Development Of Mouse Submandibular Gland Research Articles

NFIB Regulates Embryonic Development of Submandibular Glands

NFIB (nuclear factor I B) is a NFI transcription factor family member, which is essential for the development of a variety of organ systems. Salivary gland development occurs through several stages, including prebud, bud, pseudoglandular, canalicular, and terminal. Although many studies have been done to understand mouse submandibular gland (SMG) branching morphogenesis, little is known about SMG cell differentiation during the terminal stages. The goal of this study was to determine the role of NFIB during SMG development. We analyzed SMGs from wild-type and Nfib-deficient mice (Nfib−/−). At embryonic (E) day 18.5, SMGs from wild-type mice showed duct branching morphogenesis and differentiation of tubule ductal cells into tubule secretory cells. In contrast, SMGs from Nfib−/− mice at E18.5 failed to differentiate into tubule secretory cells while branching morphogenesis was unaffected. SMGs from wild-type mice at E16.5 displayed well-organized cuboidal inner terminal tubule cells. However, SMGs from Nfib−/− at E16.5 displayed disorganized inner terminal tubule cells. SMGs from wild-type mice at E18.5 became fully differentiated, as indicated by a high degree of apicobasal polarization (i.e., presence of apical ZO-1 and basolateral E-cadherin) and columnar shape. Furthermore, SMGs from wild-type mice at E18.5 expressed the protein SMGC, a marker for tubule secretory cells. However, SMGs from Nfib−/− mice at E18.5 showed apicobasal polarity, but they were disorganized and lost the ability to secrete SMGC. These findings indicate that the transcription factor NFIB is not required for branching morphogenesis but plays a key role in tubule cell differentiation during mouse SMG development.

Immunohistochemical localization of barx2 in the developing fetal mouse submandibular glands.

The development of mouse submandibular gland (SMG) begins at embryonic day 11.5–12 (E11.5–12), during which successive rounds of epithelial clefting and branching create complex epithelial tree-like structures. Homeobox genes regulate place-dependent morphogenesis, including epithelial-mesenchymal interactions, and control the expression patterns of signaling molecules. The Barx2 containing Homeobox exerts several key roles in development. Some studies have shown that the Barx2 plays important roles in the epithelial-mesenchymal interactions of organogenesis. However, the mechanisms of Barx2 associated with the development of SMG are obscure. In this study, we demonstrated for the first time the exact spatial and temporal Barx2 expression pattern in SMG epithelial tissue during development using immunohistochemical staining and Real-Time quantitative PCR. Barx2 was expressed in the nucleus of the epithelial cells located in the proliferative and differentiative regions of the developing SMG during the early development stages (E11.5–E13.5). After the E14.5-time period, the expression gradually decreased, and at E16.5, expression mostly disappeared despite the fact that evidence of cytodifferentiation, such as the appearance of proacinar cells, distinct lumen formation, and secretory products, was beginning to be observed. Results of Real-Time PCR demonstrated that the amount of Barx2 mRNA expression in SMG was maximal on E14.5, and gradually decreased by E18.5. These results indicate that Barx2 is associated with early stage epithelial tissue development, and can be a useful epithelial marker of the SMG during early developmental stages.

Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms.

Analyses of gene expression profiles at five different stages of mouse submandibular salivary gland development provide insight into gland organogenesis and identify genes that may be critical at different stages. Genes with similar expression profiles were clustered, and RT-PCR was used to confirm the developmental changes. We focused on fibroblast growth factor receptor 1 (FGFR1), as its expression is highest early in gland development. We extended our array results and analyzed the developmental expression patterns of other FGFR and FGF isoforms. The functional significance of FGFR1 was confirmed by submandibular gland organ culture. Antisense oligonucleotides decreased expression of FGFR1 and reduced branching morphogenesis of the glands. Inhibiting FGFR1 signaling with SU5402, a FGFR1 tyrosine kinase inhibitor, reduced branching morphogenesis. SU5402 treatment decreased cell proliferation but did not increase apoptosis. Fgfr, Fgf and Bmp gene expression was localized to either the mesenchyme or the epithelium by PCR, and then measured over time by real time PCR after SU5402 treatment. FGFR1 signaling regulates Fgfr1, Fgf1, Fgf3 and Bmp7 expression and indirectly regulates Fgf7, Fgf10 and Bmp4. Exogenous FGFs and BMPs added to glands in culture reveal distinct effects on gland morphology. Glands cultured with SU5402 were then rescued with exogenous BMP7, FGF7 or FGF10. Taken together, our results suggest specific FGFs and BMPs play reciprocal roles in regulating branching morphogenesis and FGFR1 signaling plays a central role by regulating both FGF and BMP expression.

Regulation of cadherin junctions during mouse submandibular gland development.

Submandibular gland (SMG) development involves branching morphogenesis of the salivary epithelium into the surrounding mesenchyme, accompanied by proliferation and differentiation of immature salivary cells along acinar and ductal cell lineages. During development, salivary cell sorting and cell-cell adhesion are likely to be directed by cadherin adhesion receptors. We show that two classic cadherins, N- and E-cadherin, participate in SMG development. Early in embryonic morphogenesis, both cadherins displayed diffuse staining with regionalized localization to cell-cell borders. At this stage, significant pools of N- and E-cadherins were Triton-soluble, suggesting that fractions of these molecules were not localized to stable junctional complexes associated with the actin cytoskeleton. With cytodifferentiation, cadherins became progressively Triton-insoluble, and this correlated with their organization at cell-cell interfaces. In the cytodifferentiated SMG, N-cadherin was absent, whereas E-cadherin remained at cell-cell interfaces. Early in morphogenesis, beta-catenin was also primarily Triton-soluble, and its association with the actin cytoskeleton and localization to the adherens junctions increased with cytodifferentiation. Greater recruitment of cadherins and beta-catenin to cell-cell borders was paralleled by changes in membrane association of two Rho GTPases, Cdc42 and RhoA. N-cadherin was detected only at early stages of postnatal development, whereas E-cadherin and beta-catenin became progressively Triton-insoluble during differentiation. Our results indicate that N-cadherin functions transiently in SMG development. On the other hand, E-cadherin and beta-catenin appear to play different roles during tissue organization and cytodifferentiation. In early morphogenesis, E-cadherin and beta-catenin are likely to participate in SMG remodeling, whereas during cytodifferentiation, they form stable cell-cell contacts, and may collaborate with Rho GTPases in the establishment and maintenance of salivary cell polarity.

Extracellular-matrix gene expression during mouse submandibular gland development

Early morphogenesis of mouse submandibular glands begins on late day 11 of fetal development when the epithelium begins to bud from the surrounding mandibular mesenchyme. Using total RNA collected from fetal BALB/c submandibular glands, steady-state levels of mRNA expression for extracellular matrix molecules were measured using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). By comparing the PCR amplification products of both the cellular mRNA and a synthetic template, pMATRIX, it was possible to measure the direct expression of collagens α2(I), α1(111), αl(IV), fibronectin, laminin B2, elastin and lysyl oxidase genes. There was an observed trend for an increasing concentration of collagen α2(I), collagen al(III) and lysyl oxidase mRNA molecules per cell on day 16 of development. The relative abundance of elastin mRNA was detectable only on day 16. Fibronectin and laminin B2 were more constitutively present but had their highest copy number per cell on day 16. The presence of extracellular-matrix protein was confirmed by immunohislochemistry using day-16 fetal glands and adult glands. With the construction of the pMATRIX supertemplate and the advent of quantitative, competitive RT-PCR technology, it has been possible to measure small changes in the steady-state concentrations for extracellular-matrix mRNA during salivary gland development.

Distribution of alpha 6 integrin subunit in developing mouse submandibular gland.

We examined the development of mouse submandibular gland by light and electron microscopy and determined the distribution of the alpha 6 integrin subunit and laminin in this process by immunofluorescence and immunoelectron microscopy. At Days 13.5 and 14 of gestation alpha 6 was localized over the entire cell surface of undifferentiated epithelial cells of the terminal cluster. On Day 15 the expression of alpha 6 could no longer be detected over central cells in the proximal portion of branched terminal cluster, whereas peripheral cells were stained over the entire surface. On Day 17 of gestation to day of birth, alpha 6 expression was restricted to the basolateral surface of the differentiated acinotubular structure. Its expression on acinar cells was uniformly distributed throughout the basolateral membrane, but on duct cells stronger staining towards the basal surface was noted. Similar expression was observed in adult acinar and duct cells. Expression of the alpha 6-subunit at the cell-cell contact in an early stage and its expression at the basolateral surface in an advanced stage indicate that integrin containing alpha 6 plays a significant role in cell-cell and cell-substrate interaction. Stage and cell type-specific change of integrin expression may be significant for submandibular development.

Appearance of acinar-cell-specific mucin in prenatal mouse submandibular glands

Abstract The appearance of an acinar-cell-specific mucin was studied during fetal mouse submandibular gland development. The mucin was first detected in stage 23 and was quantitated through birth by radioimmunoassay (RIA). Quantitation results showed that the mucin accumulation was biphasic. Results from Western blotting and radioimmunoassay indicated that the mucin from the prenatal glands was similar both antigenically and in size to the mucin isolated from adult mice. Observations from light microscopy revealed a continuing progression of complexity throughout prenatal development, indicative of morphogenesis charateristic of differentiating exocrine tissues. When sections from various stages were compared morphometrically, it became clear that the overall ratio of epithelial cells to mesenchymal cells increased nearly 6-fold throughout the prenatal stages observed. The study suggests that acinar cell development in the mouse submandibular gland passes through a protodifferentiated stage. The proportions of epithelial and mesenchymal cells in the submandibular gland and the sensitivity of the RIA indicate that the mucin per cell actually increased to detectable levels at the onset of protodifferentiation, and this increase does not reflect a change in the relative proportions of epithelial and mesenchymal cells.