Abstract
Lgr5+ stem cells play crucial roles in maintaining intestinal epithelial cell homeostasis. However, the cellular heterogeneity and underlying regulatory programs of Lgr5+ small intestinal stem cells (ISCs) remain elusive. In this study, we profiled gene expression and chromatin accessibility of Lgr5+ ISCs at single-cell resolution to gain a deeper understanding of the lineage specification and early fate determining mechanisms. Our analysis identified a total of 6 subsets of Lgr5+ cell populations, which exhibited heterogeneity in gene expression and chromatin structure. We found that early fate-determining processes diverged the absorptive and secretory lineages within Lgr5+ cells. We further constructed gene regulatory networks controlling lineage determination and identified Foxa3 as a key transcription factor that regulates the differentiation of the intestinal secretory precursor. In vitro knockdown of Foxa3 disrupted the differentiation of Paneth cells by modulating Peroxisome-Proliferator-Activated Receptors (PPARs). Further Foxa3-targeted CUT&Tag sequencing analysis also verified that Foxa3 predominantly drives Paneth cell differentiation in the small intestine by regulating the expression of core genes in the PPAR signaling pathway. These results provide a comprehensive reference map for advancing our understanding of intestinal epithelial development and related diseases.
Data availability
All sequencing data have been uploaded to the China National Center for Bioinformation (CNCB) Genome Sequence Archive (GSA) database under the accession number: PRJCA030631. Previously published snRNA-seq datasets originating from Hickey et al. (https://doi.org/10.1038/s41586-023-05915-x) and reanalyzed in this study are accessible via https://doi.org/10.5061/dryad.8pk0p2ns8. The numerical source data underlying all main Figures in the manuscript can be found in the Supplementary Data 5–9. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
Code availability
All analyses in this paper were performed using R (v 4.2.2) and Python (v 3.7.16). The standard procedures provided by these software packages were followed in conducting the analyses. The scripts for data analysis and visualization are available on GitHub (https://github.com/Xinran-Deng/Single-Nucleus-Multi-Omics-Analysis-of-Mouse-Small-Intestinal-Lgr5-Cells).
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Acknowledgements
This work was supported by “National Key Research and Development Program of China” (2022YFA0806200, 2021YFC2501800, and 2022YFC2009802) and “Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project” (2025ZD01902600). We are also grateful to BerryGenomics (Beijing, China) for assistance with Single Nucleus Multi-Omics sequencing.
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Xinran Deng contributed to methodology, investigation, formal analysis, and writing; Shenfei Sun contributed to methodology, investigation, validation, and writing; Chenqi Lu contributed to conceptualization, methodology, and writing; Hanxiao Du, Xiaoyu You, Yumou Gong, Runrong Li and Xingyu Yu contributed to formal analysis; fujing huang contributed validation and writing; Kai Gong and Jingxin Guo contributed to validation; Bing Zhao contributed to conceptualization; Xinhua Lin contributed to conceptualization, and funding acquisition; Ning Jiang contributed to conceptualization, methodology, writing, and funding acquisition.
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Deng, X., Sun, S., Lu, C. et al. Single-nucleus multi-omics analysis of mouse small-intestinal Lgr5+ cell populations reveals Foxa3-induced Paneth cell-lineage differentiation. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09736-2
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DOI: https://doi.org/10.1038/s42003-026-09736-2
