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In the intestine, lacteals are specialized lymphatic vessels that absorb dietary lipids and rely on surrounding villus smooth muscle to propel lymph, forming the muscular-lacteal complex (MLC). How distinct mesenchymal populations coordinate assembly of this functional lymphatic unit remains unknown. By integrating developmental single-cell profiling, genetic lineage tracing, conditional mouse genetics, and functional assays of lipid absorption, we identify Notch3 as a central organizer of MLC development that coordinates communication between distinct mesenchymal lineages. While Notch3 promotes smooth muscle differentiation within the PDGFR\u03b1\u207a lineage, PDGFR\u03b2\u207a lineage cells do not directly contribute to villus smooth muscle. Instead, they function as Notch3-dependent signaling hubs that instruct expansion and differentiation of neighboring PDGFR\u03b1\u207a smooth muscle progenitors via paracrine TGF\u03b2 signaling. Loss of Notch3 in PDGFR\u03b2\u207a cells disrupts MLC development, impairs intestinal lipid absorption, and causes postnatal growth failure and lethality. Restoration of TGF\u03b2 signaling rescues the structural, functional, and survival defects caused by Notch3 loss, identifying TGF\u03b2 as a critical downstream effector of the Notch3 pathway. Furthermore, selective inhibition of canonical Notch signaling in the PDGFR\u03b2 lineage fails to phenocopy Notch3 deletion, revealing a non-canonical mechanism of Notch3 function in intestinal mesenchymal development. Together, these findings establish PDGFR\u03b2\u207a cells as essential mesenchymal signaling organizers and define a new paradigm in which lineage-specific, non-canonical Notch3 signaling coordinates villus stromal communication to build a functional intestinal lymphatic niche.<\/jats:p>","DOI":"10.64898\/2026.08.13.742813","type":"posted-content","created":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T09:10:12Z","timestamp":1786785012000},"source":"Crossref","is-referenced-by-count":0,"title":["A NON-CANONICAL ROLE FOR NOTCH3 IN BUILDING THE INTESTINAL LYMPHATIC NICHE"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3894-6646","authenticated-orcid":false,"given":"Liqing","family":"Huang","sequence":"first","affiliation":[{"name":"Department of Biomedical and Translational Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0581-7509","authenticated-orcid":false,"given":"Bhargav","family":"Sanketi","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9844-7852","authenticated-orcid":false,"given":"Madhav","family":"Mantri","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Cornell University, Ithaca, NY 14850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-5779-1903","authenticated-orcid":false,"given":"Yanxi","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7139-2299","authenticated-orcid":false,"given":"Clare","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-4441-9423","authenticated-orcid":false,"given":"Tristan","family":"Tran","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6085-7311","authenticated-orcid":false,"given":"Iwijn","family":"De Vlaminck","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Cornell University, Ithaca, NY 14850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1251-7395","authenticated-orcid":false,"given":"Natasza A.","family":"Kurpios","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081713051228000_2026.08.13.742813v1.1","doi-asserted-by":"crossref","first-page":"5664","DOI":"10.1038\/s41598-017-05912-x","article-title":"Mimicking Paracrine TGF\u03b21 Signals during Myofibroblast Differentiation in 3D Collagen Networks","volume":"7","year":"2017","journal-title":"Scientific Reports"},{"key":"2026081713051228000_2026.08.13.742813v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2011.07.001"},{"key":"2026081713051228000_2026.08.13.742813v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.284.5415.770"},{"key":"2026081713051228000_2026.08.13.742813v1.4","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M115.655548"},{"key":"2026081713051228000_2026.08.13.742813v1.5","first-page":"15073","article-title":"\u2019Notch Signaling in Vascular Smooth Muscle Cells\u2019, Adv Pharmacol, 78: 351\u201382. 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