Salivary glands regenerate after radiation injury through SOX2‐mediated secretory cell replacement

Research Article15 January 2018Open Access Transparent process Salivary glands regenerate after radiation injury through SOX2-mediated secretory cell replacement Elaine Emmerson Elaine Emmerson orcid.org/0000-0002-5902-3368 Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Alison J May Alison J May Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Lionel Berthoin Lionel Berthoin Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Noel Cruz-Pacheco Noel Cruz-Pacheco Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Sara Nathan Sara Nathan Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Aaron J Mattingly Aaron J Mattingly Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Jolie L Chang Jolie L Chang Department of Otolaryngology, University of California, San Francisco, CA, USA Search for more papers by this author William R Ryan William R Ryan Department of Otolaryngology, University of California, San Francisco, CA, USA Search for more papers by this author Aaron D Tward Aaron D Tward orcid.org/0000-0003-4868-8732 Department of Otolaryngology, University of California, San Francisco, CA, USA Search for more papers by this author Sarah M Knox Corresponding Author Sarah M Knox [email protected] orcid.org/0000-0002-7567-083X Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Elaine Emmerson Elaine Emmerson orcid.org/0000-0002-5902-3368 Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Alison J May Alison J May Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Lionel Berthoin Lionel Berthoin Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Noel Cruz-Pacheco Noel Cruz-Pacheco Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Sara Nathan Sara Nathan Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Aaron J Mattingly Aaron J Mattingly Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Jolie L Chang Jolie L Chang Department of Otolaryngology, University of California, San Francisco, CA, USA Search for more papers by this author William R Ryan William R Ryan Department of Otolaryngology, University of California, San Francisco, CA, USA Search for more papers by this author Aaron D Tward Aaron D Tward orcid.org/0000-0003-4868-8732 Department of Otolaryngology, University of California, San Francisco, CA, USA Search for more papers by this author Sarah M Knox Corresponding Author Sarah M Knox [email protected] orcid.org/0000-0002-7567-083X Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Author Information Elaine Emmerson1,3,‡, Alison J May1,‡, Lionel Berthoin1, Noel Cruz-Pacheco1, Sara Nathan1, Aaron J Mattingly1, Jolie L Chang2, William R Ryan2, Aaron D Tward2 and Sarah M Knox *,1 1Program in Craniofacial Biology, Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA 2Department of Otolaryngology, University of California, San Francisco, CA, USA 3Present address: The MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +1 415 502 0811; E-mail: [email protected] EMBO Mol Med (2018)10:e8051https://doi.org/10.15252/emmm.201708051 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Salivary gland acinar cells are routinely destroyed during radiation treatment for head and neck cancer that results in a lifetime of hyposalivation and co-morbidities. A potential regenerative strategy for replacing injured tissue is the reactivation of endogenous stem cells by targeted therapeutics. However, the identity of these cells, whether they are capable of regenerating the tissue, and the mechanisms by which they are regulated are unknown. Using in vivo and ex vivo models, in combination with genetic lineage tracing and human tissue, we discover a SOX2+ stem cell population essential to acinar cell maintenance that is capable of replenishing acini after radiation. Furthermore, we show that acinar cell replacement is nerve dependent and that addition of a muscarinic mimetic is sufficient to drive regeneration. Moreover, we show that SOX2 is diminished in irradiated human salivary gland, along with parasympathetic nerves, suggesting that tissue degeneration is due to loss of progenitors and their regulators. Thus, we establish a new paradigm that salivary glands can regenerate after genotoxic shock and do so through a SOX2 nerve-dependent mechanism. Synopsis Salivary glands regenerate after radiation injury through SOX2-mediated secretory acinar cell replacement as shown using genetic lineage tracing and ablation methods, in combination with in vivo and ex vivo gamma radiation-induced damage models. SOX2+ stem cells are essential to acinar cell replacement in the sublingual gland (SLG) during homeostasis and after radiation-induced damage. SOX2-mediated acinar cell replacement is contingent on neuronal muscarinic signalling. In the absence of nerves, a muscarinic mimetic can drive SOX2-mediated regeneration. SOX2 function is essential for SLG regeneration following radiation-induced injury. SOX2 along with parasympathetic nerves are diminished in human salivary gland biopsies following irradiation therapy and SOX2 and the acinar lineage are upregulated in response to muscarinic activation. Introduction Therapeutic radiation continues to be a life-saving treatment for cancer patients and is utilized for a spectrum of malignancies including those of the head and neck. Indeed, the vast majority of patients suffering head and neck cancer will receive radiotherapy in addition to chemotherapy and surgery (~60,000 new patients per year in US; Siegel et al, 2015). Although this combination treatment is highly efficacious in eliminating tumors, a severe side effect is damage and/or destruction of healthy tissue lying in the field of radiation. Such organs include the salivary glands, which exhibit tissue dysfunction even after low doses of radiation (Grundmann et al, 2009). At the higher doses routinely given to patients (60 Gy), off-target radiation destroys saliva-synthesizing acinar cells (Sullivan et al, 2005; Redman, 2008) and results in a lifetime of dry mouth and co-morbidities (e.g., tooth decay, oral infections, poor wound healing (Brown et al, 1975; Dreizen et al, 1977; Dusek et al, 1996). Although there has been success with intensity modulated radiation to spare one of the three major salivary glands (parotid), the proximity of the glands to the tumor sites often prevents application of this technique, leaving 80% of head and neck cancer patients with dry mouth syndrome (Lee & Le, 2008). As with all other organs damaged by radiation, including the lungs, heart, and bladder (Emami et al, 1991), there are few, if any, treatments available to improve or restore tissue function. Current treatment options for cancer survivors suffering radiation-induced salivary dysfunction and degeneration focus on short-term relief from the symptoms, but no long-term restorative therapies are available. Regenerative strategies such as reactivating endogenous stem cells or transplanting non-irradiated stem cells have been proposed (Lombaert et al, 2008; Ogawa et al, 2013; Pringle et al, 2016). However, these applications are curtailed by the dearth of knowledge regarding the identity of adult salivary progenitor cells that contribute to acini under homeostatic or injury conditions. Although it was recently proposed that acinar cells are derived through self-duplication rather than from defined progenitors (Aure et al, 2015), an analysis of subpopulations of these cells for progenitor-like activity was not performed. It also remains to be determined whether acinar cells, either through self-duplication or through progenitor cell expansion, are capable of repopulating cells after genotoxic damage. Although a plethora of studies have utilized irradiated salivary glands as a model of degeneration (Zeilstra et al, 2000; Coppes et al, 2001, 2002), the regenerative capacity of adult salivary cells damaged by radiation has not been investigated in vivo. How acinar cells are replaced during salivary gland homeostasis is also poorly understood. Studies in adult organs over the last 150 years have clearly shown that peripheral nerves are essential for the maintenance of organ and tissue integrity (Erb, 1868). Skeletal muscle atrophies in the absence of stimulation by motor neurons (Fu & Gordon, 1995; Batt & Bain, 2013) and epithelial organs such as fungiform taste buds (Von Vintschgau & Honigschmied, 1877), prostate (Wang et al, 1991; Lujan et al, 1998) and the salivary gland degenerate after ablation of sensory and/or autonomic nerves (Schneyer & Hall, 1967; Mandour et al, 1977; Kang et al, 2010). Although it is unclear how nerves control tissue homeostasis for these organs, studies in skin indicate sensory nerves, through sonic hedgehog secretion, promote the self-renewal of adult epithelial stem cells and consequently the maintenance of the downstream cell lineage, that is, dome cells in the skin (Peterson et al, 2015; Xiao et al, 2015). In addition, studies in the salamander (Wallace, 1972) and embryonic salivary gland (Knox et al, 2013) suggest that peripheral nerves have the capacity to regenerate tissue via activation of multipotent stem cells, but evidence for this in the adult mammalian system is lacking. Using a combination of mouse genetics, ex vivo cultures, and human tissue explants, we unexpectedly discover that salivary acini are capable of regenerating after radiation and do so in response to cholinergic activation through a progenitor cell-dependent mechanism. We show that SOX2 marks the sole progenitor for the acinar lineage that can replace acinar cells during homeostasis and after radiation-induced injury, indicating that salivary progenitors can withstand, at least in the short term, genotoxic shock. Importantly, treatment of healthy and irradiated tissue with cholinergic mimetics stimulated acinar cell replenishment. Thus, our data reveal the extensive regenerative capacity of the tissue even under genotoxic shock and suggest that targeting of SOX2+ cells might be a therapeutic approach to regenerate tissue damaged by radiation therapy. Results SOX2 marks a progenitor cell that gives rise to acinar but not duct cells during salivary gland homeostasis SOX2 has been established as a progenitor cell marker in the fetal mouse submandibular and sublingual salivary glands, but whether SOX2+ cells in the adult tissue also produce acinar and duct cells is unclear (Arnold et al, 2011; Emmerson et al, 2017). Furthermore, whether these cells are also present in adult human salivary glands is not known. We found SOX2 to be expressed by a subset of acinar cells in all three of the major adult human salivary glands [Fig 1A, submandibular gland (SMG), sublingual gland (SLG), parotid gland (PG)]. In the mouse, SOX2 protein was restricted to the adult murine SLG (absent from the SMG and PG, Figs 1B and EV1A) where it was expressed by undifferentiated aquaporin (AQP)5-positive, mucin (MUC)19-negative acinar cells (21 ± 4% of all AQP5+ acinar cells; Fig 1C and D). Consistent with their potential role as a progenitor cell, ~6% of SOX2+AQP5+ cells co-expressed Ki67 (Figs 1E and EV1B) while 19 ± 4% were in the cell cycle (CyclinD1+; Fig EV1C). To determine whether SOX2+ cells contributed to acinar and duct lineages, we performed genetic lineage tracing using Sox2CreERT2 mice (Arnold et al, 2011) crossed to a Rosa26mTmG reporter strain. The Rosa26mTmG mouse is a double-fluorescent reporter which when crossed with a Cre line expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision (Muzumdar et al, 2007; Fig EV1D). As such, lineage-traced cells will express mG. As shown in Fig 1F, SOX2+ cells self-renew and produce differentiated acinar cells marked by AQP5 and MUC19 but not KRT8+ duct cells after 14 or 30 days (Figs 1F and EV1E). Thus, our lineage tracing results indicate that SOX2+ cells are lineage-restricted progenitor cells that give rise to differentiated progeny, similar to what has been observed in the epidermis, intestine, and incisor (Owens & Watt, 2003; Barker, 2014; Seidel et al, 2017). Figure 1. SOX2 marks a progenitor cell that gives rise to acinar but not duct cells in the adult salivary gland Representative image of adult human submandibular (SMG), sublingual (SLG), and parotid (PG) salivary gland (non-IR, 28–33 years) immunostained for SOX2, epithelia (E-cadherin; ECAD) or CD44, and nuclei. Single arrows indicate SOX2 expressing acinar cells. Scale bar is 20 μm. Wild-type murine SMG and SLG stained for SOX2, ECAD, and nuclei. Arrowheads indicate SOX2 expressing cells. Scale bar is 50 μm. Yellow dashed line denotes border between SMG and SLG. Sox2eGFP sublingual salivary glands (SLG) were immunostained for GFP and differentiated acinar marker mucin 19 (MUC19). White dashed lines outline Sox2eGFP+ MUC19(−) cells. Scale bar = 20 μm. AQP5+SOX2+ cells as a percentage of total AQP5+ acinar cells. SLG immunostained for SOX2, Ki67, and epithelial marker E-cadherin (ECAD). White arrow indicates proliferating Ki67+SOX2+ cell. White lines outline individual cells and nuclei. Scale bar = 10 μm. Representative images of Sox2 lineage-traced SLG. Recombination was induced in Sox2CreERT2;Rosa26mTmG mice and salivary gland traced for 24 h and 30 days before immunostaining for SOX2, acinar markers AQP5 and MUC19, and ductal marker KRT8. * indicates MUC19(−) Sox2CreERT2GFP(+) cells. Scale bars = 30 μm. mT = membrane-bound Tomato. Data information: Cells quantified in (D) were counted from three non-consecutive sections of n = 5 female adult SLGs. Data are presented as mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. SOX2 marks a subset of acinar cells that replenish acini Wild-type murine PG stained for SOX2, ECAD, and nuclei. Scale bar is 50 μm. The percentage of acinar SOX2+ and SOX2− cells that are Ki67+ were counted using FACS and shown as a percentage of total AQP5+SOX2+ or AQP5+SOX2− cells. The % of SOX2+ acinar cells that are either CyclinD1+ or CyclinD1−. Schematic of Rosa26mTmG Cre-mediated gene excision (adapted from Muzumdar et al, 2007). Representative image of Sox2 lineage-traced SLG. Cre-mediated recombination was induced in Sox2CreERT2;Rosa26mTmG mice and SLG analyzed 14 or 30 days later by immunostaining for SOX2. Scale bar = 25 μm. Representative images of Kit lineage-traced SLG and SMG. Cre-mediated recombination was induced in KitCreERT2;Rosa26mTmG mice and SMG/SLG analyzed 14 days and 6 months later. Tissue was stained with AQP5 to mark acinar cells and KRT8 to mark intercalated duct cells. Scale bar = 25 μm. mT = membrane-bound Tomato. Data information: Data in (B), SLG were pooled from n = 2 mice (85,000 events). Data in (C) were calculated from three non-consecutive fluorescent sections of each SLG from n = 3 mice with individual values plotted. Error bars show mean ± SD. Download figure Download PowerPoint Given KIT+ cells, which reside primarily in the intercalated ducts of the SLG and SMG (Andreadis et al, 2006; Nelson et al, 2013), have previously been proposed to give rise to acinar cells in adult tissue (Lombaert et al, 2008; Nanduri et al, 2013, 2014; Pringle et al, 2016), we genetically traced these cells using the KitCreERT2 promoter crossed to the Rosa26mTmG reporter at 6 weeks of age. However, no KIT+ cell-derived acinar cells (i.e., double positive for AQP5 and mG) were evident in either the SLG or SMG at 14 days or 6 months after induction (Fig EV1F). Instead, KIT+ cells contributed exclusively to the intercalated ducts in the SLG (as can be observed by co-staining for the intercalated duct marker KRT8) and intercalated and larger ducts in the SMG. Thus, these data indicate that KIT+ cells are progenitors for the ductal and SOX2+ cells for the acinar lineage. SOX2 and SOX2+ cells are essential for production of secretory acini Our lineage tracing analysis confirmed that SOX2+ cells give rise to acinar but not duct cells. However, as we also observed the presence of Ki67+SOX2− acinar cells (~6% SOX2+Ki67+ and 16.5% SOX2-Ki67+ cells, Fig EV1B), suggestive of an alternative progenitor cell or a transit-amplifying cell for the acinar lineage, we investigated the requirement of SOX2 and SOX2+ cells in SLG maintenance and repair by genetically removing Sox2 in SOX2+ cells using Sox2CreERT2; Sox2fl/fl mice (Fig 2A and C) or ablating SOX2+ cells using diphtheria toxin (DTA) expressed under the control of the inducible Sox2 promoter (Sox2CreERT2;Rosa26DTA; Fig 2B and D). In the latter assay, SOX2+ cells undergo cell death in response to intracellular production of DTA. Ablation of Sox2 from SOX2+ cells or elimination of SOX2+ cells via DTA severely depleted SOX2+ and AQP5+ cells but not KRT8+ ductal cells indicating Sox2 and SOX2+ cells were necessary for maintaining functional acini (Fig 2A–D; efficiency of Sox2 or SOX2+ cell ablation is shown in Fig EV2A). In the absence of Sox2, acinar but not ductal cells exited the cell cycle, as shown by the decrease in cyclin D1 (CCND1)+ acinar cells (Fig EV2D; arrowheads indicate CCND1+ cells and dotted white lines highlight ductal cells). Furthermore, ablation of SOX2+ cells resulted in few remaining acini by 8 days (Figs 2B and D, and EV2A), as shown by large regions of the ductal network completely devoid of AQP5+ cells (ducts are marked by dashed lines or KRT8 in Fig 2B). To exclude the possibility that tissue degeneration was solely due to destabilization of the tissue rather than loss of acinar cell replacement, we examined SLG after a short-term ablation. As shown in Appendix Fig S1, at day 4 or 5 (3 or 4 days of tamoxifen treatment), few SOX2+ cells remained in the gland of both the Sox2CreERT2; Sox2fl/fl and Sox2CreERT2;Rosa26DTA SLG (Appendix Fig S1A and B) and Sox2 transcripts were substantially reduced (Appendix Fig S1B). However, acini were present albeit disorganized and atrophic in appearance. Furthermore, we did not observe an increase in SOX2+ cells (or Sox2 transcripts), indicating that SOX2 is not ectopically expressed in acinar cells in response to tissue damage. We also determined whether alterations in tissue composition were due to reduced innervation, an essential regulator of tissue function. However, we measured similar innervation in Sox2CreERT2; Sox2fl/fl SLG to wild-type controls and a significant increase in axon bundles in Sox2CreERT2; Rosa26DTA SLG (Fig EV2B and C). The latter finding suggests ablation of cells triggers the release of factors that promote innervation but that, even with increased innervation, regeneration is not possible without SOX2+ cells. In sum, these results indicate that SOX2+ cells, at least under the conditions tested, are the sole acinar progenitors in the SLG and that acini do not arise from the self-duplication of fully differentiated acinar cells, as suggested previously (Aure et al, 2015). Similar to studies in the epidermis, intestine, and incisor (Owens & Watt, 2003; Barker, 2014; Seidel et al, 2017), our data also suggest the presence of a transit-amplifying population derived from SOX2+ cells that may be involved in rapidly repopulating the acinar compartment. Figure 2. SOX2 and SOX2+ cells are essential for the replenishment of salivary acinar cells A, B. Sox2 or SOX2+ cells were ablated in SLG of Sox2CreERT2;Sox2fl/fl;Rosa26mTmG/+ mice (A; see schematic) or Sox2CreERT2Rosa26DTA;Rosa26mTmG/+ mice (B; see schematic). Sections were immunostained for AQP5, KRT8, or ECAD and nuclei. Scale bars = 50 μm. Dashed white lines outline ducts. n = 3 per genotype. C, D. Quantification of ductal area in Sox2CreERT2;Sox2fl/fl (C) or Sox2CreERT2Rosa26DTA;Rosa26mTmG/+ SLG (D) expressed as a percentage of total epithelial area. In (C), right graph, the number of SOX2+ cells in Sox2CreERT2;Sox2fl/fl SLG expressed as a percentage of total cells. In (D), right graph, the total number of KRT8+ ductal, AQP5+ and SOX2+ acinar cells in wild-type and Sox2CreERT2Rosa26DTA;Rosa26mTmG/+ mice was counted. Data information: Calculations of cell numbers/duct areas were performed on three non-consecutive fluorescent sections of each SLG from n = 3 mice/genotype. Data in (C and D) (n = 3) are means + SD and were analyzed by Student's t-test. In (C), *P = 0.011 and **P = 0.0041, and in (D), left graph **P = 0.0015 and right graph ***P = 0.0007 and **P = 0.0018. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Ablation of Sox2 or SOX2+ cells reduces acinar cell replacement despite the presence of nerves A–C. Sox2 or SOX2+ cells were ablated in SLG of Sox2CreERT2; Sox2fl/fl; Rosa26mTmG/+ mice (Fig 2A; see schematic) or Sox2CreERT2; Rosa26DTA; Rosa26mTmG/+ mice (Fig 2B; see schematic). (A, B) Sections of WT, Sox2CreERT2;Sox2fl/fl, and Sox2CreERT2;Rosa26DTA SLG were immunostained for SOX2 or TUBB3 and nuclei. White arrowheads indicate SOX2+ cells. White dotted square is magnified in the image to the right to highlight that there are few SOX2+ cells remaining in tissue and that non-nuclear (green) staining is suggestive of debris. Scale bar = 50 μm. (C) Raw integrated density of nerves was calculated using ImageJ. D. WT or Sox2CreERT2;Sox2fl/fl SLG immunostained for cyclin D1 (CCND1) and nuclei. Dashed lines = ducts; arrowheads = CCND1+ acinar cells. Scale bar = 50 μm. Data information: Data in (C), WT n = 4, Sox2fl/fl n = 4, DTA n = 3. Individual values were plotted, as means + SD, and data were analyzed using a one-way analysis of variance with post hoc Dunnett's test. **P = 0.0091. Data in (D) are a representative image from n = 4 mice. Download figure Download PowerPoint Parasympathetic nerves preserve SOX2+ progenitors and promote SOX2-mediated acinar cell replacement Adult murine and human salivary glands atrophy after removal of parasympathetic activity. However, the effect of denervation on acinar cell replacement and progenitor cells has not been investigated (Garrett et al, 1999; Raz et al, 2013). To this end, we denervated one of the two pairs of murine SLGs by transecting the chorda tympani (Fig 3A; contralateral glands were used as internal controls). After 7 days, transcript levels of neuronal genes Tubb3, Vip, and Vacht (Fig 3B, red bars) and GFRα2+ or TUBB3+ nerves (Figs 3C and EV3C) were severely reduced, indicating successful denervation. We did not observe a concurrent loss of the cholinergic muscarinic receptors Chrm1 and Chrm3 transcripts (Fig 3B, red bars); however, it is possible that in the absence of parasympathetic innervation a compensatory mechanism may maintain Chrm1 and Chrm3 transcription. Although the SLG is predominately served by the parasympathetic branch with very little sympathetic innervation in comparison (Emmelin et al, 1965), we did observe a reduction in sympathetic innervation following chorda tympani transection (Fig EV3A). As such, although the levels of sympathetic nerves are minor, we cannot rule out that some of the effects of denervation may be due to a loss of sympathetic input. Figure 3. Parasympathetic nerves are necessary for maintaining SOX2+ cells and promoting SOX2-mediated acinar cell replacement A. Schematic shows time course of denervation and location of chorda tympani (CT) in adult mice. B. Gene expression (qPCR) analysis of intact (uninjured contralateral gland) and nerve transected SLG 7 and 30 days (D7 or D30) after surgery. Gene expression was normalized to Rsp18 and intact controls for each time point. C–F. Control and nerve transected SLG were immunostained 7 days after denervation for nerves (GFRα2), acinar cells (AQP5 and MIST1), ductal cells (KRT8), and epithelial cells (ECAD). The number of SOX2+, AQP5+, MIST1+, KRT8+, and KRT5+ cells in control and transected SLG was counted and represented as a percentage of the number of cells in control SLG (F). Scale bars in (C, D and E) = 25 μm. G, H. Recombination was induced in Sox2CreERT2;Rosa26mTmG mice 3 days after nerve transection and SLG traced for 11 days before being immunostained for TUBB3. The percentage of GFP+ and mT+ acinar cells in control and transected glands are shown in (G). Scale bar in (H) = 25 μm. Data information: Data in (B) (n = 5) are means + SEM and were analyzed using a one-way analysis of variance with a post hoc Dunnett's test. Sox2 (D7) *P = 0.0455, Tubb3 (D7) **P = 0.0082, Tubb3 (D30) **P = 0.0091, Vip (D7) **P = 0.0098, Vip (D30) **P = 0.0063, Vacht (D7) **P = 0.0071, Muc19 (D7) *P = 0.0419, Aqp5 (D7) *P = 0.0468. Data in (F and G) were calculated from three non-consecutive fluorescent sections of each SLG from n = 5 mice/group or genotype, are means + SD, and were analyzed by Student's t-test. SOX2+ *P = 0.0197, AQP5+ *P = 0.0106, %GFP+ ***P = 0.0000096, %mT+ ***P = 0.0000096. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Transection of the chorda tympani depletes acinar cells at 7 days A–G. (A, C, D, G) Control and nerve transected SLG were immunostained 7 days after denervation for tyrosine hydroxylase (TH; A), SOX2 and TUBB3 (C), KRT5 (D), caspase-3 (CASP3; G), epithelial cells (ECAD), and nuclei. Quantification of the size of acinar cells (B) in adult wild-type (WT) SLG with intact or transected chorda tympani (CT) 7 days after denervation. Scale bars in (A, C, D, G) = 25 μm. (E, F) Recombination was induced in Sox2CreERT2;Rosa26mTmG mice 24 h before nerve transection and SLG traced for 15 days before being immunostained for TUBB3. The percentage of GFP+ and mT+ acinar cells in control and transected glands are shown in (F). Scale bar in (E) = 25 μm. (H) Fold change in expression of genes involved in cell cycle and apoptosis 7 days after denervation, compared to intact control. Dashed line denotes the intact control. Data information: Data in (B, F, and H) n = 5. Data in (B) are a box and whisker plot of n = 5 mice, showing means (horizontal line), upper and lower quartiles (box) and upper and lower values (whiskers) and were analyzed using Student's t-test. ***P = 0.00000347. Data in (F) are means + SD and were analyzed by Student's t-test. %GFP+ ***P = 0.0000208, %mT+ ***P = 0.0000208. Data in (H) were normalized to Rsp18 and the intact control (dashed line). Ccnd1 *P = 0.0477. Download figure Download PowerPoint Similar to the effect of radiation therapy on tissue structure (Sullivan et al, 2005; Redman, 2008) adult acinar cells, as well as SOX2+ progenitors, were more sensitive to the loss of innervation than ducts. Denervation resulted in reduced acinar cell size (as observed previously; Patterson et al, 1975; Fig EV3B) decreased AQP5 protein and transcript levels of the differentiated acinar cell marker Muc19 (Fig 3B and D). Interestingly, transcript and protein levels of MIST1 were unchanged following denervation (Fig 3B, E and F), suggesting that while functional markers of acinar cells are disrupted in the absence of innervation, acinar cell identity is not adversely affected. Strikingly, SOX2+ cells lose expression of Sox2 (demonstrated using the Sox2eGFP mouse) and the levels of SOX2 protein and transcript were greatly reduced (Figs 3B, C and F, and EV3C), indicating SOX2 maintenance requires innervation. To determine whether SOX2+ cells remained capable of repopulating the tissue after denervation, we performed genetic lineage tracing where Cre driven by the endogenous Sox2 promoter (Sox2CreERT2;Rosa26mTmG) was activated 3 days after denervation and traced until day 14. As shown in Fig 3G and H, acinar cell replacement by SOX2+ progenitors was significantly reduced (~50%) 14 days after transection. Similarly, in SLG in which recombination was induced before nerve transection (tamoxifen 1 day prior to transection), acinar cell replacement by SOX2+ progenitors was sign