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. Author manuscript; available in PMC: 2019 Aug 7.
Published in final edited form as: Cell Rep. 2018 Aug 7;24(6):1464–1470.e3. doi: 10.1016/j.celrep.2018.07.016

Limited regeneration of adult salivary glands after severe injury involves cellular plasticity

Pei-Lun Weng 1, Marit H Aure 2,4, Takamitsu Maruyama 2, Catherine E Ovitt 2,3,5,*
PMCID: PMC6350767  NIHMSID: NIHMS1000329  PMID: 30089258

SUMMARY

In the adult salivary glands, the origin of replacement and regenerated acinar cells remains unclear. Although many reports describe the identification of stem cells in adult salivary glands, we have shown that differentiated acinar cells can be maintained and regenerated through self-duplication. Here, we have used genetic mouse models to further investigate acinar cell replacement and regeneration during homeostasis and after injury. Under normal conditions or after duct ligation, replacement of duct and acinar cells occurs through lineage-restricted progenitors. In contrast, after irradiation, in vivo lineage tracing shows that acinar, as well as duct cells, contribute to acinar cell regeneration, revealing that cellular plasticity is involved in salivary gland repair. Our results also indicate that even after radiation damage, several cell populations have regenerative potential for restoring salivary gland function.

INTRODUCTION

Severe loss of salivary gland function is a side effect of radiation therapy used to treat head and neck cancer patients. Decreased saliva secretion leads to dry mouth, tooth decay, oral infection, and impaired taste and speech (Vissink et al., 2010). Radiation-induced dry mouth results from the gradual loss of secretory acinar cells (Vissink et al., 2010).

The promise of stem cells for regenerative therapy has spurred an intense hunt for cells in the salivary gland with the capacity to replace secretory acinar cells lost due to radiation. Several cell populations in the adult salivary glands have been designated as stem cells based on various criteria (Pringle et al., 2013), such as the expression of general stem cell markers. Keratin 5 (K5) marks stem cells in several tissues (Leung et al., 2007; Rock et al., 2009) and is expressed by embryonic salivary gland progenitors (Knox et al., 2010). In the adult salivary gland, K5 is co-expressed with Keratin 14 (K14) in both intercalated and excretory duct cells (Kwak et al., 2016). A population of intercalated duct cells also expresses Axin2 (Hai et al., 2010), a direct downstream target of Wnt signaling (Clevers et al., 2014). Axin2 reporter mice were used to show that Wnt-responsive cells in various tissues are stem cells (Clevers et al., 2014). Salivary gland excretory duct cells have been shown to be Wnt responsive in vivo (Maimets et al., 2016). Both K5- and Wnt-responsive duct cells have been proposed as bipotent stem cells capable of generating both duct and acinar cells (Knox et al., 2010; Maimets et al., 2016).

Under normal homeostasis, we recently showed that acinar cells are replenished by self-duplication and following duct ligation injury, surviving acinar cells proliferate to regenerate the gland (Aure et al., 2015b). Additionally, K14-expressing duct cells contribute only to ducts but not to acinar cells under normal conditions in the adult mouse submandibular gland (SMG) (Kwak et al., 2016). In contrast to the stem cell proposal, these results suggest that the duct and acinar cell lineages are maintained separately, as has been established in other tissues with relatively slow turnover (Kopp et al., 2016).

To investigate the mechanism of endogenous cell replacement, we have tracked the contributions of duct and acinar cells to normal salivary gland turnover and to regeneration after injury. In vivo fate mapping shows that intercalated duct cells labeled by K5 or by Axin2 are fate-restricted ductal progenitor cells. However, after irradiation, acinar cell regeneration is traced to both acinar and duct cells. Our data demonstrate that cellular plasticity contributes to endogenous regeneration in injured salivary glands.

RESULTS

K5+ cells are duct progenitors under homeostatic conditions

In adult salivary glands, K5 expression is restricted to intercalated and basal excretory ducts, and myoepithelial cells. To investigate the lineage potential of K5-expressing intercalated duct cells, we crossed the KRT5CreER mouse, carrying a tamoxifen-inducible Cre recombinase (CreER), with the R26tdTomato reporter strain. In KRT5CreER;R26tdTomato mice, persistent and heritable tdTomato (RFP) expression is induced exclusively in K5-expressing cells by Cre-mediated removal of a transcriptional stop sequence. Due to the sexual dimorphism of murine salivary glands, analysis was performed in both sexes. Adult KRT5CreER;R26tdTomato mice (6-8 weeks) were given a single tamoxifen dose and the fate of K5+ cells was examined under homeostatic conditions (Figures 1A-1B). Three days after tamoxifen induction, RFP expression was observed in intercalated, and basal excretory duct cells, as well as myoepithelial cells, but was not detected in acinar cells, as determined by co-staining with antibodies to K5, Keratin 7 (K7; duct cells), Mist1 (acinar cells), Nkcc1 (acinar and a subset of duct cells), and smooth muscle actin (Sma; myoepithelial cells) (Figures 1C-G, S1A-E, S1P and S4A, B). After 90 (Figures 1H-L, S1F-J and S4A, B) and 180 days (Figures 1M-Q, S1K-O and S4A, B), analysis showed RFP+ cell expansion exclusively into duct cells, but no co-localization of RFP with the acinar cell marker Mist1 (Figures S4A, B) (Table S1). After 180 days, we observed that K5-expressing cells in the basal excretory ducts generate luminal duct cells (Figure S1P). RFP expression was undetectable in mice not treated with tamoxifen (Figure S1Q), ruling out non-specific activation of the reporter gene. Thus, K5+ cells in the intercalated and excretory ducts of adult mouse SMG are lineage restricted duct progenitor cells under homeostatic conditions.

Figure 1. K5-expressing cells generate duct but not acinar cells in the female SMG under homeostatic conditions.

Figure 1.

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(A) KRT5CreER was crossed with the R26tdTomato reporter strain. LoxP sites are marked by black triangles. (B) Experimental timeline of tamoxifen (TAM) induction and tissue harvest. (C-G) After a 3-day chase, RFP-labeled cells in female SMG are co-localized with K5 (C) and K7 (D) within the intercalated ducts, and with Sma in myoepithelial cells (G). RFP-positive cells are not co-localized with acinar cells marked by Mist1 (E) or Nkcc1 (F). (H-Q) After 90-day (H-L) and 180-day (M-Q) chase periods, the expanded number of RFP-labeled cells in female SMG remain co-localized with specific duct (H, I, M and N) or myoepithelial cell (L and Q) markers, but not with acinar cell (J, K, O and P) markers. Nuclei are stained with DAPI (blue). Scale bars: 25 μm.

Wnt responsive Axin2+ cells are duct progenitors under homeostatic conditions

Axin2, a direct downstream target of Wnt signaling, is expressed in intercalated duct cells of adult mouse SMG (Hai et al., 2010). We used the Axin2CreERT2 mouse model, crossed with the R26tdTomato reporter strain to investigate the lineage potential of Axin2+ cells under homeostatic conditions (Figures 2A, B). In Axin2CreERT2;R26tdTomato mice, co-labeling with antibodies to K5 and RFP at three days after tamoxifen induction showed that K5 and Axin2 mark overlapping, but non-identical, sets of intercalated duct cells (Figures 2C and S2A). There was no co-localization of RFP with Mist1 or Sma (Figures 2E-G, S2C-E and S4C, D). Over 90 and 180 days, Axin2+ contributed to duct cell turnover, as confirmed by co-staining with antibodies to RFP and the duct cell-specific marker K7 (Figures 2I, 2N, S2G and S2L). In contrast, there was no co-localization of RFP with acinar or myoepithelial cell markers (Figures 2J-L, 2O-Q, S2H-J, S2M-O and S4C, D) (Table S1). Fate mapping was also performed using the conditional doxycycline-induced mouse line Axin2rtTA;TRECre;R26tdTomato. Mice were fed doxycycline for three days to induce Cre recombinase, and activate RFP expression (Figures S2P, Q). Analysis of the Axin2rtTA;TRECre;R26tdTomato mice gave results identical to lineage tracing with the Axin2CreERT2;R26tdTomato strain. Wnt-responsive cells present in the intercalated ducts were labeled with RFP following 3 days of induction (Figures S2R and S4E), and a major contribution to duct turnover was evident after 1-year chase (Figures S2R and S4E) (Table S1). However, no contribution to acinar cells in the SMG was found. Axin2CreERT2;R26tdTomato and Axin2rtTA;TRECre;R26tdTomato mice not treated with tamoxifen or doxycycline, respectively, were analyzed as negative controls to rule out non-specific expression of RFP (Figures S2S, T). We conclude that under homeostatic conditions, Wnt-responsive Axin2+ cells located in intercalated ducts of the SMG contribute to duct turnover (Figure S2U).

Figure 2. Axin2-expressing cells generate duct but not acinar cells in the female SMG under homeostatic conditions.

Figure 2.

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(A) Axin2CreERT2 was crossed with the R26tdTomato reporter strain. LoxP sites are marked by black triangles. (B) Experimental timeline of TAM induction and tissue harvest. (C-D) After a 3-day chase, RFP-labeled cells in female SMG are co-localized with K5 and K7 in intercalated ducts. (E-G) RFP-labeled cells do not co-localize with acinar cells marked by Mist1 (E) or Nkcc1 (F), or the myoepithelial cell marker Sma (G). (H-Q) After 90-day (H-L) and 180-day (M-Q) chase periods, the RFP-labeled cells are co-localized with duct (H, I, M and N), but not acinar (J, K, O and P) or myoepithelial (L and Q) markers. Nuclei are stained with DAPI (blue). Scale bars: 25 μm.

Acinar regeneration after duct ligation is independent of K5+ and Axin2+ lineages

To determine the potential of K5+ or Axin2+ duct cells during regeneration, we used the duct ligation injury model (Tamarin, 1971). KRT5CreER;R26tdTomato and Axin2CreERT2;R26tdTomato mice were treated with tamoxifen and after three days, the main excretory duct of the left SMG was ligated. The contralateral gland served as untreated control (Figures 3A, B and S3A, B). The ligation was removed after 14 days, and the SMG was allowed to regenerate for 14 days. H&E staining showed the dilation of duct lumens and loss of acinar cell population after 14 days of ligation in comparison to control. After ligation release and 14 days of regeneration, acinar cells were replaced in the regenerated gland (Figure 3C). To analyze lineages, sections of SMG from all three time points were labeled with antibody to RFP. Figures 3D-G show RFP staining in control, non-ligated SMG. After 14 days of duct ligation, RFP+ cells were present in the duct cells of KRT5CreER;R26tdTomato mice (Figures 3H, I). Staining with Mist1 and Aquaporin 5 (Aqp5) revealed fewer acinar cells in the ligated glands (Figures 3J, K). After 14 days of regeneration, RFP positive cells were increased within the K7+ ducts (Figure 3M). However, no RFP expression was observed in regenerated acinar cells labeled by Mist1 or Aqp5 (Figures 3N, O and S4F) (Table S2). Similar results were obtained using Axin2CreERT2;R26tdTomato mice (Figures S4D-O). In the ligated and regenerated SMG, RFP positive cells were co-stained with K5 and K7 (Figures S3H, I, L, M), but did not co-localize with Mist1 or Aqp5 after ligation (Figures S3J, K and S4G) or after regeneration (Figures S3N, O and S4G) (Table S2). Together, these results demonstrate that K5+ and Wnt-responsive Axin2+ cells are not the primary source of acinar cell regeneration in ligated glands.

Figure 3. K5-expressing cells generate only duct but not acinar cells following duct ligation injury.

Figure 3.

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(A and B) Experimental model (A) and timeline of TAM induction (B), duct ligation, and de-ligation. (C) H&E staining of sections from control (left) and ligated SMG (middle) at 14 days after ligation; and of ligated SMG (right) at 14 days after de-ligation following regeneration. Scale bars: 100 μm. (D-K) Confocal images of control (D-G) and ligated (H-K) SMG sections at 14 days after ligation stained with antibodies to RFP and duct cell markers, K5 (D and H) and K7 (E and I), or acinar cell markers, Mist1 (F and J) and Aqp5 (G and K). (L-O) Confocal images of ligated SMG sections at 14 days after deligation show RFP expression co-localized with K5 (L) and K7 (M), but not with Mist1 (N) and Aqp5 (O) acinar cell markers. Enclosed boxes show enlarged images to emphasize absence of co-localization between Mist1+ nuclei and surrounding RFP+ cells. Nuclei are stained with DAPI (blue). Scale bars: 25 μm.

Both duct and acinar cells contribute to limited acinar cell regeneration after radiation injury

In contrast to duct ligation, irradiation of mouse salivary glands causes irreversible loss of almost all acinar cells. To investigate the regenerative potential of duct cells after irradiation, KRT5CreER;R26tdTomato and Axin2CreERT2;R26tdTomato mice were treated with tamoxifen 3 days before receiving a single dose of 15 Gy radiation to the neck. SMGs were analyzed at 2, 30 or 90 days after irradiation (Figures 4A and S3P). H&E staining of SMG sections showed a change in morphology by 30 days, and acute loss of acinar cells after 90 days (Figures 4B and S3Q). Notably, clusters of acinar-like cells were observed at 90 days in mice of both strains (Figures 4B and S3Q, dotted lines). At 2 days after irradiation, RFP+ cells traced in KRT5CreER;R26tdTomato and Axin2CreERT2;R26tdTomato mice were localized in intercalated ducts (Figures 4C1 and S3R1). At 30 days after irradiation, acinar cells were still present (Figures 4D2 and S3S2). RFP+ duct cells had contributed to ducts but not to acinar cells, as determined by double labeling with cell type-specific markers and RFP (Figures 4D1-D3 and S3S1-S3). At 90 days after irradiation, many duct cells were labled with RFP and co-stained with K7 (Figures 4E1 and S3T1). However, using H&E staining, we also observed irregular cell clusters that co-labeled with RFP and acinar cell markers Mist1, Nkcc1, Aqp5 and Inositol 1,4,5-triphosphate receptor 3 (IP3R3) (Figures 4E2, E3, F, G, S3T2, T3, U, V and S4H, I) (Table S3). The clusters not only expressed four acinar cell-specific markers, but were also labeled with Ki67 (Figures 4H and S3W). These results suggest that following severe injury, both K5+ and Axin2+ intercalated duct cells have the capacity to generate mitotically active secretory acinar cells. This is the first in vivo evidence that duct cells can generate acinar cells in adult SMG after radiation-induced injury.

Figure 4. Both duct and acinar cells regenerate acinar cells after radiation-induced injury.

Figure 4.

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(A) Experimental model and timeline of TAM induction, irradiation, and tissue harvest using KRT5CreER;R26tdTomato mice. (B) H&E staining of SMG sections at 2, and 30 days after a single dose (15 Gy) of radiation shows altered morphology, but acinar cells are still present. After 90 days, cell clusters are present (circled by dotted line). Scale bars: 100 μm. (C1-C3) At 2 days after irradiation, images of sections stained with antibodies to RFP and cell type specific markers K7 (C1), Mist1 (C2) and Nkcc1 (C3) show RFP+ cells are located in intercalated ducts. (D1-D3) At 30 days, RFP+ cells are co-localized with duct cell markers (D1), but not acinar cell markers (D2-D3). (E1-E3) At 90 days after irradiation, antibodies to RFP stain clusters of cells that co-localize with both duct (E1) and acinar cell markers (E2-E3). (F and G) At 90 days after irradiation, clusters of RFP+ cells co-localize with acinar cell markers Aqp5 (F) and IP3R3 (G). (H) At 90 days after irradiation, Ki67-positive cells (arrowheads) within the RFP+ Nkcc1+ clusters show that acinar cells in the cluster continue to proliferate. (I) Experimental model and timeline of TAM administration, irradiation and tissue harvest using Mist1CreERT2;R26tdTomato mice. (J) H&E staining of SMG at 2 and 30 days after irradiation shows altered morphology, but acinar cells are still present. At 90 days after irradiation, cell clusters are observed (circled with dotted line). Scale bars: 100 μm. (K-L) At 2 days (K1-K3) and 30 days (L1-L3) post-irradiation, SMG sections from Mist1CreERT2;R26tdTomato mice were stained with antibodies to RFP and K7 (K1 and L1), Mist1 (K2 and L2) and Nkcc1 (K3 and L3) (M1) At 90 days after irradiation, colocalization of RFP and K7 (arrowheads) suggests that Mist1+ acinar cells undergo acinar-to-ductal transition. (M2, M3, N, O) Large clusters of RFP+ cells co-localized with acinar cell markers Mist1 (M2), Nkcc1 (M3), Aqp5 (N) and IP3R3 (O). (P) Ki67-positive cells (arrowheads) show that the RFP+ Nkcc1+ cell clusters derived from Mist1-expressing cells continue to proliferate. Nuclei are stained with DAPI (blue). Scale bars: 25μm.

We previously showed that surviving acinar cells can regenerate the SMG after duct ligation (Aure et al., 2015b). However, irradiation results in permanent loss of most acinar cells, so regeneration by these cells may be impaired. To test this hypothesis, similar radiation experiments were done using the Mist1CreERT2;R26tdTomato mice. To label the majority of acinar cells, the mice were given tamoxifen daily for 3 days, and 15 Gy radiation was administered to the neck after a subsequent 3 days (Figure 4I). At 2 and 30 days after irradiation, H&E staining and co-labeling with antibody to Mist1 and Nkcc1 showed that RFP+ acinar cells were still present (Figures 4J, K1-3, L1-3 and S4J). From 2 to 30 days, approximately 90% of Mist1+ acinar cells were co-labeled with RFP (Table S3). Notably, as observed using the KRT5CreER;R26tdTomato and Axin2CreERT2;R26tdTomato mice, irregular cell clusters were observed at 90 days after irradiation (Figures 4J, M1-M3). Of these cell clusters, 58% were RFP+ (Table S3), indicating that they were generated from acinar cells labeled prior to irradiation in the Mist1CreERT2;R26tdTomato mice. The RFP+ cell clusters co-localized with Mist1, Nkcc1, Aqp5 and IP3R3, but not with K7 (Figures 4M1-M3, N, O and S4J). Like the K5- and Axin2-derived cell clusters, cells within these RFP+ clusters were labeled with Ki67, indicating the ability to proliferate at 90 days after irradiation (Figure 4P) These results suggest that both acinar and duct cells contribute to acinar cell regeneration after radiation-induced injury. In addition to acinar cell clusters, we also observed RFP+ cells at 90 days that unexpectedly co-localized with the duct cell-specific marker K7 (Figure 4M1, arrowheads). This apparent transition from acinar to duct cell phenotype is further evidence of cellular plasticity occurring as a result of radiation injury (Aure et al., 2015a).

DISCUSSION

Loss of acinar cells after duct ligation is reversible through regeneration, and cell transplantation can rescue secretory function in damaged salivary glands (Aure et al., 2015b; Lombaert et al., 2008a), but the mechanisms of acinar cell repair and regeneration after injury remain unclear. We have used three genetic models to trace the fate of intercalated duct and acinar cells following injury to investigate the mechanisms of endogenous cell replacement and regeneration. Under normal conditions, we find that intercalated duct cells expressing either K5 or Axin2 have limited potential, and generate duct, but not acinar cells. These results are consistent with an earlier study that found K14+ duct cells generate only duct cells (Kwak et al., 2016). In agreement with our finding that regeneration can be accomplished by acinar cell duplication if there are surviving acinar cells present (Aure et al., 2015b), we find no detectable contribution to acinar cells from K5- or Axin2-expressing intercalated duct cells following duct ligation injury.

In contrast to duct ligation, irradiation of the salivary glands causes permanent loss of acinar cells (Konings et al., 2005). Although histological analysis and immunohistochemistry show that Mist1-positive acinar cells are still present at 30 days after irradiation (Figure 4D2) (Varghese et al., 2018), few remain at 90 days (Figure 4E2). However, large acinar cell-like clusters were observed in irradiated SMG in all three genetic models analyzed (Figures 4B, J and S3Q). Similar clusters have been noted in salivary glands of irradiated rats, and minipigs (Konings et al., 2006; Stramandinoli-Zanicotti et al., 2013). The clusters expressed differentiated acinar cell markers, including Mist1, Nkcc1, Aqp5 and IP3R3, suggesting that they may be functional. These results are supported by several clinical studies, which have reported increased salivary flow rates in head and neck cancer patients over time following radiation therapy (Hey et al., 2011; Murdoch-Kinch et al., 2008).

These lineage tracing data provide the first in vivo evidence that duct cells can generate acinar cells in the salivary glands following severe injury. Approximately 50% of the acinar clusters are derived from K5 or Axin2 duct cells. The remaining 50% are generated from acinar cells, presumably by the mechanism of self-duplication known to occur during salivary gland regeneration (Aure et al., 2015b). Thus, both acinar and duct cell types can contribute to endogenous regeneration after severe cell loss.

The evidence that duct cells serve as facultative progenitors in the salivary gland is consistent with the demonstrated ability of these cells to rescue secretory function (Lombaert et al., 2008a; Maimets et al., 2016; Xiao et al., 2014). Lineage tracing of duct cells to acinar cells was observed only under conditions of severe cell loss. The ability of lineage-restricted cells to either change phenotype or to generate other cell types to compensate for tissue loss is frequently recognized as a mechanism involved in regeneration (Raven et al., 2017; Thorel et al., 2010). Cellular plasticity is induced in several tissues as part of the normal injury response (Merrell and Stanger, 2016; Tata and Rajagopal, 2016). Our data suggest that Mist1+ acinar cells also undergo cell type transitions in irradiated glands, consistent with reports documenting the plasticity of other differentiated secretory cells (Willet et al., 2018; Yu et al., 2018). This suggests that several cell populations have regenerative potential useful for restoring salivary gland function.

The finding that acinar cells within the clusters continue to proliferate in the irradiated gland (Figures 4H, P) has exciting translational implications. Acinar cell proliferation and self- duplication under normal conditions play a major role in maintaining the secretory cells (Aure et al., 2015b). Several studies have shown that radiation-induced salivary gland damage can be mitigated by administering growth factors directly to the glands (Limesand et al., 2009; Lombaert et al., 2008b; Xiao et al., 2014). Providing stimulatory factors, while controlling fibrosis and inflammation, which may interfere with complete regeneration, could activate endogenous regeneration of functional secretory acinar cells in irradiated salivary glands.

This study provides new insight into the mechanisms of salivary gland regeneration. In vivo lineage tracing demonstrates that acinar cells can be regenerated through either acinar or duct cells, following severe cell loss, and reveals a role for cellular plasticity in repairing the salivary gland. Our data show that acinar cells can retain proliferative activity long after radiation injury and suggest the exciting possibility that stimulation of endogenous cells, which has been demonstrated in various tissues (Wells and Watt, 2018), may be a potential strategy for engineering the regeneration of salivary glands in head and neck cancer patients with radiation-induced dry mouth.

Contact for Reagent and Resource Sharing

Requests for further information, resources and reagents should be directed to and will be fulfilled by the Lead Contact, Catherine E. Ovitt (Catherine_Ovitt@urmc.rochester.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

All mouse strains were maintained on a C57BL/6L background. Mist1CreERT2 (Shi et al., 2009), KRT5CreER (Jovov et al., 2011), Axin2rtTA (Maruyama et al., 2016) and TRECre (Maruyama et al., 2016) have been previously described. Axin2CreERT2 (Axin2tm1(cre/ERT2)Rnu/J) and R26tdTomato (Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) were obtained from Jackson Laboratory. Standard genotyping was performed. Mice were maintained on a 12-hour light/dark cycle. Food and water were provided ad libitum. All procedures and protocols were approved and conducted in accordance with the University Committee on Animal Resources at the University of Rochester Medical Center.

METHOD DETAILS

Tamoxifen

Tamoxifen working solution was prepared by mixing 100 μl ethanol and 60 mg Tamoxifen (MP Biomedicals) before adding 900 μl of corn oil (Sigma-Aldrich). For lineage tracing studies, KRT5CreERT;R26tdTomato, Axin2CreERT2;R26tdTomato and Mist1CreERT2;R26tdTomato mice, 6-8 weeks of age, were administered tamoxifen (0.25 g/gm body weight) by gavage.

Doxycycline

The Axin2rtTA;TRECre;R26tdTomato mouse strain was created by crossing Axin2rtTA, TRECre and R26tdTomato reporter mice. Axin2rtTA;TRECre;R26tdTomato mice, 4 weeks of age, were treated with doxycycline (2 mg/ml plus 50 mg/ml sucrose) for 3 days, as described (Maruyama et al., 2016).

Tissue processing

Tissue was isolated and fixed in 4% paraformaldehyde at 4°C overnight. The tissue was processed using a Tissue-Tek VIPTM processing machine (Sakura Finetek USA, Inc.) before paraffin embedding. Sections (5 μm) were cut using a Leica RM2125 rotary microtome and collected on Superfrost Plus slides (Thermo Fisher Scientific).

Immunohistochemistry

Paraffin sections were deparaffinized and rehydrated. Sections were heated in antigen retrieval buffer (10 mM Tris base, 1 mM EDTA) for 10 minutes. After cooling, sections were blocked with 10% normal donkey or goat serum in PBS containing 0.1% bovine serum albumin (PBSA), before incubating in primary antibody overnight at 4°C. Primary antibodies: Aqp5 (sc-9890, Santa Cruz, 1:100), Cytokeratin 5 (905501, Biolegend, 1:300), Cytokeratin 7 (ab9021, Abcam, 1:200), DsRed (632392, Takara, 1:250), IP3R3 (610312, BD Biosciences, 1:400), Ki67 (550609, BD Biosciences, 1:100), Mist1 (sc-324133, Santa Cruz, 1:100), Mist1 (ab187978, Abcam, 1:200), Nkcc1 (sc-21545, Santa Cruz, 1:100), RFP (600-401-379, Rockland, 1:500), Sma (MS-113-P, Lab Vision, 1:300). The following day, sections were rinsed in PBS-tween (1 × 5min) and PBS (2 × 5min), then incubated with secondary antibody. For double labeling with same species primary antibodies, extra blocking using 10% normal rabbit serum and unconjugated donkey anti-rabbit Fab fragment (Jackson ImmunoResearch) in PBSA was added before incubation with the second primary antibody, followed by incubation in second secondary antibody. Dilution for the Alexa Flour fluorescent secondary antibodies (Invitrogen) is at 1:500. Nuclei were stained with DAPI (Thermo Fisher Scientific). Sections were mounted in Shandon Immu-Mount (Thermo Fisher Scientific). Fluorescent images were acquired using a Leica TCS SP5 confocal system, with a 40x oil immersion objective and a zoom of 1, 2 or 2.5, and were processed using Adobe Photoshop.

H&E staining

Paraffin sections were deparaffinized and rehydrated. Sections were stained with hematoxylin and eosin (H&E), dehydrated through ethanol gradient and mounted using PermountTM (Thermo Fisher Scientific). Images were acquired using an Olympus DX41 microscope with a DP71 camera, analyzed on DP-BSW-3.2 software, and processed using Adobe Photoshop.

Duct ligation

KRT5CreER;R26tdTomato and Axin2CreERT;R26tdTomato male and female mice (6-8 weeks, n≥5) were given tamoxifen (0.25 g/gm body weight) by gavage 3 days before ductal ligation. On the day of surgery, mice were pre-treated with Buprenorphine SR™ (1 mg/kg, Wildlife Pharmaceuticals) analgesic, and subsequently anesthetized with ketamine HCL (80 mg/kg, JPH pharmaceuticals) and xylazine (8 mg/kg, Lloyd Laboratories) through intraperitoneal injection. An incision was made on the left side of the neck, and the main duct of the left submandibular gland was isolated and ligated using a titanium hemostatic clip (#R9180, Vitalitec Int). The right contralateral submandibular gland served as sham control. After 14 days, mice were re-anesthetized, the incision was opened, and the clip was removed without injury to the main duct. Following the de-ligation, the submandibular glands were either isolated immediately or the incision was closed, and the glands were allowed to regenerate for 14 days. Tissues were isolated and fixed in 4% paraformaldehyde at 4°C overnight. Fixed tissues were placed in 70% ethanol at 4°C prior to processing. Sections were used for immunohistochemistry as described above.

Irradiation

KRT5CreERT;R26tdTomato, Axin2CreERT2;R26tdTomato mice (6-8 weeks, n≥3/ group) were given tamoxifen (0.25 g/gm body weight) by gavage 3 days before irradiation. Mist1CreERT2;R26tdTomato mice (6-8 weeks, n=3/ group) were administered tamoxifen on 3 consecutive days (0.25 g/gm body weight) by gavage and were irradiated after another 3 days. Mice were anesthetized with ketamine HCL (100 mg/kg, JPH pharmaceuticals) and xylazine (10 mg/kg, Lloyd Laboratories) through intraperitoneal injection. Mice were positioned over the slit of a custom-built collimator, which limited radiation exposure to the neck region and allowed shielding of the whole body. Mice were administered a single dose of 15Gy from a Cs137 gamma radiation source (Shepard). Tissues were isolated at 2, 30 or 90 days after irradiation and fixed in 4% paraformaldehyde at 4°C overnight. Fixed tissues were placed in 70% ethanol at 4°C prior to processing. Sections were used for immunohistochemistry as described above.

Quantification and Statistical Analysis

For quantification of double labeled cells, five random areas (430 × 330 μm) were chosen within each gland, from at least three biological replicates (n≥3).

Supplementary Material

Supplementary File

ACKNOWLEDGEMENTS

The authors would like to thank Dr. D. Bohmann and M. J. Tsai for comments and critical reading of the manuscript, Dr. W. Hsu for providing the Axin2rtTA;TRECre;R26tdTomato mice, and J. J. Varghese and I. L. Schmale for technical expertise. This work was supported by R56DE025098 and R01DE022949 (CEO) from NIH/NIDCR, and by a generous gift from Mrs. Holly Elwell.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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