PDFGRα+ Stromal Cells Promote Salivary Gland Proacinar Differentiation Through FGF2-dependent BMP7 Signaling

Stromal cells can direct epithelial differentiation during organ development; however, these pathways remain poorly defined. FGF signaling is essential for submandibular salivary gland development, and FGF2 can regulate proacinar cell differentiation in organoids through autocrine signaling in stromal cells. We performed scRNA Seq and identified stromal cell subsets expressing Fgf2 and Fgf10 that also express Pdgfrα. When combined with epithelial cells in organoids, MACS-sorted PDGFRα+ cells sufficiently promoted proacinar differentiation. Gene expression analysis revealed FGF2 activates the gene Bmp7 in the stroma. BMP7 could replace stromalsignaling and stimulate epithelial acinar differentiation but not branching. However, in the absence of FGF2, pathway analysis revealed that the stromal cells differentiated into myofibroblasts. Myofibroblast differentiation was induced when we treated organoids with TGFβ1, which also prevented proacinar differentiation. Conversely, FGF2 reversed TGFβ’s effects. Dissecting pathways driving acinar differentiation will facilitate development of regenerative therapies. 2.0 Summary Statement Embryonic salivary glands contain multiple stromal cell populations. FGF2 maintains the stromal Pdgfrα+ population in-vitro. The PDGFRα+ stromal cells drive early epithelial secretory cell differentiation using BMP7.

[1]  Derek C. Liberti,et al.  Genomic, epigenomic, and biophysical cues controlling the emergence of the lung alveolus , 2021, Science.

[2]  Michael C. Kelly,et al.  Generation of a Single-Cell RNAseq Atlas of Murine Salivary Gland Development , 2020, iScience.

[3]  Victor G. Puelles,et al.  Decoding myofibroblast origins in human kidney fibrosis , 2020, Nature.

[4]  Guocheng Yuan,et al.  Distinct Mesenchymal Cell Populations Generate the Essential Intestinal BMP Signaling Gradient. , 2020, Cell stem cell.

[5]  R. Sekiguchi,et al.  Single-Cell RNA-seq Identifies Cell Diversity in Embryonic Salivary Glands , 2019, Journal of dental research.

[6]  C. Ponting,et al.  Resolving the fibrotic niche of human liver cirrhosis using single-cell transcriptomics , 2019, bioRxiv.

[7]  D. Nelson,et al.  ROCK inhibitor increases proacinar cells in adult salivary gland organoids , 2019, bioRxiv.

[8]  Hyung-Seok Kim,et al.  BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis , 2019, bioRxiv.

[9]  H. Roderick,et al.  Myofibroblast Phenotype and Reversibility of Fibrosis in Patients With End-Stage Heart Failure. , 2019, Journal of the American College of Cardiology.

[10]  Alireza Hadj Khodabakhshi,et al.  Metascape provides a biologist-oriented resource for the analysis of systems-level datasets , 2019, Nature Communications.

[11]  S. Ichinose,et al.  Stem cell competition orchestrates skin homeostasis and ageing , 2019, Nature.

[12]  Lucy X. Fan,et al.  A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition. , 2019, American journal of physiology. Renal physiology.

[13]  Seidai Sato,et al.  Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice , 2018, PloS one.

[14]  S. Preissl,et al.  Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response , 2018, eLife.

[15]  G. Carpenter,et al.  Salivary secretion in health and disease , 2018, Journal of oral rehabilitation.

[16]  E. Morrisey,et al.  Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung , 2017, Cell.

[17]  Hannah A. Pliner,et al.  Reversed graph embedding resolves complex single-cell trajectories , 2017, Nature Methods.

[18]  M. Gaete,et al.  Fgf10 and Sox9 are essential for the establishment of distal progenitor cells during mouse salivary gland development , 2017, Development.

[19]  M. Clarke,et al.  Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells , 2017, Science.

[20]  Brittany E. Schutrum,et al.  Encapsulation of primary salivary gland cells in enzymatically degradable poly(ethylene glycol) hydrogels promotes acinar cell characteristics. , 2017, Acta biomaterialia.

[21]  D. Nelson,et al.  Endothelial cell regulation of salivary gland epithelial patterning , 2017, Development.

[22]  E. Fuchs,et al.  Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweat versus hair fate decision , 2016, Science.

[23]  Luzhe Sun,et al.  TGF-β1 promotes acinar to ductal metaplasia of human pancreatic acinar cells , 2016, Scientific Reports.

[24]  J. Camden,et al.  Increased Expression of TGF-β Signaling Components in a Mouse Model of Fibrosis Induced by Submandibular Gland Duct Ligation , 2015, PloS one.

[25]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[26]  B. Ebert,et al.  Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis. , 2015, Cell stem cell.

[27]  J. Plemons,et al.  Managing xerostomia and salivary gland hypofunction: executive summary of a report from the American Dental Association Council on Scientific Affairs. , 2014, Journal of the American Dental Association.

[28]  V. P. Eswarakumar,et al.  Combined KIT and FGFR2b Signaling Regulates Epithelial Progenitor Expansion during Organogenesis , 2013, Stem cell reports.

[29]  Qing Li,et al.  Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue , 2013, Proceedings of the National Academy of Sciences.

[30]  J. Jones,et al.  Xerostomia and salivary hypofunction in vulnerable elders: prevalence and etiology. , 2012, Oral surgery, oral medicine, oral pathology and oral radiology.

[31]  James Castracane,et al.  The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds. , 2012, Biomaterials.

[32]  Richard A. Lang,et al.  Differential Interactions of FGFs with Heparan Sulfate Control Gradient Formation and Branching Morphogenesis , 2009, Science Signaling.

[33]  Michael Elkin,et al.  Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis , 2007, Development.

[34]  R. Jonsson,et al.  Salivary dysfunction associated with systemic diseases: systematic review and clinical management recommendations. , 2007, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[35]  Melinda Larsen,et al.  FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis , 2005, Development.

[36]  A. Hampl,et al.  FGF2 inhibits proliferation and alters the cartilage-like phenotype of RCS cells. , 2004, Experimental cell research.

[37]  M. Rastaldi,et al.  Epithelial-mesenchymal transition of tubular epithelial cells in human renal biopsies. , 2002, Kidney international.

[38]  S. Kato,et al.  FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. , 2000, Biochemical and biophysical research communications.

[39]  G. Martin,et al.  The roles of FGFs in the early development of vertebrate limbs. , 1998, Genes & development.

[40]  M. Kusakabe,et al.  A pituitary-salivary mixed gland induced by tissue recombination of embryonic pituitary epithelium and embryonic submandibular gland mesenchyme in mice. , 1985, Developmental biology.

[41]  T. Sakakura,et al.  Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland. , 1976, Science.

[42]  A. Friedenstein,et al.  THE DEVELOPMENT OF FIBROBLAST COLONIES IN MONOLAYER CULTURES OF GUINEA‐PIG BONE MARROW AND SPLEEN CELLS , 1970, Cell and tissue kinetics.

[43]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[44]  森川 暁.  Development of mesenchymal stem cells partially originate from the neural crest , 2009 .

[45]  M. Melnick,et al.  Submandibular gland morphogenesis: stage-specific expression of TGF-alpha/EGF, IGF, TGF-beta, TNF, and IL-6 signal transduction in normal embryonic mice and the phenotypic effects of TGF-beta2, TGF-beta3, and EGF-r null mutations. , 1999, The Anatomical record.