Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.

The seromucous acinar cells of the adult rat submandibular gland secrete a characteristic mucin glycoprotein and a family of unusual glutamine/glutamic acid-rich proteins (GRP). Monoclonal antibodies to the mucin and GRP localized in a very few Type III cells in glands of newborn and 1 day-old rats, using light and electron microscopic immunocytochemistry. Both mucin and GRP reactivities were present in the polymorphic Type IIIP granules during the 1st postnatal week. By 9 days after birth, the granules contained both mucin and GRP and were mucous-like in appearance. At earlier stages, however, cells containing only GRP or mucin could be found, indicating that the initiation of GRP and mucin biosynthesis may not be coordinately regulated. No reactivity was seen in the neonatal Type I cells or in duct cells at any age. Northern and Western blot analysis showed GRP mRNA and protein levels to be barely detectable at birth, with marked increases during the first 2 postnatal weeks. In contrast, Western blots of B1-immunoreactive proteins (B1-IP) showed levels highest in the 1st week and markedly decreased in the adult. Immunocytochemical colocalization, using gold particles of different sizes, showed that the B1-IP, mucin, and GRP colocalized in the granules. These results strengthen the hypothesis that the adult acinar cells develop from the neonatal Type III cells. No evidence was obtained for the involvement of Type I cells in the pathway of acinar cell development.

Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP-1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous-like apical granules that labeled with anti-mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti-SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP-1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti-SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti-mucin, and serous cells had numerous dense granules containing CSP-1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti-SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well-formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells. Anat Rec 266: 30 -42, 2002.

The B1-immunoreactive proteins (B1-IPs) are major secretory products of rat submandibular gland acinar-cell progenitors, and are also produced by neonatal and adult rat sublingual and parotid glands. In order to characterize the B1-IPs, we have previously isolated cDNA clones encoding rat parotid secretory protein (PSP; the predominant parotid B1-IP) and the related clone ZZ3, which is developmentally regulated in the neonatal submandibular gland. The remainder of the B1-IPs were uncharacterized. This report demonstrates that all of the B1-IPs are derived from the PSP and ZZ3 transcripts. Molecular cloning and Western-blot analyses using PSP- and ZZ3-specific antisera show that, of the B1-IPs, only PSP and neonatal submandibular gland protein A (SMGA) are products of the Psp gene. This finding corrects our previous assertion that SMGA is derived from ZZ3. Neonatal submandibular gland proteins B1 and B2, as well as apparent Mr 26000-28000 and Mr 18000-20000 forms in submandibular, sublingual and parotid glands, are derived from the gene encoding ZZ3 by differential N-glycosylation and by proteolytic cleavage. The apparent Mr 18000-20000 proteolytic products are significant in secretion product collected in vitro, but rare in gland homogenate and submandibular/sublingual saliva. The gene encoding ZZ3 has been named Smgb. Psp and Smgb are regulated similarly in the developing submandibular gland, but differently in the sublingual and parotid glands. The expression pattern of Psp is conserved between rat and mouse. However, no evidence for proteins derived from an Smgb-like gene was observed in neonatal mouse submandibular or sublingual glands.

Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 20-22,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.