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 experimental 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. Over the past year, we have made advances in understanding the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis. Surprisingly, the parasympathetic nerve function also influences the stem/progenitor cells within the epithelium. Both parasympathetic and sympathetic branches of the autonomic nervous system innervate the adult SMG, but it is the parasympathetic axons that extend from the parasympathetic submandibular ganglion (PSG) and innervate the embryonic SMG. We have determined that removal of the PSG in ex vivo SMG organ culture by tissue recombination techniques, as well as chemical and genetic approaches to decrease nerve function, unexpectedly alter the epithelial keratin profile within the progenitor cell population and decrease epithelial morphogenesis. Chemical inhibition of acetylcholine (ACh) release, chemical antagonism of muscarinic receptor activity, and gene knockdown of muscarinic receptors in the epithelium all have a similar effect on growth as removing the PSG. In gain-of-function studies, ACh analogues increase epithelial branching in an epidermal growth factor (EGFR)-dependent manner. These data suggest that neuronal-epithelial communication via ACh signaling provides a mitogenic and morphogenic signal to the SMG epithelium. We demonstrate that the ganglion maintains the epithelial progenitor cells via muscarinic receptor and EGFR signaling, which also directs ductal lineage commitment. Thus, parasympathetic innervation during embryogenesis controls epithelial progenitor maintenance and directs ductal cell fate during branching morphogenesis. Our ongoing studies will identify how the neuronal-derived ACh and epithelial muscarinic receptor signals crosstalk with the mesenchymal FGF10 and FGFR2b-mediated proliferative signals in the epithelium. Given that the PSG axons migrate in a unidirectional manner along ducts towards the end buds we hypothesized that a molecule secreted by the epithelial end buds was guiding axon migration. Microarray analysis of intact SMGs at early stages of development identified upregulation of multiple neurotrophic growth factors and receptors as SMG branching began. We separated epithelium from the mesenchyme and used Agilent whole genome microarray analysis to compare the gene expression in both tissue compartments. The neurotrophic factors and their receptors were expressed in the epithelium and mesenchyme, respectively. The neurotrophic factors were also expressed in the epithelial end buds rather than the ducts. We added function-blocking neurotrophic factor antibodies to SMG organ culture, which not only decreased axon outgrowth but also reduced epithelial growth and branching during culture. Addition of recombinant neurotrophic factors to the culture medium increased epithelial bud morphogenesis;however, axon bundles migrated aberrantly into the mesenchyme, suggesting that the directional neurotrophic factor gradient towards the end buds was disrupted. Our results suggest that epithelial-neuronal communication regulates both neuronal and epithelial morphogenesis during SMG development. To date, no lineage tracing studies identifying early embryonic SG stem/progenitor cells have been reported and few molecular markers have been identified in the epithelial compartment of the developing gland. We recently began to analyze embryonic SMG cells by fluorescence activated cell sorting (FACS) analysis to identify progenitor/stem cell subpopulations. Early cell fate decisions are coordinated by transcription factors (TFs), which control expression of genes involved in self-renewal and differentiation. Over the past year, we have begun to characterize the expression of known stem/progenitor cell-related genes during embryonic SMG development. 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, were evaluated by Agilent microarray and qPCR. 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. However, we were unable to detect Oct3/4 levels in the SMG at the beginning of development by qPCR, suggesting the SMG cells were already committed along a lineage pathway and had made at least one cell fate decision with the loss of Oct3/4 expression. Primary salivary epithelia were treated with growth factors (2 hours) to detect direct transcriptional changes and to investigate the molecular basis of cell fate specification. Growth factors previously shown to influence SMG development were used and the expression of stem/progenitor cell markers were evaluated by qPCR. 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, a proliferative marker, Sox9, an end bud marker, and the TFs Etv4 and Etv5. None of the growth factors tested influenced Sox10 or delta-Np63 expression, suggesting they were downstream of the signaling pathway or controlled by other factors. Taken together, 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 cellular processes involved in tissue morphogenesis is critical to regenerating tissue. A future goal is to regenerate irradiated salivary gland tissue utilizing autologous stem/progenitor cells.
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