Parasympathetic Innervation Maintains Epithelial Progenitor Cells During Salivary Organogenesis

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Science  24 Sep 2010:
Vol. 329, Issue 5999, pp. 1645-1647
DOI: 10.1126/science.1192046


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.

Organogenesis involves the coordinated growth of epithelium, mesenchyme, nerves, and blood vessels, which use common sets of genes, guidance cues, and growth factor–signaling pathways (15). Research on epithelial organogenesis has focused on epithelial-mesenchymal and endothelial-epithelial cell interactions. However, the function of the peripheral nervous system during epithelial organogenesis is less clear.

Pavlov’s seminal experiments on dogs demonstrated that neuronal input controls salivary gland function (6), and more recent work showed that parasympathetic innervation of salivary glands is essential for regeneration after injury (7). Because parasympathetic innervation occurs in parallel with salivary gland development (8), we hypothesized that parasympathetic innervation is required for epithelial progenitor cell function during organogenesis.

To test this hypothesis, we used mouse embryonic submandibular gland (SMG) explant culture and mechanically removed the parasympathetic submandibular ganglion (PSG) before the gland developed (9). SMG development begins at embryonic day 11 (E11), when the oral epithelium invaginates into neural crest–derived mesenchyme (10). The neuronal bodies of the PSG condense around the epithelium at E12 (fig. S1A) and could be separated from epithelium and mesenchyme in explant culture. When the separated tissues were recombined in culture, the growth of the SMG epithelium was reduced, with a significant decrease in the number of end buds in the absence of the PSG (Fig. 1, A and B, and fig. S2A). The PSG axons have abundant varicosities (fig. S2B, box) that contain the neurotransmitter acetylcholine (ACh) (8), and express the ACh synthetic enzyme (Chat) (fig. S1, B and C). ACh activates epithelial muscarinic (M) receptors, and M1 (Chrm1) is the major muscarinic receptor in the embryonic SMG epithelium (fig. S1, B and C), whereas M1 and M3 (Chrm3) stimulate saliva secretion in the adult (7). Alternatively, we perturbed ACh/M1 signaling using the chemical inhibitors, 4-DAMP (DAMP; N-2-chloroethyl-4-piperidinyl diphenylacetate), an irreversible M1/M3 inhibitor (Fig. 1C); atropine, a competitive muscarinic antagonist (fig. S2D); beta-bungarotoxin (Btx), which depletes neuronal ACh stores (fig. S2E); and small interfering RNA (siRNA) to M1 (Chrm1) (fig. S2, H to I). All treatments reduced the number of end buds (fig. S2, C to I). In contrast, inhibition of α2-adrenergic receptors with idazoxan had no effect (fig. S2F). These experiments demonstrate that epithelial morphogenesis requires PSG, ACh, and M1 activity.

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 hours, (A) with or (B) without the PSG or (C) treated with a muscarinic inhibitor (DAMP). Whole-mount images of the nerves (β3-tubulin, Tubb3) and the epithelium (peanut agglutinin, PNA) are shown. Scale bar, 200 μm. (D) Quantitative polymerase chain reaction (QPCR) analysis of gene expression. Means ± SEM of three experiments. Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Epithelial morphogenesis may also depend on the size of the epithelial progenitor pool and growth factor–mediated proliferation (11). To distinguish between these two possibilities, we measured expression of epithelial progenitor markers and growth factor signaling pathways present during SMG development. We found that removal of the PSG reduced gene expression of the epithelial progenitor cell markers cytokeratins-5 (Krt5) and -15 (Krt15), as well as aquaporin 3 (Aqp3) (Fig. 1D), and did not affect genes involved in fibroblast growth factor (FGF) and epidermal growth factor (EGF) signaling. Cytokeratin-5 protein (referred to as K5) is a basal epithelial cell marker in adult salivary glands (1214). Developing SMGs contain 9.6 ± 1.3% of K5+ cells by fluorescence-activated cell sorting (FACS) analysis, which are present in the end buds and ducts (fig. S3, A to C). K5+ cells are progenitor cells in the trachea (15) and prostate (16), and we confirmed that they are progenitor cells in the SMG by lineage tracing analysis (fig. S3, D to H). Cytokeratin-15 protein (K15) can physically pair with K5 (17); and Aqp3 is an epithelial progenitor cell marker in the lung (15, 18). Krt5, Krt15, and Aqp3 were down-regulated in intact SMGs after only 4 hours of DAMP treatment (Fig. 1D), which indicates that they are regulated by ACh/M1 signaling. Taken together, these data support our hypothesis that the PSG neurons modulate epithelial morphogenesis by affecting epithelial progenitor cells via ACh/M1 signaling.

To investigate how ACh directly influences the epithelium, we cultured isolated SMG epithelia in a three-dimensional extracellular matrix with FGF10 (19). We hypothesized that carbachol (CCh), an ACh analog, would increase epithelial morphogenesis and proliferation by increasing the K5+ progenitor cell population. Because ACh/M1 signaling transactivates epidermal growth factor receptor (EGFR) by matrix metalloproteinase (MMP)–mediated release of heparin-binding (HB)–EGF in prostate epithelium (20), we predicted that (i) HBEGF would increase K5+ cell proliferation, and (ii) an EGFR antagonist (PD168393, referred to here as PD) would inhibit CCh-induced K5+ cell proliferation. As expected, CCh and HBEGF increased epithelial morphogenesis, proliferation, and K5 staining, in an EGFR-dependent manner (Fig. 2, A to F, and fig. S4, A to B). Furthermore, CCh increased EGFR protein expression (fig. S4, C to F), and CCh-mediated morphogenesis was inhibited by PD (fig. S4G), which suggests that muscarinic-induced morphogenesis requires endogenous EGFR activity. When CCh and HBEGF were combined, they had a greater-than-additive effect on morphogenesis and proliferation, which were both inhibited by PD (Fig. 2, D to F). However, the combination did not have an additive effect on K5 staining. Therefore, CCh and HBEGF operate in the same pathway to maintain K5, and they increase proliferation of cells that do not express K5.

Fig. 2

Activation of muscarinic receptors maintains K5+K19– progenitor cells in an EGFR-dependent manner. Epithelia were cultured (A) in control media, or with (B) CCh, (C) HBEGF, (D) CCh+HBEGF, or (E) CCh+HBEGF+PD. 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, K19, and E-cadherin. Yellow cells expressing both K5 and K19 are marked by white asterisks. Images are 2 μm confocal sections; scale bar, 10 μm. (H) Quantification of the number of proliferating cells; see fig. S5 for images. Means ± SEM of three experiments. Analysis of variance (ANOVA) with post hoc Dunnett’s test; *P < 0.05, **P < 0.01, ***P < 0.001.

To investigate this further, we analyzed differentiation of the K5+ cell population. K5+ progenitor cell differentiation occurs in a manner similar to that described for the prostate (16); as the basal K5+K19– progenitor cells differentiate toward the developing lumen, they coexpress K19 (K5+K19+), and as differentiation proceeds, they lose K5 (K5–K19+) (Fig. 2G and fig. S5). Thus, the K5+ cell population (Fig. 2, A to F) includes both basal K5+K19– cells and K5+K19+ cells. To identify differences in the response to either CCh or HBEGF, we counted the proliferating cells (5-ethynyl-2′-deoxyuridine or EdU+) that were K5+K19– (green), K5+K19+ (yellow), or K5–K19+ (red) (Fig. 2H and fig. S5). An important finding was that CCh doubled the percentage of proliferating basal K5+K19– progenitor cells from 20 to 40% of the EdU+ cells. CCh also increased K5+K19+ cell proliferation, which suggested that these cells are still responsive to muscarinic activation. HBEGF increased the proliferation of both the K5+K19+ and K5–K19+ cells, which suggests that HBEGF promotes differentiation along the K19+ lineage. The combination of CCh and HBEGF resulted in similar amounts of proliferation of K5+K19–, K5+K19+, and K5–K19+ cells (Fig. 2F). Finally, CCh- and HBEGF-mediated proliferation of the K5+ and K19+ cells was completely inhibited by PD (Fig. 2H), although proliferating cells that were not K5+ or K19+ were still present (Fig. S5). Taken together, these data suggest that CCh/M1 signaling maintains the K5+ progenitor cell population in an EGFR-dependent manner, and that HBEGF/EGFR increases differentiation of the K19+ cell lineage.

We confirmed these findings in the intact SMG, by using a loss-of-function approach treating SMGs with DAMP or PD. We hypothesized that DAMP treatment would inhibit K5+ cell proliferation and result in fewer K5+ cells. We used FACS analysis of the E-cadherin+ epithelial population, measuring the number of cells expressing K5, K19, and Ki67 for proliferating cells (fig. S6, A to D). As expected, the total K5+ cell population was reduced by both DAMP and PD treatment. The reduction in K5+ cell number was not due to an increase in apoptosis (fig. S7). Note that both PD and DAMP completely inhibit proliferation of the K5+K19– basal progenitor cells. In sum, the inhibition of M1 and EGFR signaling in the intact SMG reduces the number of K5+K19– progenitor cells by inhibiting their proliferation. These data demonstrate that the maintenance of K5+ cells in an undifferentiated state (K5+K19–) in the intact SMG is dependent on M1 and EGFR signaling.

These findings suggest that K5+ progenitor cells express both M1 and EGFR. Using immunostaining and FACS analysis, we demonstrated that the majority of K5+ cells (68%) expressed both EGFR and M1 (fig. S8, A to H), which suggests that the effects of CCh and HBEGF are cell-autonomous. Furthermore, the combination of HBEGF and CCh increased the amount of both M1 and EGFR in the epithelium (fig. S8, J to N), which shows that positive feedback increases receptor expression.

We then predicted that CCh would rescue epithelial morphogenesis and K5 expression in the PSG-free SMG explants in an EGFR-dependent manner. As expected, treatment of SMG with PD, or removal of the PSG, decreased the number of end buds and the amount of K5 staining compared with the control (Fig. 3, A to E, and fig. S9). Treatment of the PSG-free explants with CCh increased the number of end buds and K5 expression, which were inhibited by PD (Fig. 3, C to E). Furthermore, PD reduced K19 expression in the absence of the PSG, which demonstrates that endogenous HBEGF maintains the K19+ cells. HBEGF increases SMG morphogenesis by inducing membrane type 2–MMP and FGF receptor (FGFR) expression (21). Thus, addition of PD to PSG-free explants reduces the EGFR-dependent morphogenesis, and the remaining growth is likely FGFR2b-dependent (10, 19).

Fig. 3

CCh rescues branching morphogenesis and K5+ progenitor cells in an EGFR-dependent manner. SMG explants, recombined (A) with or (B to D) without the PSG, were cultured with (C) CCh or (D) CCh+PD. Images are single confocal sections (2 μm); scale bar, 50 μm. (E) Quantification of K5 and K19 fluorescence. Means ± SEM of three experiments. ANOVA with post hoc Dunnett’s test; *P < 0.05, **P < 0.01.

Our data demonstrate that parasympathetic innervation maintains K5+ progenitor cells during epithelial organogenesis. A therapeutic implication is that postnatal epithelial regeneration of salivary glands may require muscarinic stimulation of the K5+ progenitor cells. Indeed, culture of denervated lobules of adult SMGs with CCh increased K5 and Krt5 expression, which were both reduced with a combination of DAMP and PD (Fig. 4, A to D, and fig. S10A). In addition, K5+ cells increase during regeneration of adult SMGs after duct ligation when the innervation to the gland is intact (22). Furthermore, other organs that contain K5+ progenitor cells, such as the prostate (16), the skin and its appendages (23), the airway epithelium and trachea (15), and taste buds (24), are innervated by the peripheral parasympathetic nervous system (25) during development. We analyzed the developing ventral prostates of P6 mice, which contain basal K5 cells and innervation from the pelvic ganglion neurons (fig. S10B). The addition of DAMP and PD to prostate organ culture also decreased K5 expression (Fig. 4, E to F, and fig. S10C), and reduced Krt5, Krt14, and Aqp3 (Fig. 4G). Thus, we report a mechanism by which K5+ epithelial progenitor cells are maintained during development, which has implications for understanding how tissue-specific epithelial progenitor cells could be targeted for organ repair or regeneration.

Fig. 4

Muscarinic receptor and EGFR signaling controls K5+ progenitor cell maintenance in the adult SMG and developing prostate. Denervated adult SMGs (A), cultured with (B) CCh or (C) DAMP+PD, were immunostained for K5 and perlecan, and (D) analyzed by QPCR. Scale bar, 20 μm, see fig. S10 for quantification. Ventral prostates from P6 mice were cultured (E) without or (F) with DAMP+PD and immunostained for K5, K19, and E-cadherin, and (G) gene expression was analyzed by QPCR. Scale bars, 50 μm. Means ± SEM of three experiments. Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Supporting Online Material

Materials and Methods

Figs. S1 to S10


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. The authors would like to thank V. N. Patel, I. T. Rebustini, L. M. Angerer, K. G. Ten-Hagen, K. M. Yamada, and S. Powers for discussions and critical reading of this manuscript. The study was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, NIH.
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