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Expression of the transcription factor, Ascl3, marks a population of adult progenitor cells, which can give rise to both acinar and duct cell types in the murine salivary glands. Using a previously reported Ascl3EGFP-Cre/+ knock-in strain, we demonstrate that Ascl3-expressing cells represent a molecularly distinct, and proliferating population of progenitor cells located in salivary gland ducts. To investigate both the role of the Ascl3 transcription factor, and the role of the cells in which it is expressed, we generated knockout and cell-specific ablation models. Ascl3 knockout mice develop smaller salivary glands than wild type littermates, but secrete saliva normally. They display a lower level of cell proliferation, consistent with their smaller size. In the absence of Ascl3, the cells maintain their progenitor function and continue to generate both acinar and duct cells. To directly test the role of the progenitor cells, themselves, in salivary gland development and regeneration, we used Cre-activated expression of diphtheria toxin (DTA) in the Ascl3-expressing (Ascl3+) cell population, resulting in specific cell ablation of Ascl3+ cells. In the absence of the Ascl3+ progenitor cells, the mice developed morphologically normal, albeit smaller, salivary glands able to secrete saliva. Furthermore, in a ductal ligation model of salivary gland injury, the glands of these mice were able to regenerate acinar cells. Our results indicate that Ascl3+ cells are active proliferating progenitors, but they are not the only precursors for salivary gland development or regeneration. We conclude that maintenance of tissue homeostasis in the salivary gland must involve more than one progenitor cell population.
The salivary glands are essential for swallowing, the preliminary steps of digestion, antimicrobial activity, immunological defense, mucosal maintenance, and dental surface mineralization. Comprised of two predominant cell types, the ductal and the secretory acinar cells, three major pairs of salivary glands in mammals produce saliva that passes through the ducts to the oral cavity. Salivary gland secretion is severely decreased under certain pathological or therapeutic conditions, including radiation treatment of patients with head and neck cancers, and autoimmune diseases such as Sjögren’s syndrome. There is currently no satisfactory treatment to reverse this damage, which is often accompanied by a permanent loss of the fluid-secreting acinar cells. In order to develop approaches for the treatment of salivary gland hypofunction, an understanding of the molecular and cellular mechanisms involved in salivary gland maintenance is required.
Maintenance of the salivary glands in adults involves limited cell turnover and replacement of aging cells (Chang, 1974; Dardick and Burford-Mason, 1993; Denny et al., 1993). Replacement cells are most likely derived from a source of undifferentiated cells, and there is considerable evidence for a stem or progenitor cell population in the salivary glands (Denny et al., 1993; Denny et al., 1990; Denny and Denny, 1999; Man et al., 2001; Schwartz-Arad et al., 1988; Zajicek et al., 1985). Several stem cell-associated markers are expressed by subpopulations of duct cells, pointing to the ducts as the presumptive location of the progenitors (Hai et al., 2010; Kimoto et al., 2008; Knox et al., 2010; Lombaert et al., 2008; Man et al., 2001; Raimondi et al., 2006). Furthermore, duct cells distinguished by expression of the stem cell marker, cKit, have been shown to promote the restoration of secretory output and tissue regeneration in radiation-damaged salivary glands of mice (Lombaert et al., 2008). However, the interactions and pathways that lead to lineage commitment and differentiation of these cells toward secretory acinar and duct cells are poorly understood.
In an earlier report, we identified a small population of progenitor cells, also found in the ducts of mouse salivary glands, which proved, by lineage tracing, to be precursors of both acinar and duct cell types (Bullard et al., 2008). These progenitor cells are characterized by the expression of Ascl3 (achaete scute-like homolog 3), a basic helix-loop-helix transcription factor (GenBank accession no. NM_020051). Ascl3 expression is limited to a subset of cells found in the ducts of all three major salivary glands (Yoshida et al., 2001). During prenatal development of the glands, only a small number of duct cells express Ascl3. However, in mature salivary glands, we found that a significant number of labeled descendents were generated from Ascl3-expressing progenitor cells (Bullard et al., 2008). We proposed that Ascl3 marks progenitor cells that are involved in the maintenance of normal gland homeostasis.
Ascl3, also known as Sgn1, is a member of the mammalian achaete scute homolog (Asc) gene family of basic helix-loop-helix (bHLH) transcription factors (Yoshida et al., 2001). Related members of the same gene family are expressed by tissue-specific progenitor cells, and are implicated in specification of cell fate and in promoting differentiation. Ascl1, also known as Mash1, is transiently expressed in neuronal progenitor cells, and loss of Ascl1 function results in the absence of specific populations of differentiated neurons in the mouse (Battiste et al., 2007; Guillemot et al., 1993). A second member of the gene family, Ascl2 functions in intestinal stem cell maintenance (van der Flier et al., 2009), and promotes terminal differentiation of epidermal precursor cells (Moriyama et al., 2008). Based on the specific expression of Ascl3 in salivary gland progenitor cells, the Ascl3 transcription factor may play a similar role in stem or progenitor cell differentiation.
In order to examine the molecular and cellular properties of the Ascl3+ progenitors, we generated an Ascl3 knockout, as well as an Ascl3+ cell-specific ablation mouse model. Using these models, we have investigated the contribution of the Ascl3+ progenitor cell population to salivary gland maintenance and regeneration.
Ascl3EGFP-Cre/+ knock-in mice were generated as previously reported (Bullard et al. 2008), and two separate lines have been maintained on a C57Bl/6 background for more than 10 generations. Heterozygotes were crossed to generate homozygous Ascl3 EGFP-Cre/EGFP-Cre knockout animals. For lineage tracing in homozygotes, mating was carried out with Ascl3EGFP-Cre/+ heterozygous females carrying the Rosa26R reporter locus derived from 129S-Gt(ROSA)26Sortm1Sor/J (Jackson Laboratory)(Soriano, 1999). Genotyping was performed as described (Bullard et al., 2008). The Ascl3EGFP-Cre/+/R26DTA/+ mice were generated from crosses of Ascl3EGFP-Cre/+ to heterozygotes of Gt(ROSA)26Sortm1(DTA)Jpmb/J strain (called R26DTA/+) (Jackson Laboratory)(Ivanova et al., 2005). Double Ascl3EGFP-Cre/+/R26DTA/+ heterozygotes were genotyped by PCR using primers for the Ascl3 knock-in (Bullard et al., 2008)and primers oIMR8052, oIMR8545, and oIMR8546 (Jackson Laboratory). Double heterozygote Ascl3EGFP-Cre/+/R26DTA/+ mice were used as experimental animals. Single heterozygous Ascl3EGFP-Cre/+/R26+/+ or Ascl3+/+/R26DTA/+ littermates were used as controls. Mice were maintained on a 12-h light, 12-h dark schedule with ad libitum access to food and water. The University Committee on Animal Resources at the University of Rochester approved all procedures and protocols.
Labeling experiments with BrdU were performed on paired male and female Ascl3EGFP-Cre/+ heterozygous and Ascl3 EGFP-Cre/EGFP-Cre knockout mice at ages P16, P42 (6 weeks), and P112 (4 months). Mice were injected intraperitoneally with BrdU (0.1mg/gm body weight; Roche, Indianapolis, IN) in PBS. At 2 hours, mice were euthanized and the salivary glands were isolated and fixed overnight in Carnoy’s fixative. Fixed glands were embedded in paraffin, and sectioned. Sections were subjected to antigen retrieval, followed by immunohistochemistry with antibody to Cre (1:600 dilution; Covance Princeton, NJ) as described (Bullard et al., 2008). BrdU was detected using the BrdU detection kit II protocol (Roche) according to the manufacturer’s instructions. Cell counts were performed on sections from 4 animals of each genotype, at the ages of P16, P42 (6 weeks), and P112 (4 months). The number of BrdU-positive cells was determined by manually counting six 200 μm2 fields on each section. Statistical analysis was performed using the Students’ t-test, 2-tailed.
For long-term labeling experiments, two litters of Ascl3EGFP-Cre/+ postnatal day 16 – 23 pups (n=14) were injected daily with BrdU (0.05 mg/gm body weight; Roche) for 5 (experiment 1) or 7 (experiment 2) days, to increase the probability of labeling potential stem cells. Following an 8-week chase period, salivary glands were isolated and incubated overnight in Carnoy’s fixative. Double immunohistochemistry with antibodies to Cre recombinase and BrdU was performed on paraffin sections, as described above.
The ductal ligation procedure was performed on pairs of sex-matched mice, aged 2–5 months. The procedure was performed on double heterozygote Ascl3EGFP-Cre/+/R26DTA/+ mice and single heterozygous Ascl3EGFP-Cre/+/R26+/+ or Ascl3+/+/R26DTA/+ littermates, as controls. Mice were anesthetized with ketamine (100 mg/kg; Bioniche Pharma, Lake Forest, IL) and xylazine (5–10 mg/kg; Lloyd Laboratories, Shenandoah, IA) administered intraperitoneally. An incision was made on the ventral left side of the neck, and the main secretory duct of the left submandibular gland was isolated away from the parallel blood vessel. A microclamp (3.5mm × 1mm, Fine Science Tools, Foster City, CA) was used to occlude the duct. The right submandibular gland served as the sham-operated internal control.
After 10 days, the animals were re-anesthetized and the incision reopened. The submandibular gland was first examined microscopically. Only those mice in which the gland had visibly decreased by 35–40% were used for subsequent regeneration analysis. The microclamp was removed without injury to the duct, and the incision was again closed. Following removal of the clamp, the glands were either taken immediately (day = 0) or allowed to recover for 7, 14 or 21 days (each time point n=3). Mice were then euthanized and the salivary glands were fixed overnight in Carnoy’s fixative, embedded in paraffin, and sectioned. The degree of atrophy or recovery was determined by hematoxylin and eosin staining, and by immunohistochemical analysis.
Submandibular glands were processed, fixed, embedded in OCT, and cryosections were stained for LacZ activity as previously described (Bullard et al., 2008). Periodic acid-Schiff and hematoxylin and eosin staining was performed on paraffin-embedded sections, after dewaxing and hydration, using standard protocols.
Salivary glands were harvested and fixed overnight in 4% paraformaldehyde at 4°C. Tissues were processed, embedded in paraffin and sectioned. Sections of 5μm thickness were dewaxed and hydrated before immunostaining. For Nkcc1 immunohistochemistry, endogenous and pseudoperoxidase activities were eliminated by treatment with 10% hydrogen peroxide. Before immunostaining, tissues were subjected to antigen retrieval by microwaving with TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 9.5) for 3.5 min at maximum power (1550 Watts) and then for 8 min at 20% of the maximum power (model JES1139BL01, General Electric Co., KY). Forty minutes after treatment, sections were washed with three changes of TBS pH 7.8 and then the endogenous biotin was blocked using an Avidin/Biotin blocking kit (Vector Labs, Burlingame, CA). After biotin blocking, sections were incubated overnight with rabbit anti-peptide polyclonal antibody directed to the C-terminus of rat Nkcc1. Nkcc1 antibody was a gift from Dr. R. James Turner (NIDCR/NIH), and was used at a dilution of 1:2500 in TBS pH 7.8 containing 0.5% immunoglobulin-free bovine serum albumin (Sigma Aldrich, St. Louis, MO). Bound immunoglobulins were detected with the Vectastain Elite ABC kit (Vector Labs) and peroxidase was visualized by incubation with 3,3′-diaminobenzidine (Vector Labs) for 5 min. When immunostaining was complete, the sections were rinsed with distilled water and contrasted with hematoxylin for 15 sec (Richard-Allan Scientific, MI). Finally, the sections were dehydrated in ethanol, cleared with xylene, and mounted using Permount (Fisher Scientific, Pittsburgh, PA). Controls were carried out using tissues from Nkcc1 knockout mice. Immunohistochemistry with all other antibodies was performed on paraffin sections as described (Bullard et al. 2008). Briefly, tissue sections were deparaffinized, and subjected to antigen retrieval in 1 mM EDTA buffer, pH: 8.0 for 10 min using a pressure cooker. Sections were allowed to cool, washed 3 times in PBS and then blocked in 10% normal donkey serum with 5% milk in PBS for 1 hour at room temperature and then incubated with the primary antibodies: rabbit anti-Cre recombinase (Covance) diluted 1:800; goat anti-cytokeratin 19 (Santa Cruz Biotechnology Inc, CA) diluted 1:50; rabbit anti-Mist1 (a gift from Dr. Stephen Konieczny, Purdue University) diluted 1:2000; rabbit anti-KCa1.1 (1:500, Santa Cruz). Secondary antibodies used included: Cy3-conjugated donkey anti-rabbit diluted 1:500 (Jackson Immunoresearch, West Grove, PA); Cy2-conjugated donkey anti-rabbit diluted 1:500 (Jackson); Cy2-conjugated donkey anti-goat diluted 1:500 (Jackson); Alexa fluor 546 goat anti-rabbit diluted 1:500 (Invitrogen, Carlsbad, CA). Antibodies were diluted in antibody diluent (DAKO Cytomation, CA). Blocking for double immunolabeling of Nkcc1 and KCa1.1 or Cre was done using donkey anti-rabbit unconjugated Fab fragments (Jackson) diluted 1:10 in 5% normal goat serum for 1 hour before addition of the second primary antibody. Immunostaining with antibody to caspase 3 was performed on paraffin sections from both wild type and Ascl3 knockout mice using the Signal Stain Cleaved Caspase-3 kit (Cell Signaling, Danvers, MA), according to the manufacturer’s instructions.
Brightfield and fluorescent images were made using an Olympus DX41 microscope connected to a DP71 camera, with DP-BSW-V3.2 software (Olympus America Inc., Center Valley, PA). Confocal images were taken on a Leica TCS SPII confocal microscope, using Leica confocal software (Leica Microsystems, Mannheim, Germany). Surface area measurements to compare duct area to total surface area were made on 9 sections each of submandibular gland from wild type, and knockout, and 12 sections from cell ablation model mice, respectively. ImageJ (NIH) software was used for analysis.
Saliva was collected from the submandibular glands of wild type, knockout, and cell ablation model mice. Salivary flow rates were measured during ex-vivo perfusion of the isolated submandibular glands as previously described (Romanenko et al., 2007). Salivation was stimulated by perfusion with the cholinergic receptor agonist carbachol (0.3 μm CCh; Sigma) and the beta-adrenergic agonist isoproterenol (5 μm IPR; Sigma). Following the experiment, the submandibular glands were removed, blotted and weighed.
To confirm the hypothesis that Ascl3+ cells are proliferating progenitor cells, we conducted BrdU labeling experiments. Heterozygous mice carrying one copy of the Ascl3EGFP-Cre allele are phenotypically normal. In these animals, cells that express Ascl3 can be detected through EGFP fluorescence or by immunostaining, using an antibody to Cre recombinase. Salivary glands were isolated from postnatal day 16 (P16) mice two hours after BrdU injection, and double immunohistochemistry was performed on sections using antibodies to BrdU and Cre. Co-localization of BrdU was observed in approximately 50% of the Ascl3-expressing cells (Fig. 1A–C). This result demonstrates that Ascl3+ progenitor cells are mitotically active, consistent with their characterization as salivary gland progenitor cells.
To further characterize the Ascl3-expressing progenitor cells, we examined the expression of cell type-specific markers. Nkcc1 is a sodium-potassium-chloride co-transporter critical for saliva secretion, which is localized to the basolateral membranes of all acinar cells in the submandibular, sublingual and parotid glands (Evans et al., 2000). A subset of duct cells in all three glands also expresses Nkcc1 on the membrane surface (He et al., 1997) (Fig. 1D–F). As this expression pattern is notably similar to that of Ascl3, we examined whether the two proteins are co-localized. Double immunohistochemistry on salivary gland sections showed that all duct cells expressing Nkcc1 are co-localized with the progenitor cells expressing Ascl3 (Fig. 1F). The Ascl3+/Nkcc1+ cells are present throughout the ductal system of submandibular, sublingual and parotid glands, and are clearly molecularly distinct from the majority of the epithelial duct cells.
Underscoring this conclusion that the Ascl3+ progenitors are molecularly distinct, we have also found that expression of the large conductance calcium-activated potassium channel (KCa1.1) is specifically co-localized to these duct cells positive for both Ascl3 (data not shown) and Nkcc1 (Fig. 1E). The discovery that at least two acinar cell markers normally absent from duct cells are specifically expressed in the Ascl3+ cell type is intriguing. Expression of Nkcc1 and KCa1.1 in the subset of duct cells known to generate acinar cell progeny (Bullard et al., 2008) supports the idea that Ascl3+ cells are an intermediate progenitor cell type.
The initial identification of Ascl3+ cells as bipotent progenitor cells (Bullard et al., 2008), raised the question of their relationship to salivary gland stem cells. The onset of Ascl3 expression at embryonic day 15.5 argues against a function of Ascl3+ cells as early salivary gland stem cells, since the glands begin development at embryonic day 11 (Patel et al., 2006). We wished to investigate whether the Ascl3+ pool of cells might instead be related to a previously proposed, but not yet clearly defined, adult salivary gland stem cell. Label-retaining cells have been identified in rat salivary gland ducts, following BrdU labeling and an 8-week chase period, and are presumed to be slowly cycling stem cells (Kimoto et al., 2008).
We performed long-term BrdU labeling experiments to test whether the Ascl3+ progenitor cells in the ducts are label-retaining cells. Mice were given daily BrdU injections from P16 through P23, followed by an 8-week chase period. The salivary glands were then removed, processed, and analyzed by double immunohistochemistry, using antibodies for BrdU and Cre recombinase. Both antibodies label a subset of duct cells (Fig. 2A–C). However, there was no evidence of co-localization of the two antigens in one cell (n=14 animals; 2 sections each). We conclude that Ascl3+ progenitor cells are not label-retaining cells, and are not likely to be slowly cycling stem cells.
The insertion of EGFP-Cre into the Ascl3 locus creates a loss-of-function allele, as the entire coding sequence of the transcription factor has been replaced (Bullard et al., 2008). To investigate the function of the Ascl3 transcription factor, we generated Ascl3 null mice homozygous for this allele. Ascl3 EGFP-Cre/EGFP-Cre mice were born at the expected Mendelian frequencies, and knockout mice were viable and fertile. Complete absence of intact Ascl3 mRNA in the knockout glands was verified both by RT-PCR and on Northern blots (data not shown).
Ascl3 EGFP-Cre/EGFP-Cre mice develop salivary glands, indicating that patterning and differentiation pathways are not impaired by absence of the transcription factor. However, the submandibular glands of the knockout mice exhibit a consistently reduced size compared to those of wild type littermates (Fig. 3A). This difference, observed in both male and female submandibular glands, is detectable at P16 (female Ascl3+/+ 16.38±2.87 mg; Ascl3 EGFP-Cre/EGFP-Cre 12.51±2.24 mg; P=0.018; n=6 for both genotypes) and is maintained with age. At 12 months, the submandibular gland weight for female Ascl3+/+ is 45.94±4.93 mg, while female Ascl3 EGFP-Cre/EGFP-Cre glands are 33.60±2.05 mg (P<0.001; n=7 for both genotypes).
To assess whether the glands of Ascl3 EGFP-Cre/EGFP-Cre knockout mice are altered in their ability to maintain normal cell numbers, we used BrdU labeling to measure cell proliferation. At P16, wild type and knockout submandibular glands showed equivalent numbers of BrdU pulse-labeled cells (Fig. 3B) [P16: median Ascl3EGFP-Cre/+ = 38.5; median Ascl3 EGFP-Cre/EGFP-Cre = 30.0]. However, at P42, the number of BrdU+ cells in knockout glands was decreased to 65% of the number found in wild type glands [median Ascl3EGFP-Cre/+ = 82.5; median Ascl3 EGFP-Cre/EGFP-Cre = 54.0; P<0.01]. By P112, overall levels of cell proliferation are very low in the adult glands (Chang, 1974; Denny and Denny, 1999). However, the knockout glands continued to show lower numbers of BrdU-positive cells within the parenchyma compared to wild type controls [median Ascl3EGFP-Cre/+ = 10.0; median Ascl3 EGFP-Cre/EGFP-Cre= 7.0; P<0.05].
A lower number of proliferating cells could be explained by higher rates of cell death. We therefore examined mutant and wild type salivary glands for differences in levels of apoptosis. Immunohistochemistry was performed with an antibody to caspase 3, a marker for apoptotic cells. We tested submandibular sections from wild type, heterozygous, and knockout mice at 2–3 months of age. However, no difference in the level of apoptosis based on genotype was found (data not shown).
Staining with an antibody to Cre revealed that the cells in which Ascl3 would have been expressed remain present in the ducts of salivary glands from Ascl3 EGFP-Cre/EGFP-Cre knockout mice (Fig. 3C). Moreover, lineage tracing in the Ascl3 EGFP-Cre/EGFP-Cre knockout animals demonstrated that these progenitor cells retain the ability to generate both acinar and duct cells in the absence of the Ascl3 transcription factor, itself (Fig. 3D). Thus, bipotency of the progenitor cells is not altered in the absence of Ascl3. We tested the function of the Ascl3 EGFP-Cre/EGFP-Cre salivary glands by measuring saliva secretion. The level of salivary secretion from glands of Ascl3 EGFP-Cre/EGFP-Cre animals was not significantly different from that measured in wild type littermates, nor were changes found in ionic composition of the saliva in the knockout animals (data not shown).
Acinar cell number and morphology appeared normal on sections from Ascl3 EGFP-Cre/EGFP-Cre knockout mice stained with antibodies to the acinar cell-specific proteins, aquaporin 5 or the transcription factor, Mist1 (data not shown). Likewise, cytokeratin 19, a marker of differentiated duct cells (Hisatomi et al., 2004; Kishi et al., 2006), showed identical staining patterns in knockout (Fig. 3C) and control (data not shown) submandibular glands. Only one gene product showed a distinct change in expression in the Ascl3 knockout glands. Nkcc1 expression is dramatically reduced in the ducts of Ascl3 EGFP-Cre/EGFP-Cre salivary glands, although expression by the acinar cells remains unaffected (Fig. 1G). RT-PCR analysis of knockout glands failed to detect significant changes in the expression of additional markers, including the duct cell-specific transcription factor, grainyhead Cp2L1 (Yamaguchi et al., 2006), as well as several stem cell-associated factors: keratin 5, cKit, Sca-1, and Sox2 (data not shown).
As the bipotency of Ascl3+ progenitor cells is not lost in Ascl3 knockout mice, the role of these cells in salivary gland development and regeneration may be independent of Ascl3 function. To directly test the role of the Ascl3+ progenitor cells, we generated a cell-specific ablation model, using the Gt(ROSA)26Sortm1(DTA)Jpmb/J mouse strain (called R26DTA/+), in which the gene encoding diphtheria toxin A subunit (DTA) is inserted at the ubiquitously expressed Rosa locus (Ivanova et al., 2005). Expression of the DTA coding sequence is prevented by a strong transcriptional stop signal (Fig. 4A). When bred to Ascl3EGFP-Cre/+ heterozygotes, the loxP-flanked stop sequence is removed, and DTA expression is activated to produce conditional deletion of Cre-expressing cells. Double heterozygous animals (Ascl3EGFP-Cre/+/R26DTA/+) carry an intact copy of Ascl3 and the inserted EGFP-Cre cassette, as well as a single copy of the DTA gene at the Rosa locus. Single heterozygous (Ascl+/+/R26DTA/+) littermates carrying the inactive DTA gene were used as controls.
Double heterozygous Ascl3EGFP-Cre/+/R26DTA/+ mice were born at the expected Mendelian frequencies, although they were smaller and showed a high rate of morbidity. This was often manifested at the age of 2–3 weeks, although many animals survived to adulthood. As Ascl3 expression is not confined to the salivary gland (unpublished observations), we attribute the lower body mass and increased morbidity to other systemic effects.
Double heterozygous Ascl3EGFP-Cre/+/R26DTA/+ mice developed morphologically normal salivary glands, although the glands were significantly smaller in size than those of wild type littermates [Female Ascl+/+/R26DTA/+ 29.4 ± 5.3 mg (n=6; age = 2 month); Female Ascl3EGFP-Cre/+/R26DTA/+ 23.3 ± 2.9 mg (n=6; age = 2 month); P<0.05]. Measurements of duct surface area to total area were made on sections of submandibular gland from wild type, knockout and ablation mice. Although the Ascl3+ progenitor cells should be ablated, no significant difference in the duct cell to total cell ratio of the area was detected [Ascl+/+ 27.0 ± 5.1%; Ascl3EGFP-Cre/EGFP-Cre 25.4 ± 6.1%; Ascl3EGFP-Cre/+/R26DTA/+ 24.7 ± 6.4%; (n=9 measurements per group)].
To ascertain that the DTA locus is efficiently activated in Ascl3-expressing cells, we performed immunohistochemical analysis, taking advantage of the specific localization of the Nkcc1 and KCa1.1 transporters in Ascl3+ progenitor cells. Immunohistochemistry with antibody to Nkcc1 confirmed that activation of the DTA gene in the double heterozygotes caused nearly complete ablation of the Ascl3+ progenitor cells (Fig. 4B and C). In addition, nearly all KCa1.1-positive cells are absent from the ducts of double heterozygous Ascl3EGFP-Cre/+/R26DTA/+ mice (Fig. 4Dand E ). Although we have shown that duct-specific expression of Nkcc1 may be dependent on the presence of the Ascl3 transcription factor (Fig. 1G), the Ascl3EGFP-Cre/+/R26DTA/+ mice retain one intact copy of the Ascl3 locus (Fig. 4A), so Nkcc1 expression should be unaffected. We conclude that the absence of Nkcc1+ cells in the ducts of Ascl3EGFP-Cre/+/R26DTA/+ mice is due to the specific ablation of these cells by Cre-mediated activation of the DTA gene. Cre-positive cells were not found in Ascl3EGFP-Cre/+/R26DTA/+ animals, but were readily detected in control Ascl3EGFP-Cre/+/R26+/+ littermates (data not shown).
The acinar cells in salivary glands of Ascl3EGFP-Cre/+/R26DTA/+ mice appear to be normally differentiated, as determined by immunohistochemistry for several cell-specific markers, including aquaporin 5 (data not shown), and Nkcc1 (Fig. 4C). Furthermore, RT-PCR to test expression levels of the duct cell markers Cp2L1, cKit, Keratin 5, and Sca-1 did not reveal significant changes (data not shown).
To test the function of the glands in Ascl3EGFP-Cre/+/R26DTA/+ mice, ex vivo submandibular gland perfusion was performed, following stimulation with isoproterenol and carbachol, as described earlier (Romanenko et al., 2007). In comparison to wild type mice, both the flow rate and levels of total secretion were significantly reduced in the glands of Ascl3EGFP-Cre/+/R26DTA/+ mice (Fig. 5A and B ). However, when fluid secretion data were normalized to body weight, the total secretion was not significantly different from wild type levels (data not shown).
Through lineage tracing, we previously demonstrated that Ascl3+ progenitors are precursors of serous demilune cells in the sublingual gland (Bullard et al., 2008). These cells are distinguished by expression of the secreted demilune cell and parotid protein (Dcpp) (Bekhor et al., 1994). To examine the fate of serous demilune cells in the absence of Ascl3+ progenitors, an antibody to Dcpp was used to probe sublingual glands from the Ascl3EGFP-Cre/+/R26DTA/+ mice. Although the Ascl3+ progenitors have been ablated, we find only a slight decrease in the number of Dcpp-positive serous demilune cells in Ascl3EGFP-Cre/+/R26DTA/+ mice compared to control littermates (data not shown). This result implies that serous demilune cells can arise from at least two separate progenitor populations.
We next asked if Ascl3+ cells are essential for stress-induced tissue repair. To test the regenerative potential of these progenitor cells, a tissue injury model caused by ductal ligation was employed. Obstruction of the salivary gland excretory duct in rodents results in a near total loss of acinar cells, and atrophy of the gland (Tamarin, 1971; Walker and Gobe, 1987). If the obstruction is removed, the acinar cells are gradually replaced and the gland appears to completely recover, providing a model system for salivary gland regeneration (Takahashi et al., 2004; Tamarin, 1971; Walker and Gobe, 1987).
To test whether the Ascl3+cells might be essential for the regeneration of lost acinar cells, ductal ligation was performed on matched pairs of Ascl3EGFP-Cre/+/R26DTA/+ mice, and Ascl3EGFP-Cre/+/R26+/+ or Ascl3+/+/R26DTA/+ control littermates. Only one gland in each was ligated, and the contra-lateral glands served as controls. In all mice, the ductal ligation was removed after 10 days, and the atrophied gland was allowed to recover. Recovery periods ranged from 0 to 21 days. Glands taken on the day of ligation release (Release day=0) were analyzed to confirm that acinar cells were depleted (Fig. 5B, C). Sections taken at day =14 of recovery (Fig. 5E) show that the glands of Ascl3EGFP-Cre/+/R26DTA/+ mice are repopulated with acinar cells at a level comparable to that seen in control Ascl3EGFP-Cre/+/R26+/+ littermates (Fig. 5D). After 3 weeks of regeneration, the ligated glands of each genotype had regained the weight of the contra-lateral control gland. No significant difference in the acinar to duct cell ratio was detected. Thus, salivary gland regeneration following ductal ligation can proceed in the absence of the Ascl3+ progenitor cells. Taken together, our results suggest that salivary gland development and regeneration involve more than one progenitor-like cell type and that there is functional compensation in both mechanisms.
Maintenance of tissues in adult organisms is generally dependent on a population of tissue-specific stem or progenitor cells. We previously reported the identification of a progenitor cell population in the salivary gland that is characterized by expression of the bHLH transcription factor, Ascl3 (Bullard et al., 2008). These progenitor cells are located in the ducts of all three major salivary glands, and are precursors of both acinar and duct cells (Bullard et al., 2008). We have investigated the cellular and molecular properties of the Ascl3+ progenitor cells using both knockout and cell ablation mouse models. The relatively late onset of Ascl3 expression at embryonic day 15.5 argues against a function of Ascl3+ cells as early salivary gland stem cells. However, based on BrdU labeling studies and immunohistochemical data, we confirm that Ascl3+ cells represent a proliferating, bipotential cell population in the postnatal salivary gland. Furthermore, as Ascl3 expression is not detected in the differentiated progeny (Bullard et al., 2008), our results suggest that these cells are intermediate and transient progenitor cells that have either just divided or are undergoing division. However, with little or no self-renewal capacity, they do not conform to a strict definition of stem cells.
The Ascl3+ cells display an expression pattern that is molecularly distinct from surrounding duct cells, including at least two additional proteins, Nkcc1 and KCa1.1, both of which are normally found in acinar cells (Evans et al., 2000; Park et al., 2001). This could suggest direct regulation of the Nkcc1 and KCa1.1 genes by the Ascl3 transcription factor. The presence of four highly conserved E-box motifs in the Nkcc1 upstream genomic sequence (unpublished observations) supports this possibility, as does the fact that Nkcc1 (but not KCa1.1) expression is lost in the duct cells of Ascl3 knockout mice. However, the Ascl3 transcription factor is reported to be a transcriptional repressor (Yoshida et al., 2001), potentially making the regulation more complex, and further investigation will be needed to define the interactions of this factor.
Given the activities of the mammalian Ascl gene family proteins, Ascl1 and Ascl2, (Battiste et al., 2007; Guillemot et al., 1993; Moriyama et al., 2008; van der Flier et al., 2009), we investigated whether the Ascl3 transcription factor is required for the progenitor function of the Ascl3+ cells in the salivary gland. We found that in Ascl3 knockout mice, the salivary glands develop normally but are smaller than those of wild type littermates. However, the Ascl3 transcription factor is not required for bipotency of the progenitor cells, as both acinar and duct cell progeny are generated. The reduced size of the glands is associated with a decrease in cell proliferation, such as has been reported for olfactory bulb progenitors lacking Ascl1 (Murray et al., 2003). The mechanism, by which cell proliferation is reduced in these models, is not clear. Taken together, our data on the Ascl3 knockout indicate that the Ascl3 transcription factor is involved, but not essential, for the development and maintenance of functional salivary glands.
To directly investigate the role of the Ascl3+ progenitor cells within the salivary glands, we genetically ablated the progenitor cell population, using conditional activation of DTA in the Ascl3-expressing cells. The strict localization of Nkcc1 and KCa1.1 to Ascl3+ duct cells proves an advantage in confirming the efficiency of the ablation model. We have demonstrated the complete absence of duct cells expressing either protein in the Ascl3EGFP-Cre/+/R26DTA/+ mice. In the absence of the Ascl3+ progenitor cells, the Ascl3EGFP-Cre/+/R26DTA/+ mice develop functional salivary glands, although with a greater size defect than that observed in the knockout mice. The manifestation of this defect in both models supports our hypothesis that the Ascl3+ progenitor cells are involved in the maintenance of normal gland homeostasis.
However, the development of morphologically normal glands in the absence of the Ascl3+ progenitor cells indicates that more than one progenitor cell type must contribute to salivary gland development and maintenance. Our observation that the majority of serous acinar cells in the submandibular and parotid glands are not derived from Ascl3+ progenitor cells are consistent with this result (Bullard et al., 2008). We further tested the requirement for the Ascl3+ progenitor cells in an induced model of salivary gland injury and repair (Tamarin, 1971; Walker and Gobe, 1987). Surprisingly, although the glands of Ascl3EGFP-Cre/+/R26DTA/+ mice lack Ascl3+ progenitor cells, acinar cell regeneration occurred within 14 days and the extent of recovery was similar to that of controls. These results clearly demonstrate that the ablation of the entire Ascl3+ progenitor cell population does not impair gland development, function, or repair.
Our data substantiate the characterization of Ascl3+ cells as intermediate progenitors with the developmental plasticity to give rise to duct and acinar cells in the adult salivary gland. However, although Ascl3+ cells are active proliferating progenitors, they are not essential for salivary gland development, maintenance or repair. We propose that ablation of Ascl3+ progenitor cells is most likely tolerated through the compensatory action of additional distinct progenitor cell populations. The co-existence of subpopulations of stem or progenitor cells has been proposed to account for the observation that some tissues appear to harbor more than one stem cell-like population (Li and Clevers, 2010). The redundancy of multiple progenitor cell pools could provide a flexible mechanism for ensuring efficient maintenance and adequate repair of the salivary gland.
The authors would like to thank Dr. Tetsuji Nakamoto for establishing the ductal ligation surgical procedure, and Michael Rogers, Marilyn Elliot, Yasna Jaramillo, Laurie Koek, and Jennifer McLaughlin for excellent technical assistance. We thank Dr. Christopher Kaufman for assistance with experimental design. In addition we thank Drs. R. James Turner (NIDCR/NIH) and Stephen Konieczny (Purdue University) for the gifts of Nkcc1 and Mist1 antibodies, respectively. We are grateful to Drs. James Melvin, Dirk Bohmann and Matthew Hoffman for helpful discussions, and to Dirk Bohmann for critical reading of the manuscript. This work was supported by grants R01 DE008921 and R01 DE018896 from NIDCR (CEO).
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