Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.

Institut Curie Laboratory of Genetics and Developmental Biology INSERM U934 CNRS UMR3215 PSL Université Paris Paris France

Masaryk University Faculty of Medicine Department of Histology and Embryology Brno Czech Republic

Sorbonne Université Collège Doctoral Paris France

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Affolter M, Zeller R, Caussinus E. Tissue remodelling through branching morphogenesis. Nat Rev Mol Cell Biol. 2009;10:831–842. doi: 10.1038/nrm2797 PubMed DOI

Goodwin K, Nelson CM. Branching morphogenesis. Development. 2020;147:dev184499. doi: 10.1242/dev.184499 PubMed DOI

Wang S, Sekiguchi R, Daley WP, Yamada KM. Patterned cell and matrix dynamics in branching morphogenesis. J Cell Biol. 2017;216:559–570. doi: 10.1083/jcb.201610048 PubMed DOI PMC

Paine IS, Lewis MT. The Terminal End Bud: the Little Engine that Could. J Mammary Gland Biol Neoplasia. 2017;22:93–108. doi: 10.1007/s10911-017-9372-0 PubMed DOI PMC

Koledova Z, Zhang X, Streuli C, Clarke RB, Klein OD, Werb Z, et al.. SPRY1 regulates mammary epithelial morphogenesis by modulating EGFR-dependent stromal paracrine signaling and ECM remodeling. Proc Natl Acad Sci U S A. 2016;113:E5731–5740. doi: 10.1073/pnas.1611532113 PubMed DOI PMC

Kouros-Mehr H, Werb Z. Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis. Dev Dyn. 2006;235:3404–3412. doi: 10.1002/dvdy.20978 PubMed DOI PMC

Sumbal J, Koledova Z. FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis. Development. 2019;146. doi: 10.1242/dev.185306 PubMed DOI

Wiseman BS, Werb Z. Stromal effects on mammary gland development and breast cancer. Science. 2002;296:1046–1049. doi: 10.1126/science.1067431 PubMed DOI PMC

Zhao C, Cai S, Shin K, Lim A, Kalisky T, Lu W-J, et al.. Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells. Science. 2017;356. doi: 10.1126/science.aal3485 PubMed DOI

Brownfield DG, Venugopalan G, Lo A, Mori H, Tanner K, Fletcher DA, et al.. Patterned collagen fibers orient branching mammary epithelium through distinct signaling modules. Curr Biol. 2013;23:703–709. doi: 10.1016/j.cub.2013.03.032 PubMed DOI PMC

Hammer AM, Sizemore GM, Shukla VC, Avendano A, Sizemore ST, Chang JJ, et al.. Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth. Neoplasia. 2017;19:496–508. doi: 10.1016/j.neo.2017.04.004 PubMed DOI PMC

Jones CE, Hammer AM, Cho Y, Sizemore GM, Cukierman E, Yee LD, et al.. Stromal PTEN Regulates Extracellular Matrix Organization in the Mammary Gland. Neoplasia. 2019;21:132–145. doi: 10.1016/j.neo.2018.10.010 PubMed DOI PMC

Nerger BA, Jaslove JM, Elashal HE, Mao S, Košmrlj A, Link AJ, et al.. Local accumulation of extracellular matrix regulates global morphogenetic patterning in the developing mammary gland. Curr Biol. 2021. [cited 2021 Mar 19]. doi: 10.1016/j.cub.2021.02.015 PubMed DOI PMC

Peuhu E, Kaukonen R, Lerche M, Saari M, Guzmán C, Rantakari P, et al.. SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland. EMBO J. 2017;36:165–182. doi: 10.15252/embj.201694387 PubMed DOI PMC

Sumbal J, Belisova D, Koledova Z. Fibroblasts: The grey eminence of mammary gland development. Semin Cell Dev Biol. 2020. doi: 10.1016/j.semcdb.2020.10.012 PubMed DOI

Shyer AE, Rodrigues AR, Schroeder GG, Kassianidou E, Kumar S, Harland RM. Emergent cellular self-organization and mechanosensation initiate follicle pattern in the avian skin. Science. 2017;357:811–815. doi: 10.1126/science.aai7868 PubMed DOI PMC

Shyer AE, Tallinen T, Nerurkar NL, Wei Z, Gil ES, Kaplan DL, et al.. Villification: how the gut gets its villi. Science. 2013;342:212–218. doi: 10.1126/science.1238842 PubMed DOI PMC

Goodwin K, Mao S, Guyomar T, Miller E, Radisky DC, Košmrlj A, et al.. Smooth muscle differentiation shapes domain branches during mouse lung development. Development. 2019;146. doi: 10.1242/dev.181172 PubMed DOI PMC

Kim HY, Pang M-F, Varner VD, Kojima L, Miller E, Radisky DC, et al.. Localized Smooth Muscle Differentiation Is Essential for Epithelial Bifurcation during Branching Morphogenesis of the Mammalian Lung. Dev Cell. 2015;34:719–726. doi: 10.1016/j.devcel.2015.08.012 PubMed DOI PMC

Palmer MA, Nerger BA, Goodwin K, Sudhakar A, Lemke SB, Ravindran PT, et al.. Stress ball morphogenesis: How the lizard builds its lung. Sci Adv. 2021. [cited 2022 Jan 15]. doi: 10.1126/sciadv.abk0161 PubMed DOI PMC

Ewald AJ, Brenot A, Duong M, Chan BS, Werb Z. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev Cell. 2008;14:570–581. doi: 10.1016/j.devcel.2008.03.003 PubMed DOI PMC

Huebner RJ, Lechler T, Ewald AJ. Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells. Development. 2014;141:1085–1094. doi: 10.1242/dev.103333 PubMed DOI PMC

Huebner RJ, Neumann NM, Ewald AJ. Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration. Development. 2016;143:983–993. doi: 10.1242/dev.127944 PubMed DOI PMC

Panciera T, Azzolin L, Cordenonsi M, Piccolo S. Mechanobiology of YAP and TAZ in physiology and disease. Nat Rev Mol Cell Biol. 2017;18:758–770. doi: 10.1038/nrm.2017.87 PubMed DOI PMC

Gouon-Evans V, Rothenberg ME, Pollard JW. Postnatal mammary gland development requires macrophages and eosinophils. Development. 2000;127:2269–2282. doi: 10.1242/dev.127.11.2269 PubMed DOI

Gouon-Evans V, Lin EY, Pollard JW. Requirement of macrophages and eosinophils and their cytokines/chemokines for mammary gland development. Breast Cancer Res. 2002;4:155–164. doi: 10.1186/bcr441 PubMed DOI PMC

Gyorki DE, Asselin-Labat M-L, van Rooijen N, Lindeman GJ, Visvader JE. Resident macrophages influence stem cell activity in the mammary gland. Breast Cancer Res. 2009;11:R62. doi: 10.1186/bcr2353 PubMed DOI PMC

Lilla JN, Werb Z. Mast cells contribute to the stromal microenvironment in mammary gland branching morphogenesis. Dev Biol. 2010;337:124–133. doi: 10.1016/j.ydbio.2009.10.021 PubMed DOI PMC

Parsa S, Ramasamy SK, De Langhe S, Gupte VV, Haigh JJ, Medina D, et al.. Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling. Dev Biol. 2008;317:121–131. doi: 10.1016/j.ydbio.2008.02.014 PubMed DOI

Sferruzzi-Perri AN, Robertson SA, Dent LA. Interleukin-5 transgene expression and eosinophilia are associated with retarded mammary gland development in mice. Biol Reprod. 2003;69:224–233. doi: 10.1095/biolreprod.102.010611 PubMed DOI

Nguyen-Ngoc K-V, Ewald AJ. Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix. J Microsc. 2013;251:212–223. doi: 10.1111/jmi.12017 PubMed DOI PMC

Dvorak P, Bednar D, Vanacek P, Balek L, Eiselleova L, Stepankova V, et al.. Computer-assisted engineering of hyperstable fibroblast growth factor 2. Biotechnol Bioeng. 2018;115:850–862. doi: 10.1002/bit.26531 PubMed DOI

Koledova Z, Sumbal J, Rabata A, de La Bourdonnaye G, Chaloupkova R, Hrdlickova B, et al.. Fibroblast Growth Factor 2 Protein Stability Provides Decreased Dependence on Heparin for Induction of FGFR Signaling and Alters ERK Signaling Dynamics. Front Cell Dev Biol. 2019;7:331. doi: 10.3389/fcell.2019.00331 PubMed DOI PMC

Sumbal J, Vranova T, Koledova Z. FGF signaling dynamics regulates epithelial patterning and morphogenesis. bioRxiv; 2020. p. 2020.11.17.386607. doi: 10.1101/2020.11.17.386607 DOI

Heisenberg C-P, Bellaïche Y. Forces in tissue morphogenesis and patterning. Cell. 2013;153:948–962. doi: 10.1016/j.cell.2013.05.008 PubMed DOI

Bonnans C, Chou J, Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol. 2014;15:786. doi: 10.1038/nrm3904 PubMed DOI PMC

Li R, Li X, Hagood J, Zhu M-S, Sun X. Myofibroblast contraction is essential for generating and regenerating the gas-exchange surface. J Clin Invest. 2020;130:2859–2871. doi: 10.1172/JCI132189 PubMed DOI PMC

Li CM-C, Shapiro H, Tsiobikas C, Selfors LM, Chen H, Rosenbluth J, et al.. Aging-Associated Alterations in Mammary Epithelia and Stroma Revealed by Single-Cell RNA Sequencing. Cell Rep. 2020;33:108566. doi: 10.1016/j.celrep.2020.108566 PubMed DOI PMC

Yoshitake R, Chang G, Saeki K, Ha D, Wu X, Wang J, et al.. Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium. Front Cell Dev Biol. 2022;10:850568. doi: 10.3389/fcell.2022.850568 PubMed DOI PMC

Li R, Bernau K, Sandbo N, Gu J, Preissl S, Sun X. Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response. Morrisey E, Dietz HC, editors. Elife. 2018;7:e36865. doi: 10.7554/eLife.36865 PubMed DOI PMC

Branchfield K, Li R, Lungova V, Verheyden JM, McCulley D, Sun X. A three-dimensional study of alveologenesis in mouse lung. Dev Biol. 2016;409:429–441. doi: 10.1016/j.ydbio.2015.11.017 PubMed DOI PMC

McCarthy N, Manieri E, Storm EE, Saadatpour A, Luoma AM, Kapoor VN, et al.. Distinct Mesenchymal Cell Populations Generate the Essential Intestinal BMP Signaling Gradient. Cell Stem Cell. 2020;26:391–402.e5. doi: 10.1016/j.stem.2020.01.008 PubMed DOI PMC

Powell DW, Pinchuk IV, Saada JI, Chen X, Mifflin RC. Mesenchymal cells of the intestinal lamina propria. Annu Rev Physiol. 2011;73:213–237. doi: 10.1146/annurev.physiol.70.113006.100646 PubMed DOI PMC

Xiang J, Guo J, Zhang S, Wu H, Chen Y-G, Wang J, et al.. A stromal lineage maintains crypt structure and villus homeostasis in the intestinal stem cell niche. BMC Biol. 2023;21:169. doi: 10.1186/s12915-023-01667-2 PubMed DOI PMC

Heitman N, Sennett R, Mok K-W, Saxena N, Srivastava D, Martino P, et al.. Dermal sheath contraction powers stem cell niche relocation during hair cycle regression. Science. 2020;367:161–166. doi: 10.1126/science.aax9131 PubMed DOI PMC

Ahlers JMD, Falckenhayn C, Holzscheck N, Solé-Boldo L, Schütz S, Wenck H, et al.. Single-Cell RNA Profiling of Human Skin Reveals Age-Related Loss of Dermal Sheath Cells and Their Contribution to a Juvenile Phenotype. Front Genet. 2022;12:797747. doi: 10.3389/fgene.2021.797747 PubMed DOI PMC

Shoshkes-Carmel M, Wang YJ, Wangensteen KJ, Tóth B, Kondo A, Massasa EE, et al.. Subepithelial telocytes are an important source of Wnts that supports intestinal crypts. Nature. 2018;557:242–246. doi: 10.1038/s41586-018-0084-4 PubMed DOI PMC

Hughes AJ, Miyazaki H, Coyle MC, Zhang J, Laurie MT, Chu D, et al.. Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme. Dev Cell. 2018;44:165–178.e6. doi: 10.1016/j.devcel.2017.12.004 PubMed DOI PMC

Silberstein GB, Daniel CW. Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct. Dev Biol. 1982;90:215–222. doi: 10.1016/0012-1606(82)90228-7 PubMed DOI

Feinberg TY, Zheng H, Liu R, Wicha MS, Yu SM, Weiss SJ. Divergent Matrix-Remodeling Strategies Distinguish Developmental from Neoplastic Mammary Epithelial Cell Invasion Programs. Dev Cell. 2018;47:145–160.e6. doi: 10.1016/j.devcel.2018.08.025 PubMed DOI PMC

Fata JE, Werb Z, Bissell MJ. Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes. Breast Cancer Res. 2004;6:1–11. doi: 10.1186/bcr634 PubMed DOI PMC

Hannezo E, Clgj S, Moad M, Drogo N, Heer R, Sampogna RV, et al.. A Unifying Theory of Branching Morphogenesis. Cell. 2017;171. doi: 10.1016/j.cell.2017.08.026 PubMed DOI PMC

Joshi PA, Waterhouse PD, Kasaian K, Fang H, Gulyaeva O, Sul HS, et al.. PDGFRα+ stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland. Nat Commun. 2019;10:1760. doi: 10.1038/s41467-019-09748-z PubMed DOI PMC

Macias H, Moran A, Samara Y, Moreno M, Compton JE, Harburg G, et al.. SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number. Dev Cell. 2011;20:827–840. doi: 10.1016/j.devcel.2011.05.012 PubMed DOI PMC

Labernadie A, Kato T, Brugués A, Serra-Picamal X, Derzsi S, Arwert E, et al.. A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nat Cell Biol. 2017;19:224–237. doi: 10.1038/ncb3478 PubMed DOI PMC

Omelchenko T, Fetisova E, Ivanova O, Bonder EM, Feder H, Vasiliev JM, et al.. Contact interactions between epitheliocytes and fibroblasts: formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization. Proc Natl Acad Sci U S A. 2001;98:8632–8637. doi: 10.1073/pnas.151247698 PubMed DOI PMC

Barbazan J, Pérez-González C, Gómez-González M, Dedenon M, Richon S, Latorre E, et al.. Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction. Nat Commun. 2023;14:6966. doi: 10.1038/s41467-023-42382-4 PubMed DOI PMC

Sternlicht MD, Sunnarborg SW, Kouros-Mehr H, Yu Y, Lee DC, Werb Z. Mammary ductal morphogenesis requires paracrine activation of stromal EGFR via ADAM17-dependent shedding of epithelial amphiregulin. Development. 2005;132:3923–3933. doi: 10.1242/dev.01966 PubMed DOI PMC

Neumann NM, Kim DM, Huebner RJ, Ewald AJ. Collective cell migration is spatiotemporally regulated during mammary epithelial bifurcation. J Cell Sci. 2023;136:jcs259275. doi: 10.1242/jcs.259275 PubMed DOI PMC

Wang S, Matsumoto K, Lish SR, Cartagena-Rivera AX, Yamada KM. Budding epithelial morphogenesis driven by cell-matrix versus cell-cell adhesion. Cell. 2021;184:3702–3716.e30. doi: 10.1016/j.cell.2021.05.015 PubMed DOI PMC

Scheele CLGJ, Hannezo E, Muraro MJ, Zomer A, Langedijk NSM, van Oudenaarden A, et al.. Identity and dynamics of mammary stem cells during branching morphogenesis. Nature. 2017;542:313–317. doi: 10.1038/nature21046 PubMed DOI PMC

Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593–605. doi: 10.1002/dvg.20335 PubMed DOI

Wendling O, Bornert J-M, Chambon P, Metzger D. Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse. Genesis. 2009;47:14–18. doi: 10.1002/dvg.20448 PubMed DOI

Riedl J, Flynn KC, Raducanu A, Gärtner F, Beck G, Bösl M, et al.. Lifeact mice for studying F-actin dynamics. Nat Methods. 2010;7:168–169. doi: 10.1038/nmeth0310-168 PubMed DOI

Conti MA, Even-Ram S, Liu C, Yamada KM, Adelstein RS. Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice. J Biol Chem. 2004;279:41263–41266. doi: 10.1074/jbc.C400352200 PubMed DOI

Koledova Z. 3D Coculture of Mammary Organoids with Fibrospheres: A Model for Studying Epithelial-Stromal Interactions During Mammary Branching Morphogenesis. Methods Mol Biol. 2017;1612:107–124. doi: 10.1007/978-1-4939-7021-6_8 PubMed DOI

Koledova Z, Lu P. A 3D Fibroblast-Epithelium Co-culture Model for Understanding Microenvironmental Role in Branching Morphogenesis of the Mammary Gland. Methods Mol Biol. 2017;1501:217–231. doi: 10.1007/978-1-4939-6475-8_10 PubMed DOI

Kasid A, Lippman ME, Papageorge AG, Lowy DR, Gelmann EP. Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity. Science. 1985;228:725–728. doi: 10.1126/science.4039465 PubMed DOI

Sumbal J, Koledova Z. Single Organoids Droplet-Based Staining Method for High-End 3D Imaging of Mammary Organoids. Methods Mol Biol. 2022;2471:259–269. doi: 10.1007/978-1-0716-2193-6_14 PubMed DOI

Susaki EA, Tainaka K, Perrin D, Kishino F, Tawara T, Watanabe TM, et al.. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 2014;157:726–739. doi: 10.1016/j.cell.2014.03.042 PubMed DOI

Lloyd-Lewis B, Davis FM, Harris OB, Hitchcock JR, Lourenco FC, Pasche M, et al.. Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods. Breast Cancer Res. 2016;18:127. doi: 10.1186/s13058-016-0754-9 PubMed DOI PMC