells and their derived signals in the AGM region at the single-cell level, which largely corroborated the results obtained from the Slide-seq data. Specifically, the MERFISH data indicates that N-cad ⁺ MSCs are in direct contact with aortic endothelial cells and are the primary cellular source of Jag1 and Cxcl12 around the aorta.

Given the unique position of N-cad ⁺ MSCs and their intricate interactions with pre-HSCs, we formulated the hypothesis that N-cad ⁺ MSCs play a crucial role in embryonic hematopoiesis. To functionally validate our hypothesis, we are conducting a series of experiments using Ncad ^CreER; Cxcl12 ^flox/floxand Ncad ^CreER; Jag1 ^flox/flox mouse models. We anticipate that embryos lacking N-cad ⁺ MSC derived Jag1 or Cxcl12 will exhibit a substantial decline in intra-aortic hematopoietic clusters, phenotypic pre-HSCs, and functional HSCs.

In summary, our study employed sequencing-based (Slide-seq) and imaging-based (MERFISH) spatial transcriptomics technologies to successfully characterize dynamic, spatially resolved profiles of niche components and associated signaling in the AGM region at the single cell level. This work significantly enhances our understanding of niche signaling and its role in regulating embryonic hematopoiesis and may aid the development of novel methods for inducing the transition from pluripotent stem cells to HSCs.