Neuronal and Progenitor/Stem Cell Function During Salivary Gland Development

National Institute Of Dental & Craniofacial Research

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Abstract

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. 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. The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. However, the mechanisms by which epithelial progenitor cells are maintained during salivary organogenesis are poorly understood. 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 reported that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number of keratin 5-positive epithelial progenitor cells which decreased epithelial morphogenesis. These effects were rescued with an acetylcholine analog. In addition, chemical inhibition of acetylcholine 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. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and EGFR, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Our analysis of the keratin-5 positive cells in salivary gland development involved genetic lineage tracing experiments to prove that the keratin-5 positve cells were epithelial progenitor cells. Therefore, the parasympathetic innervation maintained the epithelial keratin 5-positive progenitor cell population in an undifferentiated state, which was required for organogenesis. This mechanism for epithelial progenitor cell maintenance may be targeted for organ repair or regeneration. Early cell fate decisions are coordinated by transcription factors (TFs), which control expression of genes involved in self-renewal and differentiation. We have characterized 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, Sox9, 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 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.