Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis

Abstract

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 organogenesis are poorly understood. We report that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number and morphogenesis of keratin 5-positive epithelial progenitor cells. These effects were rescued with an acetylcholine analog. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and epidermal growth factor receptor, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Parasympathetic innervation maintained the epithelial 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.

Fig 1. Removal of the PSG in mouse SMG explant culture decreases branching morphogenesis and expression of basal progenitor cell markers

SMGs were cultured for 44 hrs with (A) or without (B) the PSG or treated with a muscarinic inhibitor (DAMP) (C). Whole-mount images of the nerves (beta-3 tubulin, Tubb3) and the epithelium (peanut agglutinin, PNA) are shown. Scale bar = 200 μm. (D) qPCR analysis of gene expression. Mean ± SEM of three experiments. Students t-test; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 2. Activation of muscarinic receptors maintains K5+K19- progenitor cells in an EGFR-dependent manner

Epithelia were cultured in control media (A), with CCh (B), HBEGF (C), CCh+HBEGF (D), CCh+HBEGF+PD (E). Immunostaining of proliferation (EdU) and K5+ cells is shown. Images are 3 μM confocal sections; scale bar = 50 μm. (F) Quantification of epithelial morphogenesis, proliferation, and K5 protein. AU = arbitrary units × 100. (G) Epithelia were immunostained for K5 and K19. Yellow cells expressing both K5 and K19 are marked by white*. Images are 2 μM confocal sections; scale bar = 10 μm. (H) Quantification of the number of proliferating cells; see fig S5 for images. Mean ± SEM of three experiments. ANOVA with post hoc Dunnetts test; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3. CCh rescues branching morphogenesis and K5+ progenitor cells in an EGFR-dependent manner

SMG explants, recombined with (A) or without (B-D) the PSG, were cultured with CCh (C) or CCh+PD (D). Images are single confocal sections (2 μM); scale bar = 50 μm (E) Quantification of K5 and K19 fluorescence. Mean ± SEM of three experiments. ANOVA with post hoc Dunnetts test; *P < 0.05, **P < 0.01.

Fig 4. Muscarinic receptor/EGFR signaling controls K5+ progenitor cell maintenance in the adult SMG and developing prostate

Denervated adult SMGs (A) cultured with CCh (B) or DAMP+PD (C), were immunostained for K5 and perlecan, and analyzed by qPCR (D). Scale bar = 20 μm, see fig S10 for quantification. Ventral prostates from P6 mice (E) were cultured ± DAMP+PD (F) and stained for K5, K19 and E-cadherin, and gene expression was analyzed by qPCR (G). Scale bars = 50 μm. Mean ± SEM of three experiments. Students t-test; *P < 0.05, **P < 0.01, ***P < 0.001.