Identifying factors that maintain epithelial stem/progenitor cells and drive their cell fate decisions within a developing organ is essential to understand development and has implications for organ regeneration. This project has two approaches, one investigating the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis, and the second identifying embryonic mouse salivary gland stem/progenitor cell populations within the gland. The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. We have made advances in understanding the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis. Surprisingly, parasympathetic nerve function also influences the stem/progenitor cells within the epithelium. We found that parasympathetic innervation of the SMG epithelium stimulates basal keratin 5-positive (K5+) progenitor cell proliferation, thus maintaining a pool of undifferentiated progenitor cells necessary for organ growth and development. This mechanism could be targeted during organ repair or regeneration. We demonstrated that acetylcholine signaling, via the muscarinic M1 receptor and EGFR, increased morphogenesis and the number of K5+ cells by increasing K5+ cell proliferation. Genetic lineage tracking experiments proved that K5+ cells are progenitor cells in the SMG. As K5+ cells differentiate, they co-express keratin 19 (K5+K19+) and, as differentiation proceeds, cells retain K19 but not K5 (K5-K19+). Therefore, the parasympathetic innervation maintained the epithelial keratin 5-positive progenitor cell population in an undifferentiated state, which was required for organogenesis. We have also made advances in understanding the function of KIT signaling during development. The cell surface receptor KIT has been used to isolate stem/progenitor cells from adult SMGs. These KIT+ progenitor/stem cells can be transplanted into irradiated adult SMGs to regenerate the damaged tissue and restore secretory function. The mechanisms of how KIT receptor function influences or controls KIT+ progenitor cell-mediated regeneration are not understood. We are investigating how KIT signaling influences progenitor cell function. Since FGF10/FGFR2b signaling is also required for SMG development, we hypothesize that both KIT and FGFR2b signaling are required for the survival of SMG progenitor cells during development and regeneration. During development, the SMG epithelium expresses KIT while stem cell factor (SCF), the ligand for KIT, is produced by both the mesenchyme and the epithelium. SCF alone does not support SMG growth, and in combination with FGF10 it does not increase morphogenesis or proliferation. In loss-of-function experiments, siRNA knockdown of KIT in the SMG significantly downregulated the expression of transcription factors (TFs) downstream of FGFR2b signaling. We propose that FGFR2b and KIT signaling regulate progenitor cell proliferation, which is important for development and regeneration after injury. We have begun to investigate how cell fate decisions are coordinated by transcription factors (TFs), which control expression of genes involved in self-renewal and differentiation. We characterized the expression of known stem/progenitor cell-related genes during embryonic SMG development. This analysis included TFs involved in stem cell self-renewal (Sox2, Nanog, Oct3/4, cMyc, and the Etv family), progenitor differentiation (Sox family), basal progenitor cell markers (cytokeratins), and proliferation markers for transit amplifying cells. Embryonic stem (ES) cells are maintained by four important TFs (Sox2, Nanog, Oct3/4, cMyc) whose expression is influenced by different growth factors. The TFs Nanog, Sox2, and Klf4 were detectable in E13 SMGs. Growth factors previously shown to influence SMG development were used to stimulate epithelium and the expression of stem/progenitor cell markers were evaluated. Nanog, Sox2, and Klf4 expression decreased after FGF10 treatment, suggesting FGF10 drives the cells along a more differentiated end bud fate. FGF10 upregulated the expression of cMyc, Sox9, and the TFs Etv4 and Etv5. Our data suggest that early E13 SMGs contain two distinct major epithelial cell compartments, the end bud and duct, in which different TFs and stem/progenitor cell markers influence the maintenance, differentiation and/or proliferation of these cell populations. Understanding the cell lineage of progenitor cells within the salivary glands will be important from the clinical perspective where progenitor cells of specific lineages may be more appropriate than pluripotent stem cells for clinical transplantation to regenerate irradiation-damaged salivary glands.

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National Institute of Dental & Craniofacial Research
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Patel, Vaishali N; Pineda, Dallas L; Hoffman, Matthew P (2017) The function of heparan sulfate during branching morphogenesis. Matrix Biol 57-58:311-323
Knosp, Wendy M; Knox, Sarah M; Lombaert, Isabelle M A et al. (2015) Submandibular parasympathetic gangliogenesis requires sprouty-dependent Wnt signals from epithelial progenitors. Dev Cell 32:667-77
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