The salivary glands produce fluid that contains enzymes to help us to digest our food. These glands contain a tree-like network of cells – known as acinar cells – that produce the fluid, and cells that form ducts to transport the fluid out of the glands. Both types of cells form from stem cells as animal embryos develop. Like all developing organs, the salivary glands receive many different signals that guide how they grow. However, the identity of the cues that instruct a stem cell to produce a new acinar cell or duct cell are not known.

Emmerson et al. studied how the salivary glands develop in mouse embryos. The experiments show that a protein called SOX2 – which is an essential regulator of stem cells in embryos – is required for acinar cells to form. Loss of SOX2 inhibited the production of acinar but not duct cells. Furthermore, nerves that surround the gland provide support to cells that produce SOX2 and promote the formation of acinar cells.

Further experiments suggest that the nerves also play the same role in humans. Adult organs often use developmental signals to repair or regenerate tissue. As such, understanding how an organ develops may lead to new therapies that can stimulate salivary glands and other organs to regenerate after they have been damaged in adults.

Acinar cells are essential to the function of exocrine organs including the pancreas, tracheonasal glands and salivary glands. These cells produce a mucin and/or protein-rich fluid that is transported through an interconnected ductal network to the external surface where it serves multiple roles including the protection and function of the epithelial mucosa. Although there have been extensive studies on the mechanisms of branching morphogenesis (reviewed in Mattingly et al., 2015), the process by which most mammalian exocrine organs develop, very little is known about the mechanisms that control acinar cell development. Moreover, the factors required for the establishment of the acinar lineage in the developing salivary gland are not known.

As for the majority of exocrine organs, salivary gland acinar and duct cells form through the expansion and differentiation of a simple cuboidal epithelium. In the submandibular salivary gland (SMG), the most characterized of the three major salivary glands (considerably less is known about the development of the sublingual (SLG) and parotid (PG)), this begins with invagination of the epithelium into the condensing mesenchyme to form a single end bud containing SOX10-positive pre-acinar cells by embryonic day (E)12 (Lombaert et al., 2013). Branching of the single bud into 3–5 KIT-positive end buds occurs between E12.5 and E13.0 and is followed by rapid expansion and further differentiation between E13.5-E15.0 (Lombaert et al., 2013; Walker et al., 2008 and Figure 1A). Acinar cell differentiation can be monitored by temporal expression kinetics of genes including aquaporin (AQP) 5 at E14 (Figure 1B), Basic Helix-Loop-Helix Family Member A15 (MIST1) (Pin et al., 2001) and demilune cell and parotid protein (DCPP) at E15.0; submandibular gland protein C (SMGc)/mucin 19 (MUC19) and parotid secretory protein (PSP) at E16.0, and MUC10 at E17.0 (Nelson et al., 2013). In conjunction with acinar cell development is formation of the ductal lineage, which is marked initially by KRT19 at E12.0, followed by KRT7 at E13.5 (before lumenization) and prominin-1 at E14.0 (Nedvetsky et al., 2014; Walker et al., 2008). Both acinar and duct cells are derived from a reservoir of undifferentiated basal KRT5+ progenitors that are essential to SG development (Knox et al., 2010). However, the mechanisms regulating specification of KRT5+ cells toward these two lineages or how the proportion of duct to acinar cells is controlled are unknown.

Figure 1

Download asset Open asset

In addition to the known growth factor pathways that regulate epithelial branching morphogenesis (e.g. FGF10/FGFR2b [Jaskoll et al., 2005]), we recently discovered that neurotransmitters released from intraglandular parasympathetic nerves also control multiple aspects of SG development. The SMG and SLG receive innervation from the parasympathetic submandibular ganglion that forms at E12.0 through coalescence of neuronal cell bodies around the primary duct. The ganglion begins to innervate newly forming end buds and ducts from E12.5 (Knox et al., 2010), with unidirectional axon outgrowth from the ganglion toward the end buds being mediated by neurturin/GFRa2 signaling (Knox et al., 2013). Acetylcholine is produced as soon as the ganglion forms (Coughlin, 1975) and activates muscarinic receptors on the epithelium to maintain an undifferentiated stem cell population marked by KRT5 (Knox et al., 2010). In addition to this function, muscarinic activation also induces epithelial branching (Knox et al., 2013, 2010) but whether parasympathetic nerves are specifically required for the generation of the acinar lineage is unknown.

Using the acini-ductal network of developing murine and human salivary glands in combination with in vivo and ex vivo studies, we reveal that SOX2 is specifically required to generate the acinar cells necessary for the creation of a secretory organ. We show that SOX2 is essential for the establishment and survival of acinar cells and that its expression and the proliferative expansion of SOX2-positive cells depend on neuronal acetylcholine signaling by parasympathetic nerves. As such, we identify SOX2 as a master regulator of the salivary acinar cell lineage and uncover a conserved peripheral nerve-based mechanism for selectively generating the secretory acinar cell lineage during organogenesis.

SOX2 is an important transcriptional regulator of development and maintenance in multiple organs including trachea, oesophagus and intestine (Arnold et al., 2011; Gontan et al., 2008; Okubo et al., 2006; Que et al., 2009, 2007). In the murine E13 embryo, SOX2 is expressed by the invaginating oral epithelium (Figure 2—figure supplement 1B), throughout the epithelium of the SLG (Figure 1C) and the proximal duct of the SMG and the developing parasympathetic ganglion (Figure 1C; Lombaert and Hoffman, 2010). Genetic lineage tracing at E10.5 indicated that SOX2-positive cells are progenitors giving rise to both duct and acinar cells of the SMG and SLG (Figure 1D,E Arnold et al., 2011). However, we found that SOX2 becomes restricted to a subpopulation of AQP5+ acinar cells in the postnatal and adult SLG, the most poorly understood of the three major salivary glands (Figure 1F and G), suggesting that SOX2 might function in the expansion and/or maintenance of the acinar lineage.

To study the role of Sox2 in salivary gland morphogenesis, a tamoxifen-inducible Cre under the control of the epithelial Krt14 promoter was used to ablate Sox2 prior to the onset of gland ontogenesis (E10.5; see extensive loss of SOX2 expression in epithelia but not the ganglion in Figure 2—figure supplement 1B). Unexpectedly, despite SOX2-positive cells giving rise to both acinar and duct cells, genetic ablation of Sox2 had a profound effect on the generation of acini but not ducts. The number of end buds in the SMG and SLG was significantly reduced from early stages of development and onwards, with the SLG exhibiting little to no branching (E13 to E16.5; Figure 2A–B and Figure 2—figure supplement 1A–E). Conversely, duct morphology, the number of KRT19-positive ductal cells and ductal gene transcripts Krt7, Krt19 and Egfr, which are required for ductal development (Häärä et al., 2009; Knox et al., 2010), were similar to wild-type controls (Figure 2A–D and Figure 2—figure supplement 1C). Moreover, by E16.5, the number of SOX10-positive progenitors and differentiated AQP5-positive and MIST1-positive acinar cells were severely reduced (Figure 2C–E, Figure 2—figure supplement 1C). Further analysis of changes in gene expression profiles between wild-type control and Sox2-deficient SMG+SLG by RNA-seq and qPCR validation confirmed the reduction in genes associated with differentiated acinar cells (Smgc, Pip, Spdef, Rab3d; Figure 2C, Figure 2—figure supplement 1G). To rule out the phenotype being a cause of a difference in recombination efficiency between acini and ducts, we confirmed that Sox2 ablation efficiency is comparable between AQP5+/SOX10+ acini and KRT19+ ducts (Figures 1E and 2F) using a lineage-tracing model (Krt14^CreERT2;Sox2^fl/fl;Rosa26^mTmG).

Figure 2 with 1 supplement see all

Download asset Open asset

Figure 2—source data 1

Source data relating to Figure 2C.

qPCR analysis of E16.5 Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) SMG+SLG for genes involved in acinar differentiation, ductal differentiation and innervation, with expression normalised to Rsp29 and the WT. n = 3 embryos per genotype. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.005

Download elife-26620-fig2-data1-v2.docx

Figure 2—source data 2

Source data relating to Figure 2D.

Quantification of cells expressing acinar or ductal markers, cleaved caspase-3 or Ki67 in acini of E16.5 in Krt14^CreERT2; Sox2^fl/fl and wild-type (WT). n = 2–4 glands/genotype and cells were counted in 3–4 acini/gland. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.006

Download elife-26620-fig2-data2-v2.docx

Figure 2—source data 3

Source data relating to Figure 2F.

SMG+SLG from Krt14^CreERT2; Rosa26^mTmG and Krt14^CreERT2; Rosa26^mTmG; Sox2^fl/fl were immunostained for SOX10 and AQP5 and GFP+ cells expressing SOX10 and AQP5 were quantified and expressed as a percentage of total positive cells. n = 3 glands/genotype and cells were counted in 3–4 acini/gland. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.007

Download elife-26620-fig2-data3-v2.docx

Figure 2—source data 4

Source data relating to Figure 2G.

qPCR for enrichment of Sox10 in SOX2 ChIP. n = 20 pooled SLG, average three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.008

Download elife-26620-fig2-data4-v2.docx

SOX2 co-localizes with the acinar progenitor marker SOX10 in acini of developing murine glands (Figure 2E, left panel). Given SOX10 expression was lost upon ablation of Sox2, we performed lineage tracing in conjunction with Sox2 ablation to determine if SOX10 was specifically lost in cells derived from those in which Sox2 was ablated (Krt14^CreERT2;Sox2^fl/fl;Rosa26^mTmG). In this model, cells in which Cre recombinase has been activated (and consequently Sox2 ablated) will be labeled with membrane-bound GFP+ (green), whereas cells where no recombination has occurred will retain membrane-bound Tomato (mT, red). We found that only ~5% of end bud cells deficient in Sox2 expressed SOX10 or AQP5 in comparison to control glands (Krt14^CreERT;Rosa26^mTmG) in which >90% express SOX10 or AQP5 (Figure 2E and F). We then determined whether SOX2 could directly regulate Sox10 using ChIP-qPCR analysis of E17.5 SG. This time point was chosen due to the small size of the tissue limiting our analysis at earlier time points. We found significant enrichment of SOX2 within a specific region of the Sox10 promoter (promoter region A; Figure 2G), suggesting that SOX2 may promote the generation of the acinar lineage via transcriptional control of Sox10. Combined, these data indicate that the maintenance of SOX10-positive acinar progenitor cells and the generation of differentiated AQP5-positive acinar cells are dependent on SOX2.

Previous studies in other organ systems show that a deficiency in SOX2 results in a cell fate switch (23–26]). However, we did not find any evidence that Sox2-deficient cells in the end buds divert toward a duct cell fate (Figure 3A, GFP+ cells do not express early ductal marker KRT19), indicating that the reduction in epithelial branching was not due to the accumulation of the ductal lineage. Therefore, we addressed if loss of end bud cells and reduced morphogenesis in Sox2-deficient SMG+SLG were due to apoptosis. In the absence of Sox2, there were activated Caspase-3-positive cells in the end buds (Figures 2D and 3B) and increased expression of pro-apoptotic genes (Bax, Bbc3 and Pmaip1; Figure 2—figure supplement 1G) at E16.5. Apoptosis was specific to end bud cells as no apoptosis was observed in the ducts (Figure 3B), suggesting a preferential requirement for SOX2 in end bud cell survival. In addition to increased apoptosis, the proliferation of end bud cells was decreased ~70% in E16.5 salivary glands lacking Sox2 (Figures 2D and 3B; Ki67+ cells). Apoptosis and reduced proliferation were not due to a loss in FGF signaling that has previously been shown to be required for epithelial survival and acinar cell expansion (Lombaert et al., 2013; Matsumoto et al., 2016) as the expression of downstream targets of FGF signaling transduction (Etv4, Etv5) and FGF ligands (Fgf1, Fgf10, Fgf7) were not reduced compared to wild-type controls (Figure 2—figure supplement 1G).

Figure 3

Download asset Open asset

Figure 3—source data 1

Source data relating to Figure 3E.

Quantification of the number of CASP3+ cells in acini of E11.5 Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) glands cultured for 60 hr ± Z-VAD-FMK. n = 3 glands per treatment and cells were counted in 3–4 acini per gland. Data are the mean of three biological replicates and two experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.015

Download elife-26620-fig3-data1-v2.docx

Figure 3—source data 2

Source data relating to Figure 3F.

Quantification of the number of acini of E11.5 Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) glands cultured for 60 hr ± Z-VAD-FMK. n = 3 glands per treatment. Data are means of three biological replicates and two experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.016

Download elife-26620-fig3-data2-v2.docx

As both apoptosis and reduced proliferation were apparent at E16.5, and there was reduced branching and increased apoptotic markers at E13-E13.5 (Figure 2—figure supplement 1A,B and F) to further delineate between mitosis and cell death as a cause of reduced growth, we analyzed tissue at E11.5 (Cre induction at E10.5 by injection of tamoxifen). At this stage of development, the undifferentiated oral epithelium has begun to invaginate into the condensing mesenchyme. We observed an increase in apoptotic cells in the invaginating mutant epitheliums compared to wild-type controls (arrows indicate cleaved CASP3-p cells) but proliferation was not perturbed, suggesting that cell death rather than reduced proliferation impairs organ growth (Figure 3C). To test if inhibiting apoptosis could rescue epithelial branching that is, cell death is a causative factor in the loss of morphogenesis, we cultured E11.5 mandibles from wild-type and Sox2-deficient embryos ex vivo (Knosp et al., 2015) with the pan-caspase inhibitor Z-VAD-FMK (50 µM, Nedvetsky et al., 2014); Figure 3D) for 60 hr. However, despite a significant reduction in CASP3+ cells, extensive epithelial cell survival and growth of the epithelium, we did not measure an increase in the number of acini in Sox2-deficient SMG+SLG cultured with the cell death inhibitor (Figure 3D–F). Thus, these data reveal novel, independent roles for SOX2 in regulating epithelial cell survival and generating acini.

Our previous study indicated that KRT5+ progenitors are maintained in an unspecified state by parasympathetic nerves via acetylcholine/muscarinic activation (Knox et al., 2010). As a subpopulation of KRT5+ cells of the developing SMG co-express SOX2 (Lombaert et al., 2011), we next asked if nerves also maintain SOX2+ progenitor cells by comparing SG where the ganglia had been mechanically or genetically ablated with nerve-containing controls. SMG+SLG in which the epithelium and mesenchyme was recombined without the ganglia and cultured for 48 hr (Knox et al., 2010) exhibited a severe reduction in the number of acini, SOX2 protein and SOX2+ cells. However, not only were SOX2+ progenitors adversely affected, the acinar lineage was also greatly depleted in the absence of innervation. In the ganglia-free SG, we found a significant reduction in SOX10-positive acinar progenitors and differentiated AQP5-positive acinar cells (Figure 4A–C) compared to ganglia-containing controls. Similarly, acini, SOX10+ cells, Sox2 and acinar gene expression were reduced in SMG+SLG derived from Phox2b mutant embryos that are deficient in the submandibular parasympathetic ganglion and other craniofacial ganglia (Pattyn et al., 1999) (Figure 4D–F). However, absence of innervation did not impair the expression of ductal marker KRT19 or ductal gene transcripts (Figure 4A and F). Thus, peripheral innervation not only preserves the progenitor cell pool, as previously found, but also selectively preserves those progenitors that contribute specifically to the acinar cell lineage.

Figure 4

Download asset Open asset

Figure 4—source data 1

Source data relating to Figure 4B.

E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion (nerves). The number of acini were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.018

Download elife-26620-fig4-data1-v2.docx

Figure 4—source data 2

Source data relating to Figure 4C.

E13 murine SMG+SLG cultured for 48 hr ± parasympathetic ganglion (nerves) and subjected to immunofluorescent analysis. The number of AQP5+ and SOX10+ cells were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.019

Download elife-26620-fig4-data2-v2.docx

Figure 4—source data 3

Source data relating to Figure 4E.

E11.5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr. The number of acini were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.020

Download elife-26620-fig4-data3-v2.docx

Figure 4—source data 4

Source data relating to Figure 4F.

E11.5 murine SMG+SLG deficient in Phox2b were cultured for 60 hr and qPCR performed. Data were normalized to Rsp29 and the WT. Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.021

Download elife-26620-fig4-data4-v2.docx

Next, we determined if acetylcholine/muscarinic signaling regulates SOX2 and production of acinar cells by specifically targeting muscarinic receptors. FACS analysis showed that 94% of the SOX2-positive epithelial cells in the SLG at E15.0 express the muscarinic receptor CHRM1 (Figure 5—figure supplement 1A), indicating that CHRM1 activation is a potential regulator of SOX2 cells. CHRM1 is also expressed throughout the epithelium including ~42% of SOX2-negative epithelial cells (Figure 5—figure supplement 1A and B), indicating that this receptor is not exclusive to SOX2+ cells. In support of a direct role for muscarinic receptors in expanding SOX2+ cells, treatment of isolated E14.0 SLG epithelial rudiments devoid of nerves and mesenchyme with the muscarinic receptor agonist carbachol (CCh) for 48 hr increased the number of SOX2+ cells as well as proliferating SOX2+EdU+ cells compared to the control (Figure 5A–B). Furthermore, culture of mouse E14.0 SLG with the muscarinic antagonist 4-DAMP for 4 or 24 hr reduced SOX2 expression (gene and protein), the number of SOX2+ cells and SOX2+ cell proliferation (Figure 5C–D and Figure 5—figure supplement 1C. Similar to the outcomes for SMG+SLG in which Sox2 was ablated or nerves removed, inhibition of CHRM1 decreased end bud cell proliferation, differentiated AQP5-positive acinar cells and SOX10-positive acinar progenitors (Figure 5E and Figure 5—figure supplement 1D and E), whereas KRT19-positive cells (Figure 5E and Figure 5—figure supplement 1E) or ductal lineage transcript levels were unchanged (Krt7) or increased (Krt19 and Egfr; Figure 5—figure supplement 1C). In addition, there was decreased expression of acinar differentiation markers (Aqp5, Mist1 and Chrm3; Figure 5—figure supplement 1C). Finally, to determine if muscarinic activation was sufficient to restore the acinar lineage after denervation, we treated nerve-free SMG+SLG explants (containing mesenchyme) with CCh for 48 hr and compared to untreated ganglia-containing and ganglia-free controls. As shown in Figure 5F–H, CCh was sufficient to return Sox2 expression in the SLG, and restore AQP5+ cells, and transcript levels of acinar-specific genes including Sox10, Mist1 and Aqp5 to control levels in the SMG and SLG.

Figure 5 with 1 supplement see all

Download asset Open asset

Figure 5—source data 1

Source data relating to Figure 5B.

E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. The number of SOX2+, EdU+ and SOX2+EdU+ cells were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.023

Download elife-26620-fig5-data1-v2.docx

Figure 5—source data 2

Source data relating to Figure 5C.

E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP (10 µM). The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS, normalized to control and expressed as percentage of total ECAD+ cells. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.024

Download elife-26620-fig5-data2-v2.docx

Figure 5—source data 3

Source data relating to Figure 5G.

E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and the number of AQP5+ and KRT19+ cells counted. Counts were normalized to the control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.025

Download elife-26620-fig5-data3-v2.docx

Figure 5—source data 4

Source data relating to Figure 5H.

E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and subjected to qPCR analysis. Data were normalized to Rsp29 and control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.026

Download elife-26620-fig5-data4-v2.docx

As muscarinic receptors in the salivary gland have been reported to signal via intracellular calcium and EGFR (Jiménez et al., 2001; Knox et al., 2010), we determined if either of these downstream targets was required for SOX2 and the acinar lineage. Culture of E14.0 SLG for 4 hr with the CaMK-II inhibitor KN-93 but not the EGFR inhibitor PD168393 decreased expression levels of Sox2 and Sox10 (Figure 5—figure supplement 1F). In comparison, inhibition of EGFR or calcium signaling reduced expression of more differentiated acinar markers (Mist1, Aqp5) suggesting that early and late markers of salivary gland development are differentially regulated. In contrast, ductal genes were expressed at similar or higher levels than controls upon KN-93 treatment (Figure 5—figure supplement 1F). Thus, CHRM1 and calcium signaling regulate epithelial SOX2 in the developing SLG and are required for generation of the acinar lineage.

Finally, we investigated whether neuronal signaling was an evolutionarily conserved mechanism for generating acini and preserving SOX2+ progenitors by analyzing the impact of nerves and muscarinic activation on SOX2 and the acinar lineage in human tissue. Although the murine and human glands differ in gross morphology, they undergo similar stages of morphogenesis (Teshima et al., 2011) and are highly innervated (Figure 6A and Figure 6—figure supplement 1A). Due to the paucity of expression data on human fetal salivary gland, we first characterized expression of acinar and duct markers as well as the location of CHRM1, SOX10 and SOX2. As shown in Figure 6A and Figure 6—figure supplement 1A, CHRM1 protein and proliferating SOX2+ and SOX10+cells are enriched in the acinar compartment of all major human fetal salivary glands, including the parotid gland (PG). We confirmed that human SMG/SLG/PG acinar cells are also enriched in SOX10, CHRM1 and MIST1 as well as CD44 and AQP3 protein (acini do not express AQP5 protein at these stages) and that the duct cells express KRT7 in addition to EGFR, KRT5, KIT and KRT14 (Figure 6A and Figure 6—figure supplement 1A). However, KRT19 was expressed in both acini and ducts, indicating that it is not a bona fide ductal marker in fetal human tissue (Figure 6—figure supplement 1A). To determine if parasympathetic nerves promote acinar cell formation in developing human gland, we developed a co-culture assay in which human fetal SLG explants were mixed with murine ganglia or mesenchyme (Figure 6B). Unlike mice, the innervating human ganglion resides outside the organ and the tissue is thus denervated upon dissection. Co-culture of human SLG (20–22 w) with E13.0 parasympathetic ganglia for 7 days resulted in extensive remodeling and innervation of the epithelium (Figure 6B and C). As for the murine SLG, innervation increased expression of SOX2 protein (Figure 6D) as well as of acinar markers MIST1 (~5–7 fold), AQP3 (~1.6–1.9 fold), CHRM1 (~2–10 fold) and CHRM3 (~2.5–3.4 fold) compared to mesenchyme alone (Figure 6E; n = 2 fetuses). Consistent with the hypothesis that parasympathetic nerves preferentially regulate the acinar lineage, levels of ductal gene transcripts KRT5, KRT7, KRT14, KIT and EGFR were similar to human SLG co-cultured with mesenchyme only (Figure 6E).

Figure 6 with 1 supplement see all

Download asset Open asset

Figure 6—source data 1

Source data relating to Figure 6E.

Human fetal SLG explants cultured with murine E13 PSG or mesenchyme for 7 days were subjected to gene profiling by qPCR. Data were normalized to GAPDH and control (+ mesenchyme). Data are means of six biological replicates, two individual experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.032

Download elife-26620-fig6-data1-v2.docx

Figure 6—source data 2

Source data relating to Figure 6F.

qPCR analysis of fetal human SLG (22–23 w) dissociated cells cultured ± CCh for 48 hr. Data were normalized to GAPDH and control (-CCh). Data are means of six biological replicates, two individual experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.033

Download elife-26620-fig6-data2-v2.docx

Figure 6—source data 3

Source data relating to Figure 6G.

qPCR analysis of fetal human SLG (22–23 w) explants cultured ± CCh for 72 hr. Data were normalized to GAPDH and control (-CCh). Data are means of six biological replicates, two individual experiments. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.034

Download elife-26620-fig6-data3-v2.docx

Figure 6—source data 4

Source data relating to Figure 6H.

Analysis of fetal human SLG (22–23 w) dissociated cells cultured ± CCh for 48 hr. The number of ECAD+SOX2+ and ECAD+SOX2+Ki67+ cells were measured by FACS as a percentage of total ECAD+ cells. Each # represents an independent experiment. s.d. = standard deviation.

https://doi.org/10.7554/eLife.26620.035

Download elife-26620-fig6-data4-v2.docx

To determine the impact of CHRM1 stimulation on human SOX2+ cells and the acinar lineage, we cultured human fetal SLG explants or dissociated salivary cells with CCh for 48–72 hr. As for the murine SLG, CCh treatment was sufficient to increase the expression of SOX2 and acinar cell markers in human explants as well as dissociated cells (consisting of epithelium and mesenchyme; 20–22 weeks (w) of gestation) (Figure 6F–G). Furthermore, CCh treatment promoted SOX2-positive cell proliferation (Figure 6H and Figure 6—figure supplement 1B). CCh also increased expression of KRT5 which is enriched in basal duct cells of human tissue and myoepithelial cells, suggesting these cell types are regulated by CHRM1 signaling (Figure 6F–G). Thus, muscarinic stimulation is sufficient to rescue the acinar cell lineage and SOX2 expression and to promote the proliferation of SOX2-positive cells in the developing human SLG. Combined, these data support a conserved role for cholinergic innervation in the generation of the acinar lineage from mice to humans.

Our study demonstrates a critical role for SOX2 in the generation of the acinar lineage during SG development. SOX2 acts as a master regulator of the acinar lineage by modulating the expression of lineage-specific genes and controlling both cell proliferation and survival. However, SOX2 function in acinar cell production is dependent on parasympathetic nerves that maintain its expression via acetylcholine-muscarinic-calcium signaling. The observation of similar outcomes in the human SG-nerve co-culture system provides strong evidence that the regulation of SOX2 and acini by innervation is an evolutionarily conserved mechanism that regulates the generation of acinar cells.

SOX2 also regulates cell lineage commitment in other organs such as the pituitary and skin (Andoniadou et al., 2013; Driskell et al., 2009; Goldsmith et al., 2016; Lesko et al., 2013). In the pituitary, loss of SOX2 expression in progenitors leads to a switch in cell fate between the two closely related cell types, melanotrophs and corticotrophs, in the intermediate lobe of the pituitary (Goldsmith et al., 2016). In the adult skin, SOX2 is expressed in dermal papilla cells that give rise to the three hair follicle subtypes and ablation of Sox2 alters the proportion of these subtypes (Driskell et al., 2009; Lesko et al., 2013). Despite SOX2+ progenitors contributing to acini and ducts in the SMG+SLG, we unexpectedly found that Sox2 is only required for the specification of the SOX10-positive acinar lineage. However, in contrast to the cell fate switch observed in the pituitary and skin, Sox2-deficient cells in the end buds do not divert to ductal cell fates. This suggests that acquisition of a duct cell fate requires active re-specification and that ductal identity is not the default cell fate in the absence of SOX2 or innervation. This contrasts with a previous proposal that progenitor cells, in the absence of induction, simply differentiate into duct cells (Knox et al., 2010). This conclusion is corroborated by recent genetic lineage-tracing studies in the adult SG showing that acinar cells give rise to more acinar cells but not duct cells (Aure et al., 2015). Thus, despite SOX2 being expressed by some duct cells (Lombaert et al., 2011) and SOX2-positive cells being able to contribute to ducts during development, SOX2 functions in cell specification are limited to the acinar lineage.

How salivary acinar cells are established in the SG is not known. However, acinar cell expansion in the developing SG has previously been shown to be regulated by FGF10/FGFR2b signaling (Lombaert et al., 2013; Matsumoto et al., 2016). This indicates that FGF10/FGFR2b signaling is either adversely affected by loss of SOX2 or that SOX2 acts downstream of this pathway. As we did not observe a loss of FGF signaling in Sox2-deficient SMG+SLG and FGF10/FGFR2b signaling reduces SOX2 expression in isolated SMG epithelia (Lombaert et al., 2011), we conclude that SOX2 acts downstream of FGF signaling. As such, FGF signaling most likely regulates the differentiation of SOX2-positive acinar progenitors and/or their proliferative expansion.

SOX2 has been shown to promote the survival of cancer cells in vivo and has been linked to lens cell survival in Astyanax surface fish (Boumahdi et al., 2014; Chou et al., 2013; Herreros-Villanueva et al., 2013; Ma et al., 2014), but a role for SOX2 in cell survival during mammalian organogenesis has not been reported. In this study, we reveal that genetic ablation of Sox2 but not denervation or muscarinic inhibition, which reduces but not eliminate SOX2, is sufficient to trigger cell death in the SMG+SLG. This is likely due to the levels of SOX2 remaining in epithelial cells from denervated or muscarinic receptor antagonized salivary glands being conducive to cell survival, although single-cell expression analysis is required to confirm such a mechanism. Levels of SOX2 may also influence lineage outcomes. It is possible that SOX2 may be activated in formerly SOX2-negative cells in response to cholinergic stimulation thereby specifying the acinar lineage. However, as both acinar and duct cells express SOX2, there must be other regulators at play besides SOX2 that specify this lineage. Furthermore, despite both duct and acinar cells expressing SOX2 during early gland development, apoptosis was restricted to cells in the end buds, which points to an essential and selective role of SOX2 in promoting survival of the acinar but not ductal lineage. A direct role for SOX2 in cell survival is supported by chromatin precipitation studies in skin tumor cells where a number of survival genes were directly regulated by SOX2 (Boumahdi et al., 2014).

We, and others, have previously shown that peripheral nerves maintain progenitors in an undifferentiated state, providing a pool of unspecified cells for developmental or regenerative processes (Knox et al., 2010; Xiao et al., 2015). We have also shown that nerves, through release of vasoactive intestinal peptide, regulate duct formation (Nedvetsky et al., 2014). Our current study demonstrates an unexpected role for peripheral innervation in furnishing the ductal system with acini to form a functional organ. Multiple signaling pathways such as those of the WNT and EGFR families have been reported to control epithelial differentiation and consequently tissue structure in a diverse range of organs. However, compared to neuronal signals that continuously operate during all stages of salivary gland (and organism) development, these cell/tissue intrinsic pathways tend to be transient in appearance, for example, FGF10 regulates acinar cell proliferation but is only expressed during early branching morphogenesis (Steinberg et al., 2005). As such, peripheral nerves offer a way of shaping tissue architecture throughout the development of the organism, thereby providing continuity of signals during tissue morphogenesis.

In summary, our findings support a critical role for SOX2 and nerves in establishing the secretory end units of the salivary gland, thereby selectively generating the tissue architecture necessary for organ function. Given the majority of mammalian organs receive innervation from parasympathetic ganglia, and loss of functional innervation alters epithelia and/or epithelial stem cells in multiple organs including those that express SOX2 such as taste buds, prostate and seminar vesicles, stomach and cornea (Suzuki, 2008; Tatsuta et al., 1985; Ueno et al., 2012; Wanigasekara et al., 2004; Zhao et al., 2014), our results may have significant implications for gaining insight into the molecular mechanisms underlying lineage specification and architectural outcomes in diverse systems.

1. 1. Andoniadou CL
   2. Matsushima D
   3. Mousavy Gharavy SN
   4. Signore M
   5. Mackintosh AI
   6. Schaeffer M
   7. Gaston-Massuet C
   8. Mollard P
   9. Jacques TS
   10. Le Tissier P
   11. Dattani MT
   12. Pevny LH
   13. Martinez-Barbera JP

   (2013) Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential

   Cell Stem Cell 13:433–445.

   https://doi.org/10.1016/j.stem.2013.07.004

   • PubMed
   • Google Scholar
2. 1. Arnold K
   2. Sarkar A
   3. Yram MA
   4. Polo JM
   5. Bronson R
   6. Sengupta S
   7. Seandel M
   8. Geijsen N
   9. Hochedlinger K

   (2011) Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice

   Cell Stem Cell 9:317–329.

   https://doi.org/10.1016/j.stem.2011.09.001

   • PubMed
   • Google Scholar
3. 1. Aure MH
   2. Konieczny SF
   3. Ovitt CE

   (2015) Salivary gland homeostasis is maintained through acinar cell self-duplication

   Developmental Cell 33:231–237.

   https://doi.org/10.1016/j.devcel.2015.02.013

   • PubMed
   • Google Scholar
4. 1. Boumahdi S
   2. Driessens G
   3. Lapouge G
   4. Rorive S
   5. Nassar D
   6. Le Mercier M
   7. Delatte B
   8. Caauwe A
   9. Lenglez S
   10. Nkusi E
   11. Brohée S
   12. Salmon I
   13. Dubois C
   14. del Marmol V
   15. Fuks F
   16. Beck B
   17. Blanpain C

   (2014) SOX2 controls tumour initiation and Cancer stem-cell functions in squamous-cell carcinoma

   Nature 511:246–250.

   https://doi.org/10.1038/nature13305

   • PubMed
   • Google Scholar
5. 1. Chou YT
   2. Lee CC
   3. Hsiao SH
   4. Lin SE
   5. Lin SC
   6. Chung CH
   7. Chung CH
   8. Kao YR
   9. Wang YH
   10. Chen CT
   11. Wei YH
   12. Wu CW

   (2013) The emerging role of SOX2 in cell proliferation and survival and its crosstalk with oncogenic signaling in lung Cancer

   Stem Cells 31:2607–2619.

   https://doi.org/10.1002/stem.1518

   • PubMed
   • Google Scholar
6. 1. Coughlin MD

   (1975) Early development of parasympathetic nerves in the mouse submandibular gland

   Developmental Biology 43:123–139.

   https://doi.org/10.1016/0012-1606(75)90136-0

   • PubMed
   • Google Scholar
7. 1. Driskell RR
   2. Giangreco A
   3. Jensen KB
   4. Mulder KW
   5. Watt FM

   (2009) Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis

   Development 136:2815–2823.

   https://doi.org/10.1242/dev.038620

   • PubMed
   • Google Scholar
8. 1. Goldsmith S
   2. Lovell-Badge R
   3. Rizzoti K

   (2016) SOX2 is sequentially required for progenitor proliferation and lineage specification in the developing pituitary

   Development 143:2376–2388.

   https://doi.org/10.1242/dev.137984

   • PubMed
   • Google Scholar
9. 1. Gontan C
   2. de Munck A
   3. Vermeij M
   4. Grosveld F
   5. Tibboel D
   6. Rottier R

   (2008) Sox2 is important for two crucial processes in lung development: branching morphogenesis and epithelial cell differentiation

   Developmental Biology 317:296–309.

   https://doi.org/10.1016/j.ydbio.2008.02.035

   • PubMed
   • Google Scholar
10. 1. Herreros-Villanueva M
    2. Zhang JS
    3. Koenig A
    4. Abel EV
    5. Smyrk TC
    6. Bamlet WR
    7. de Narvajas AA
    8. Gomez TS
    9. Simeone DM
    10. Bujanda L
    11. Billadeau DD

    (2013) SOX2 promotes dedifferentiation and imparts stem cell-like features to pancreatic Cancer cells

    Oncogenesis 2:e61.

    https://doi.org/10.1038/oncsis.2013.23

    • PubMed
    • Google Scholar
11. 1. Hoffman MP
    2. Kidder BL
    3. Steinberg ZL
    4. Lakhani S
    5. Ho S
    6. Kleinman HK
    7. Larsen M

    (2002) Gene expression profiles of mouse submandibular gland development: fgfr1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms

    Development 129:5767–5778.

    https://doi.org/10.1242/dev.00172

    • PubMed
    • Google Scholar
12. 1. Häärä O
    2. Koivisto T
    3. Miettinen PJ

    (2009) EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells

    Differentiation 77:298–306.

    https://doi.org/10.1016/j.diff.2008.10.006

    • PubMed
    • Google Scholar
13. 1. Jaskoll T
    2. Abichaker G
    3. Witcher D
    4. Sala FG
    5. Bellusci S
    6. Hajihosseini MK
    7. Melnick M

    (2005) FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis

    BMC Developmental Biology 5:11.

    https://doi.org/10.1186/1471-213X-5-11

    • PubMed
    • Google Scholar
14. 1. Jiménez E
    2. Pavía J
    3. Morell V
    4. Martín E
    5. Montiel M

    (2001) Muscarinic receptor subtypes and calcium signaling in Fischer rat thyroid cells

    Biochemical Pharmacology 61:337–342.

    https://doi.org/10.1016/S0006-2952(00)00565-7

    • PubMed
    • Google Scholar
15. 1. Knosp WM
    2. Knox SM
    3. Lombaert IM
    4. Haddox CL
    5. Patel VN
    6. Hoffman MP

    (2015) Submandibular parasympathetic gangliogenesis requires sprouty-dependent wnt signals from epithelial progenitors

    Developmental Cell 32:667–677.

    https://doi.org/10.1016/j.devcel.2015.01.023

    • PubMed
    • Google Scholar
16. 1. Knox SM
    2. Lombaert IM
    3. Reed X
    4. Vitale-Cross L
    5. Gutkind JS
    6. Hoffman MP

    (2010) Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis

    Science 329:1645–1647.

    https://doi.org/10.1126/science.1192046

    • PubMed
    • Google Scholar
17. 1. Knox SM
    2. Lombaert IM
    3. Haddox CL
    4. Abrams SR
    5. Cotrim A
    6. Wilson AJ
    7. Hoffman MP

    (2013) Parasympathetic stimulation improves epithelial organ regeneration

    Nature Communications 4:1494.

    https://doi.org/10.1038/ncomms2493

    • PubMed
    • Google Scholar
18. 1. Lesko MH
    2. Driskell RR
    3. Kretzschmar K
    4. Goldie SJ
    5. Watt FM

    (2013) Sox2 modulates the function of two distinct cell lineages in mouse skin

    Developmental Biology 382:15–26.

    https://doi.org/10.1016/j.ydbio.2013.08.004

    • PubMed
    • Google Scholar
19. 1. Lizama CO
    2. Hawkins JS
    3. Schmitt CE
    4. Bos FL
    5. Zape JP
    6. Cautivo KM
    7. Borges Pinto H
    8. Rhyner AM
    9. Yu H
    10. Donohoe ME
    11. Wythe JD
    12. Zovein AC

    (2015) Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition

    Nature Communications 6:7739.

    https://doi.org/10.1038/ncomms8739

    • PubMed
    • Google Scholar
20. 1. Lombaert IM
    2. Hoffman MP

    (2010) Epithelial stem/progenitor cells in the embryonic mouse submandibular gland

    Frontiers of Oral Biology 14:90–106.

    https://doi.org/10.1159/000313709

    • PubMed
    • Google Scholar
21. 1. Lombaert IM
    2. Knox SM
    3. Hoffman MP

    (2011) Salivary gland progenitor cell biology provides a rationale for therapeutic salivary gland regeneration

    Oral Diseases 17:445–449.

    https://doi.org/10.1111/j.1601-0825.2010.01783.x

    • PubMed
    • Google Scholar
22. 1. Lombaert IM
    2. Abrams SR
    3. Li L
    4. Eswarakumar VP
    5. Sethi AJ
    6. Witt RL
    7. Hoffman MP

    (2013) Combined KIT and FGFR2b signaling regulates epithelial progenitor expansion during organogenesis

    Stem Cell Reports 1:604–619.

    https://doi.org/10.1016/j.stemcr.2013.10.013

    • PubMed
    • Google Scholar
23. 1. Ma L
    2. Parkhurst A
    3. Jeffery WR

    (2014) The role of a Lens survival pathway including sox2 and αA-crystallin in the evolution of cavefish eye degeneration

    EvoDevo 5:28.

    https://doi.org/10.1186/2041-9139-5-28

    • PubMed
    • Google Scholar
24. 1. Matsumoto S
    2. Kurimoto T
    3. Taketo MM
    4. Fujii S
    5. Kikuchi A

    (2016) The WNT/MYB pathway suppresses KIT expression to control the timing of salivary proacinar differentiation and duct formation

    Development 143:2311–2324.

    https://doi.org/10.1242/dev.134486

    • PubMed
    • Google Scholar
25. 1. Mattingly A
    2. Finley JK
    3. Knox SM

    (2015) Salivary gland development and disease

    Wiley Interdisciplinary Reviews: Developmental Biology 4:573–590.

    https://doi.org/10.1002/wdev.194

    • PubMed
    • Google Scholar
26. Book

    1. Mead R

    (1988)

    The Design of Experiments

    Cambridge University Press.

    • Google Scholar
27. 1. Muench MO
    2. Suskind DL
    3. Bárcena A

    (2002) Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver

    Biological Procedures Online 4:10–23.

    https://doi.org/10.1251/bpo29

    • PubMed
    • Google Scholar
28. 1. Muzumdar MD
    2. Tasic B
    3. Miyamichi K
    4. Li L
    5. Luo L

    (2007) A global double-fluorescent cre reporter mouse

    Genesis 45:593–605.

    https://doi.org/10.1002/dvg.20335

    • PubMed
    • Google Scholar
29. 1. Nedvetsky PI
    2. Emmerson E
    3. Finley JK
    4. Ettinger A
    5. Cruz-Pacheco N
    6. Prochazka J
    7. Haddox CL
    8. Northrup E
    9. Hodges C
    10. Mostov KE
    11. Hoffman MP
    12. Knox SM

    (2014) Parasympathetic innervation regulates tubulogenesis in the developing salivary gland

    Developmental Cell 30:449–462.

    https://doi.org/10.1016/j.devcel.2014.06.012

    • PubMed
    • Google Scholar
30. 1. Nelson DA
    2. Manhardt C
    3. Kamath V
    4. Sui Y
    5. Santamaria-Pang A
    6. Can A
    7. Bello M
    8. Corwin A
    9. Dinn SR
    10. Lazare M
    11. Gervais EM
    12. Sequeira SJ
    13. Peters SB
    14. Ginty F
    15. Gerdes MJ
    16. Larsen M

    (2013) Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

    Biology Open 2:439–447.

    https://doi.org/10.1242/bio.20134309

    • PubMed
    • Google Scholar
31. 1. Okubo T
    2. Pevny LH
    3. Hogan BL

    (2006) Sox2 is required for development of taste bud sensory cells

    Genes & Development 20:2654–2659.

    https://doi.org/10.1101/gad.1457106

    • PubMed
    • Google Scholar
32. 1. Pattyn A
    2. Morin X
    3. Cremer H
    4. Goridis C
    5. Brunet JF

    (1999) The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives

    Nature 399:366–370.

    https://doi.org/10.1038/20700

    • PubMed
    • Google Scholar
33. 1. Pin CL
    2. Rukstalis JM
    3. Johnson C
    4. Konieczny SF

    (2001) The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity

    The Journal of Cell Biology 155:519–530.

    https://doi.org/10.1083/jcb.200105060

    • PubMed
    • Google Scholar
34. 1. Que J
    2. Okubo T
    3. Goldenring JR
    4. Nam KT
    5. Kurotani R
    6. Morrisey EE
    7. Taranova O
    8. Pevny LH
    9. Hogan BL

    (2007) Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm

    Development 134:2521–2531.

    https://doi.org/10.1242/dev.003855

    • PubMed
    • Google Scholar
35. 1. Que J
    2. Luo X
    3. Schwartz RJ
    4. Hogan BL

    (2009) Multiple roles for Sox2 in the developing and adult mouse Trachea

    Development 136:1899–1907.

    https://doi.org/10.1242/dev.034629

    • PubMed
    • Google Scholar
36. 1. Smith AN
    2. Miller LA
    3. Radice G
    4. Ashery-Padan R
    5. Lang RA

    (2009) Stage-dependent modes of Pax6-Sox2 epistasis regulate Lens development and eye morphogenesis

    Development 136:2977–2985.

    https://doi.org/10.1242/dev.037341

    • PubMed
    • Google Scholar
37. 1. Steinberg Z
    2. Myers C
    3. Heim VM
    4. Lathrop CA
    5. Rebustini IT
    6. Stewart JS
    7. Larsen M
    8. Hoffman MP

    (2005) FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

    Development 132:1223–1234.

    https://doi.org/10.1242/dev.01690

    • PubMed
    • Google Scholar
38. 1. Suzuki Y

    (2008) Expression of Sox2 in mouse taste buds and its relation to innervation

    Cell and Tissue Research 332:393–401.

    https://doi.org/10.1007/s00441-008-0600-1

    • PubMed
    • Google Scholar
39. 1. Tatsuta M
    2. Yamamura H
    3. Iishi H
    4. Ichii M
    5. Noguchi S
    6. Baba M
    7. Taniguchi H

    (1985)

    Promotion by vagotomy of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

    Cancer Research 45:194–197.

    • PubMed
    • Google Scholar
40. 1. Teshima TH
    2. Ianez RF
    3. Coutinho-Camillo CM
    4. Buim ME
    5. Soares FA
    6. Lourenço SV

    (2011) Development of human minor salivary glands: expression of mucins according to stage of morphogenesis

    Journal of Anatomy 219:410–417.

    https://doi.org/10.1111/j.1469-7580.2011.01405.x

    • PubMed
    • Google Scholar
41. 1. Ueno H
    2. Ferrari G
    3. Hattori T
    4. Saban DR
    5. Katikireddy KR
    6. Chauhan SK
    7. Dana R

    (2012) Dependence of corneal stem/progenitor cells on ocular surface innervation

    Investigative Opthalmology & Visual Science 53:867–872.

    https://doi.org/10.1167/iovs.11-8438

    • PubMed
    • Google Scholar
42. 1. Vasioukhin V
    2. Degenstein L
    3. Wise B
    4. Fuchs E

    (1999) The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin

    PNAS 96:8551–8556.

    https://doi.org/10.1073/pnas.96.15.8551

    • PubMed
    • Google Scholar
43. 1. Walker JL
    2. Menko AS
    3. Khalil S
    4. Rebustini I
    5. Hoffman MP
    6. Kreidberg JA
    7. Kukuruzinska MA

    (2008) Diverse roles of E-cadherin in the morphogenesis of the submandibular gland: insights into the formation of acinar and ductal structures

    Developmental Dynamics 237:3128–3141.

    https://doi.org/10.1002/dvdy.21717

    • PubMed
    • Google Scholar
44. 1. Wanigasekara Y
    2. Airaksinen MS
    3. Heuckeroth RO
    4. Milbrandt J
    5. Keast JR

    (2004) Neurturin signalling via GFRalpha2 is essential for innervation of glandular but not muscle targets of sacral parasympathetic ganglion neurons

    Molecular and Cellular Neuroscience 25:288–300.

    https://doi.org/10.1016/j.mcn.2003.10.019

    • PubMed
    • Google Scholar
45. 1. Xiao Y
    2. Thoresen DT
    3. Williams JS
    4. Wang C
    5. Perna J
    6. Petrova R
    7. Brownell I

    (2015) Neural hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium

    PNAS 112:7195–7200.

    https://doi.org/10.1073/pnas.1504177112

    • PubMed
    • Google Scholar
46. 1. Zhao CM
    2. Hayakawa Y
    3. Kodama Y
    4. Muthupalani S
    5. Westphalen CB
    6. Andersen GT
    7. Flatberg A
    8. Johannessen H
    9. Friedman RA
    10. Renz BW
    11. Sandvik AK
    12. Beisvag V
    13. Tomita H
    14. Hara A
    15. Quante M
    16. Li Z
    17. Gershon MD
    18. Kaneko K
    19. Fox JG
    20. Wang TC
    21. Chen D

    (2014) Denervation suppresses gastric tumorigenesis

    Science Translational Medicine 6:ra115.

    https://doi.org/10.1126/scitranslmed.3009569

    • PubMed
    • Google Scholar

Author details

1. Elaine Emmerson

   Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Present address

   The MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, United Kingdom

   Contribution

   EE, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-5902-3368

2. Alison J May

   Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   AJM, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Sara Nathan

   Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   SN, Formal analysis, Validation, Investigation

   Competing interests

   The authors declare that no competing interests exist.

4. Noel Cruz-Pacheco

   Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   NC-P, Data curation, Formal analysis, Validation

   Competing interests

   The authors declare that no competing interests exist.

5. Carlos O Lizama

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   COL, Data curation, Formal analysis, Methodology

   Competing interests

   The authors declare that no competing interests exist.

6. Lenka Maliskova

   Institute of Human Genetics, University of California, San Francisco, San Francisco, United States

   Contribution

   LM, Formal analysis, Methodology

   Competing interests

   The authors declare that no competing interests exist.

7. Ann C Zovein

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   ACZ, Resources, Formal analysis, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

8. Yin Shen

   Institute of Human Genetics, University of California, San Francisco, San Francisco, United States

   Contribution

   YS, Resources, Methodology

   Competing interests

   The authors declare that no competing interests exist.

9. Marcus O Muench

   Blood Systems Research Institute, San Francisco, United States

   Contribution

   MOM, Resources, Formal analysis, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-8946-6605

10. Sarah M Knox

    Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

    Contribution

    SMK, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    sarah.knox@ucsf.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-7567-083X

• 4,425

  views

• 609

  downloads

• 83

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.