Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Localization of a mouse submandibular sialomucin by indirect immunofluorescence

Summary A sialomucin from the mouse submandibular gland was localized in the gland by indirect immunofluorescence. Fluorescence was localized over the acinar cells and, to a lesser extent, in the lumen of the ducts. The mucin antiserum did not show cross-reactivity with cells from the sublingual gland or with other mucous-producing cells from the respiratory and gastrointestinal tracts of the mouse, or with salivary gland tissue of the rat. The sialomucin lacks both sulphate andl-fucose. Localization of a mucin with such a composition, within acinar cells of the mouse submandibular gland, is consistent with previous observations from histochemistry and autoradiography.     

Spatial and temporal expression of c-Kit in the development of the murine submandibular gland

The c-Kit pathway is important in the development of many mammalian cells and organs and is indispensable for the development of hematopoiesis, melanocytes, and primordial germ cells. Loss-of-function mutations in c-Kit lead to perinatal death in mouse embryos. Previously, c-Kit has been used as one of salivary epithelial stem or progenitor cell markers in mouse, its specific temporo-spatial expression pattern and function in developing murine submandibular gland (SMG) is still unclear. Here we used quantitative real-time PCR, in situ hybridization, and immunohistochemistry analysis to detect c-Kit expression during the development of the murine SMG. We found that c-Kit was expressed in the epithelia of developing SMGs from embryonic day 11.5 (E11.5; initial bud stage) to postnatal day 90 (P90; when the SMG is completely mature). c-Kit expression in the end bud epithelium increased during prenatal development and then gradually decreased after birth until its expression was undetectable in mature acini at P30. Moreover, c-Kit was expressed in the SMG primordial cord at the initial bud, pseudoglandular, canacular, and terminal end bud stages. c-Kit was also expressed in the presumptive ductal cells adjacent to the developing acini. By the late terminal end bud stage on P14, c-Kit expression could not be detected in ductal cells. However, c-Kit expression was detected in ductal cells at P30, and its expression had increased dramatically at P90. Taken together, these findings describe the spatial and temporal expression pattern of c-Kit in the developing murine SMG and suggest that c-Kit may play roles in epithelial histo-morphogenesis and in ductal progenitor cell homeostasis in the SMG.     

Changes in adhesive properties of epithelial cells during early morphogenesis of the mammary gland

Previous studies have demonstrated that cell adhesion systems are downregulated in epithelial buds at the earliest stages of submandibular gland and hair follicle development, but are restored at subsequent stages. Here it is shown that epithelial cell adhesion systems are also remodeled during early mammary gland development. Immunofluorescence and electron microscopy of the mouse mammary bud demonstrated that cell-cell adhesion systems were hardly detectable, with significant downregulation of expression of desmosomal molecules, but not of E-cadherin and beta-catenin. Hemidesmosomal structures were also rarely found, although their component molecules were expressed. Differences in cell adhesivity between cells of the mammary bud and those of the overlying epidermis were shown by the finding that the mammary cells formed smaller aggregates than the epidermal cells and were not randomly mixed with the epidermal cells. At subsequent stages, the mammary epithelium restored cell-cell adhesion systems along with de novo expression of tight junction molecules. These data, together with previous findings, indicate that remodeling of epithelial cell adhesion systems is a general feature underlying the early development of several ectoderm-derived organs and support the idea that segregation and rearrangements of cells are involved in early epithelial morphogenesis.     

Reduced levels of insulin-like growth factor-1 receptor (IGF-1R) suppress cellular signaling in experimental autoimmune sialadenitis (EAS).

The nonobese diabetic mouse (NOD) develops destruction and functional impairment of salivary and lachrymal glands, experimental autoimmune sialadenitis (EAS), resembling and representing a model for Sjogren's syndrome (SS). To investigate the mechanisms of tissue destruction in EAS, we analyzed a cell survival promoter insulin-like growth factor-1 receptor (IGF-1R) in the submandibular glands of NOD mice with this disease. We also evaluated the expression of a downstream effector of IGF-1R, BAD. Receptor-binding autoradiography revealed that the IGF-1R levels in submandibular glands from young NOD mice were lower than those in adult NOD mice. Immunofluorescence staining demonstrated that BAD expression in the epithelial cells of the submandibular gland was consistently enhanced throughout the course of EAS in NOD mice. These findings suggest that a reduction in the levels of IGF-1R induces a defective glandular homeostasis in the submandibular gland epithelial cells and triggers EAS.     

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.     

Elafin expression in human fetal and adult submandibular glands

Elafin, a bifunctional protein, has the NH(2)-terminal domain functions as a transglutaminase substrate for crosslinking to lysine-containing proteins and the COOH-terminal whey acidic protein domain as a potent anti-elastase. Human fetal submandibular glands (n=100) and adult submandibular glands (n=10) were used to elucidate the expression pattern of elafin in the developmental processes of human submandibular gland by immunohistochemistry, in situ hybridization, and western blot analysis. Elafin mRNA was expressed both in the gland epithelium and intralobular mesenchymal tissue of fetal submandibular gland in an early developmental stage (10-18 weeks) and an early intermediate developmental stage (EIDS; 19-24 weeks). The elafin antigen was also found in the intralobular mesenchyme of submandibular gland in the same stages. Thereafter, elafin was transitionally expressed in the ducts and acini of submandibular glands. In the late intermediate developmental stage (LIDS; 25-32 weeks) and the late developmental stage (LDS; 33-40 weeks), elafin became markedly positive in the excretory and striated ducts but weakly positive in the intralobular mesenchymal tissue. The elafin was heavily present in the excretory and striated ducts of adult submandibular gland, while it was sparse in the intralobular mesenchymal tissues. Western blot analysis showed the protein extracts from submandibular glands of EIDS, LIDS, and LDS, adult submandibular gland, fetal tissues (8 weeks), and adult parotid saliva migrated into multiple bands, about 25, 50, 65, and 140 kDa, which were higher than the putative size of elafin protein, 12 kDa. These data suggest that elafin, anti-elastase, is an essential component highly utilized during the morphogenetic processes of fetal salivary gland development and continuously plays a role for the protection of the tubuloalveolar structures of adult salivary gland.     

Study of the growth factors for the mammary gland: Epidermal growth factor and mesenchyme-derived growth factor

The physiological importance of EGF in the development of the mouse mammary gland during pregnancy and in spontaneous mammary tumorigenesis has been documented by a series of experimental results presented herein. In our study, we have taken a variety of experimental approaches including radioimmunoassay of EGF in the submandibular gland and plasma, mammary gland organ/cell culture, EGF receptor assay, sialoadenectomy and treatment with EGF and EGF antibodies to assess the role of EGF in the mammary gland. In particular, studies employing sialoadenectomy and EGF replacement have provided valuable information concerning the function of EGF in the body. These studies are possible in the mouse system because the submandibular gland serves as a major source of circulating EGF and also because purified mouse EGF is available commercially. Our work on the biological, endocrinological, and physiological aspects of EGF in normal and neoplastic growth of the mammary gland should be useful for the study of the regulation of mammary gland growth at the molecular level as well as for clinical investigations of mammary tumors. Finally, our findings of a mammary growth factor in embryonic mesenchymal cultures suggest the possible involvement of paracrine growth factor(s) in mammary cell growth. Further progress in this area is needed for better understanding of the complex process of mammary gland growth and development.     

Distribution of small Rho GTPases in the developing rat submandibular gland

During the rat submandibular gland (SMG) development, organogenesis and cytodifferentiation depend on the actin cytoskeleton, which is regulated by small Rho GTPases. These proteins link cell surface receptors to pathways that regulate cell motility, polarity, gene expression, vesicular trafficking, proliferation and apoptosis. The aim of this study was to evaluate, by immunohistochemistry, the distribution pattern of RhoA, RhoB, RhoC, Rac1 and Cdc42 during cytodifferentiation of the rat SMG and in male adults. All GTPases were found in epithelial and mesenchymal tissues throughout gland development. Rac1 appeared to be important for parenchyma expansion at the beginning of cytodifferentiation, while RhoC, Cdc42 and the inactive phosphorylated form of Rac1 seemed associated with lumen formation and cell polarization in terminal tubules. RhoA and RhoB labeling was evident throughout development. All GTPases were differentially expressed in the adult gland, suggesting that they play specific roles during differentiation and function of the rat SMG.     

A pituitary-salivary mixed gland induced by tissue recombination of embryonic pituitary epithelium and embryonic submandibular gland mesenchyme in mice

Renal subcapsular syngrafts of Day 9 to 11 mouse embryonic pituitary epithelium with Day 14 mouse embryonic submandibular gland mesenchyme produced mixed organs that include residual cleft structure surrounded by anterior pituitary cells some which are stained by anti-ACTH antiserum and submandibular gland-like structure with differentiated acinar cells which are stained by anti-alpha-amylase antiserum. However, when Day 8.5 or 12 embryonic pituitary epithelium was recombined with submandibular gland mesenchyme and syngrafted, development of submandibular gland-like or anterior pituitary tissues resulted, respectively. Thus, during organogenesis of the mouse anterior pituitary, there exists a developmental stage (Day 8.5-11 in utero), when prospective pituitary epithelium can respond to heterotypic submandibular gland mesenchyme with the development of a submandibular gland-like tissue.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Epithelial morphogenesis in mouse embryonic submandibular gland: Its relationships to the tissue organization of epithelium and mesenchyme

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force-generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell-cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.     

Loss of LETS protein is not a marker for salivary gland or bladder epithelial cell transformation

LETS protein was demonstrated by indirect immunofluorescence techniques in adult mouse submandibular gland and bladder epithelial cells at various stages during neoplastic transformation, after the application of a chemical carcinogen in vitro. There was no clear relationship between transformation and the loss of LETS protein; most normal and preneoplastic epithelium lacked the distinctive fibrillar distribution of fluorescent staining. In contrast, some carcinoma-producing cell lines did possess appreciable amounts of LETS protein.     

Prenatal development of the human submandibular gland

The prenatal development of the human submandibular gland has been investigated in 26 fetuses from the 10th week of gestation to full term. At 10-12 weeks, the glandular elements (primitive ducts and acini) were immature and surrounded by a loose mesenchyme. The acinar cell population increased gradually till the age of 32 weeks, and the rate of increase was diminished thereafter. At 16 weeks, intercalated and striated ducts were distinguished and their number increased till the age of 32 weeks when their number seemed to be stabilized. The development of the granular convoluted tubule cells from the proximal segments of striated ducts occupied the later stages of development. They appeared around the age of 20 weeks and proceeded till full term. At birth, the gland appeared devoid of mucous acini and fat cells and the secretory end-pieces were of the serous type. During the second trimester, periodic acid-Schiff- and alcian blue-positive secretory materials appeared in the epithelial cells of both ducts and acini, and in their lumina. This secretory activity was transitory and disappeared around the age of 28 weeks. The possible function of these secretory products is discussed.     

Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.     

Generation of a Novel MMTV-tTA Transgenic Mouse Strain for the Targeted Expression of Genes in the Embryonic and Postnatal Mammary Gland

We have generated a new and improved transgenic mouse strain that permits a temporally controlled expression of transgenes throughout mammary gland development. High expression of the tetracycline-regulatible transactivator (tTA) under control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) is restricted to mammary epithelial cells and the salivary gland. The novel MMTV-tTA mouse strain induces a sustained transactivation of responder transgenes, which can be swiftly suppressed through administration of doxycycline (Dox). An important characteristic of this strain is its expression in early progenitor cells of mammary gland anlagen beginning at day 13.5 of embryonic development. We show here that the MMTV-tTA can be used in combination with GFP reporter strains to visualize CK8/CK14-dual positive progenitors in newborn females and their derived basal and luminal epithelial cell lineages in adult females. Our observations suggest that the novel MMTV-tTA can be utilized to express exogenous proteins in multipotent mammary progenitors during the earliest stages of mammary gland development to assess their biological significance throughout mammogenesis. Moreover, we demonstrate that the expression of the MMTV-tTA is sustained during mammary gland tumorigenesis in female mice expressing wildtype ErbB2. This makes this strain particular valuable to target the expression of exogenous proteins into developing mammary tumors to assess their significance in biological processes, such as tumor cell growth and survival, metabolism, and metastasis.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.