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 KCa
1.1, 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
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 The presence of four highly conserved E-box motifs in the Nkcc1 upstream genomic sequence (unpublished observations) supports this possibility, as does the fact that Nkcc1 (but not KCa
1.1) 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.