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We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for (1) extracellular matrix structure and metabolism (TM1 subtype), (2) secreted ligand signaling to support Schlemm\u2019s canal cells (TM2), and (3) contractile and mitochondrial\/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in\n                    <jats:italic>LMX1B<\/jats:italic>\n                    cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function, and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from\n                    <jats:italic>\n                      Lmx1b\n                      <jats:sup>V265D\/+<\/jats:sup>\n                    <\/jats:italic>\n                    mutant mice (2491 TM cells). In\n                    <jats:italic>\n                      Lmx1b\n                      <jats:sup>V265D\/+<\/jats:sup>\n                    <\/jats:italic>\n                    mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. Mitochondria in TM cells of\n                    <jats:italic>\n                      Lmx1b\n                      <jats:sup>V265D\/+<\/jats:sup>\n                    <\/jats:italic>\n                    mice are swollen with a reduced cristae area, further supporting a role for mitochondrial dysfunction in the initiation of IOP elevation in these mice. Importantly, treatment with vitamin B3 (nicotinamide), which enhances mitochondrial function and metabolic resilience in other contexts, significantly protected\n                    <jats:italic>Lmx1b<\/jats:italic>\n                    mutant mice from IOP elevation.\n                  <\/jats:p>","DOI":"10.7554\/elife.107161","type":"journal-article","created":{"date-parts":[[2025,7,17]],"date-time":"2025-07-17T07:20:37Z","timestamp":1752736837000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.107161","source":"Crossref","is-referenced-by-count":4,"title":["Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 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