Cellular dynamics of epithelial clefting during branching morphogenesis of the mouse submandibular gland

We cultured the rudimental submandibular gland (SMG) of mice with a non–cell‐permeable fluorescent tracer, and observed cell behavior during epithelial branching morphogenesis using confocal time‐lapse microscopy. We traced movements of individual cells as shadowgraph movies. Individual epithelial cells migrated dynamically but erratically. The epithelial cleft extended by wiggling and separated a cluster of cells into two buds during branching. We examined the ultrastructure of the clefts in SMG rudiments treated with the laminin peptide A5G77f, which induces epithelial clefting. A short cytoplasmic shelf with a core of microfilaments was found at the deep end of the cleft. We propose that epithelial clefting involves a dynamic movement of cells at the base of the cleft, and the formation of a shelf within a cleft cell. The shelf might form a matrix attachment point at the base of the cleft with a core of microfilaments driving cleft elongation. Developmental Dynamics 239:1739–1747, 2010. © 2010 Wiley‐Liss, Inc.     

Branching morphogenesis in the embryonic mouse submandibular gland: a scanning electron microscopic study

The embryonic development of the salivary gland in the mouse was studied by scanning electron microscope (SEM). The submandibular morphogenesis was found to take place within its associated mesenchymal tissue. This stromal mesenchymal element has prevented us from observing three-dimensional details of the branching morphogenesis by SEM. In the present study, we applied a modified Evan's enzymatic digestion method to the embryonic submandibular gland. Consequently, we obtained important information about the morphogenetic events for the branching development of the mouse submandibular gland, demonstrating that a pit and shallow clefts are formed on the lateral aspect of the spherical end bud. This pit and cleft formation likely represents the initial morphogenetic movement of the epithelium that establishes primary branchings. Furthermore, SEM observation of the basal surface of the embryonic epithelial cells provided us views of the developmental change of cell projections probably involving epithelial-mesenchymal contacts.     

EGF-stimulated signaling by means of PI3K, PLCgamma1, and PKC isozymes regulates branching morphogenesis of the fetal mouse submandibular gland.

Branching morphogenesis of fetal mouse submandibular glands (SMGs) partly depends on the epidermal growth factor (EGF) receptor that triggers at least three intracellular signaling pathways involving (1) the mitogen-activated protein kinases ERK-1/2, (2) phospholipase Cgamma1 (PLCgamma1), and (3) phosphatidylinositol-3-kinase (PI3K). PLCgamma1 directly activates protein kinase C (PKC) isozymes; PI3K stimulates protein kinase B (PKB, also known as Akt), which ultimately activates PKCs and other proteins. We reported that the pattern of phosphorylation of ERK-1/2 in response to EGF in SMGs varies with fetal age and that blockade of EGF-stimulated ERK-1/2 signaling partially inhibits branching (Kashimata et al. [2000] Dev. Biol. 220:183-196). Here, we report on components of the PLCgamma1, PI3K, and PKC families of signaling molecules in fetal SMGs from the 13th day of gestation to postnatal ages. Western blotting revealed that (1) PLCgamma1 is present from E13 to E18 but drops off precipitously to negligible levels on the day of birth and thereafter, and (2) PI3K, PKB(Akt), and several PKC isozymes are expressed from E13 onward through adult life. Both PLCgamma1 and PI3K are phosphorylated in response to EGF. Inhibition of PI3K by LY294002 inhibited EGF-stimulated branching, but inhibition of PLCgamma1 by U73122 had no effect. Western blotting showed that the concentrations of 8 PKC isozymes vary with age in the fetal and postnatal SMG. However, general inhibition of PKCs by Calphostin C or specific inhibition of PKCalpha or of PKCepsilon by G?6976 or Ro-32-0432, respectively, increased EGF-stimulated branching. Calphostin C also increased EGF-stimulated phosphorylation of ERK-1/2. These findings indicate that signaling from the EGF receptor in the fetal mouse SMG varies with development and triggers stimulatory effects by means of ERK-1/2 and PI3K but inhibitory effects by means of PKC isozymes.     

Embryonic mouse submandibular salivary gland morphogenesis and the TNF/TNF-R1 signal transduction pathway

TNF is a pleiotropic cytokine that modulates cell proliferation and apoptosis. The objective of the present study was to investigate the possible function(s) of the TNF/TNF-R1 signaling pathway in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro experiments to investigate the phenotypic outcomes of enhanced and deficient ligand. The results of these experiments indicate that the TNF/TNF-R1 signal transduction pathway plays an important role in balancing cell proliferation and apoptosis during SMG duct and presumptive acini formation.     

Expression of ErbB3, ErbB4, and neuregulin-1 mRNA during tooth development.

The receptor tyrosine kinases ErbB3 and ErbB4, which bind to various variants of neuregulin (NRG), play fundamental roles in neural development and in organs, which form through epithelial-mesenchymal interactions. Here, we demonstrate that NRG-1 and the receptors ErbB3 and ErbB4 are expressed locally during rodent tooth development. However, the mRNA expression patterns of ErbB3 and ErbB4 were distinctly different during odontogenesis. Examinations of teeth in genetically heart-rescued ErbB4-/- mice did not reveal any obvious deviation from the normal phenotype. The results suggest that ErbB3 and ErbB4 may participate in tooth morphogenesis. The specific interactions between NRG isoforms and ErbB receptors during this process remain to be determined.     

Embryonic mouse submandibular salivary gland morphogenesis and the TNF/TNF‐R1 signal transduction pathway

TNF is a pleiotropic cytokine that modulates cell proliferation and apoptosis. The objective of the present study was to investigate the possible function(s) of the TNF/TNF‐R1 signaling pathway in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro experiments to investigate the phenotypic outcomes of enhanced and deficient ligand. The results of these experiments indicate that the TNF/TNF‐R1 signal transduction pathway plays an important role in balancing cell proliferation and apoptosis during SMG duct and presumptive acini formation. Anat Rec 262:318–330, 2001. © 2001 Wiley‐Liss, Inc.     

Early myoclonic encephalopathy caused by a disruption of the neuregulin-1 receptor ErbB4

The tyrosine kinase receptor ErbB4 (erythroblastic leukemia viral oncogene homolog 4) plays a crucial role in numerous neurobiological processes in the developing and adult brain. Moreover, recent molecular genetics studies implicate ErbB4 in the pathophysiology of schizophrenia. However, the phenotypic consequences of haploinsufficiency of ErbB4 are not known, as no coding mutations have been identified until now. Here, we present a patient with early myoclonic encephalopathy and profound psychomotor delay with a de novo reciprocal translocation t(2;6)(q34;p25.3), disrupting the ErbB4 gene. This patient represents the first case of haploinsufficiency for one of the ErbB family members of tyrosine kinase receptors.     

Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland

Temporary accumulation of glycogen in the epithelial cells of the developing mouse submandibular gland was examined under light microscopic histochemistry and electron microscopy. To avoid loss of water-soluble glycogen during histological tissue preparation, fixation with ethanol and embedding in hydrophilic glycol methacrylate resin was used for light microscopy, and high-pressure freezing/freeze substitution for electron microscopy. Glycogen was detected on periodic acid-Schiff stain, periodic acid-thiosemicarbazide-silver proteinate reaction, and the digestion test with alpha-amylase. On embryonic day 14, glycogen began to accumulate in the proximal portions of the developing epithelial cords. On embryonic day 17, marked glycogen particles were seen at the basal portion of the ductal epithelial cells and an abrupt increase of glycogen accumulation occurred in the secretory cells in the terminal bulbs. Ultrastructural observation indicated large clumps of glycogen particles localized in the basal portion of the terminal bulb cells. The initiation of glycogen accumulation preceded the formation of lumens in the ducts and terminal bulbs. Furthermore, proliferation analysis by bromodeoxyuridine labeling showed that this glycogen accumulation followed the cessation of the epithelial cell proliferation. Postnatally, glycogen accumulation in the terminal bulbs became gradually inconspicuous and completely disappeared by postnatal day 3, but that in the ducts was retained until around postnatal day 12. Temporary glycogen accumulation after the cell proliferation and before/during the lumen formation and secretory granule formation suggests significant involvement of the carbohydrate metabolism in the organogenesis of the submandibular gland.     

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.     

Sphingosine kinase 1 regulates differentiation of human and mouse lung fibroblasts mediated by TGF-beta1

Transforming growth factor beta (TGF-beta) contributes to the progression of pulmonary fibrosis through up-regulation of alpha-smooth muscle actin (alpha-SMA) as lung myofibroblast differentiation. Bioactive sphingosine 1-phosphate (S1P) has been shown to mimic TGF-beta signals; however, the function of S1P in lung fibrotic process has not been well documented. We found, in a mouse model of bleomycin lung fibrosis, that SPHK1 and alpha-SMA were colocalized within lung fibrotic foci and that these expressions were significantly increased in primary cultured fibroblasts. Using human lung fibroblasts WI-38, we explored the rationale of sphingosine kinase (SPHK) with TGF-beta1 stimulation. SPHK inhibitors and small interference RNA (siRNA) targeted SPHK1 decreased alpha-SMA and fibronectin expression up-regulated by TGF-beta1. In the meantime, SPHK1 inhibition did not affect smad2 phosphorylation in response to TGF-beta1. Then we examined whether S1P receptors transactivation may affect TGF-beta signals. siRNA against S1P(2) and S1P(3), but not S1P(1), reduced alpha-SMA expression as well as Y-27632, Rho kinase inhibitor. We also detected activation of Rho GTPase upon stimulation of TGF-beta1 on the cell membrane where S1P(2) or S1P(3) was overexpressed. These data suggested that SPHK1 activation by TGF-beta1 leads to Rho-associated myofibroblasts differentiation mediated by transactivated S1P receptors in the lung fibrogenic process.     

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.     

Three-dimensional observation of blood vessels supplying the gland proper and capsule of the mouse submandibular gland.

Previous studies have shown that submandibular glands suffering interruption of the blood circulation from the main supplying artery have more surviving parenchymal cells in their peripheral portion than in their central portion. Although the reason for this difference between the peripheral and central portions has not yet been clarified, the existence of collateral circulation has been suspected. The present study was designed to examine whether or not the vessels binding the gland proper to the capsule provide such collateral circulation. Silicone rubber or methacrylate was injected into the main artery supplying the mouse submandibular gland, in which the gland proper is wrapped by a capsule similar to that of the human submandibular gland, and then the gland was observed by both stereoscopic and scanning electron microscopy. Three-dimensional observations showed no communicating vessels between the gland proper and the capsule. Therefore, it is suggested that the parenchymal cells surviving in the ischemic peripheral portion of the submandibular gland are nourished by permeation of tissue fluid contained in the capsule.     

ErbB2 and ErbB3 regulate recovery from dextran sulfate sodium-induced colitis by promoting mouse colon epithelial cell survival

ErbB2 and ErbB3 receptor tyrosine kinases are key regulators of proliferation, migration, differentiation and cell survival; however, their roles in gastrointestinal biology remain poorly defined. We hypothesized that ErbB2 and ErbB3 promote colon epithelial cell survival in the context of the wound-healing response following colitis. In this study, mice bearing intestinal epithelial-specific deletion of ErbB2 or ErbB3 were treated with dextran sulfate sodium (DSS). Colon sections were examined for injury, cytokine expression, epithelial cell proliferation and apoptosis. Deletion of epithelial ErbB2 did not affect the extent of intestinal injury in response to DSS, whereas deletion of ErbB3 slightly increased injury. However, the roles of both receptors were more apparent during recovery from DSS colitis, in which ErbB2 or ErbB3 epithelial deletion resulted in greater inflammation and crypt damage during the early reparative period. Moreover, loss of ErbB3 prevented normal epithelial regeneration in the long term, with damage persisting for at least 6 weeks following a single round of DSS. Delayed recovery in mice with epithelial deletion of ErbB2 or ErbB3 was associated with increased colonic expression of tumor necrosis factor alpha and increased epithelial apoptosis. Furthermore, epithelial ErbB3 deletion increased apoptosis at baseline and during DSS injury. Additionally, epithelial cell hyperproliferation during recovery was exacerbated by deletion of either ErbB2 or ErbB3. These results suggest that ErbB2 and ErbB3 have important cytoprotective and reparative roles in the colonic epithelium following injury, by promoting colon epithelial cell survival.     

Expression of receptors for neuregulins, ErbB2, ErbB3 and ErbB4, in developing mouse cerebellum

We have examined the expression of ErbB2, ErbB3 and ErbB4 in developing mouse cerebellum. ErbB2, ErbB3 and ErbB4 were all expressed in granule cells during cerebellar development. However, the expression pattern for each ErbB receptor changed with the developmental stage. Variations of signal transduction pathway through combinations of these ErbB receptors might have important roles in controlling cerebellar postnatal development.     

Control of basement membrane remodeling and epithelial branching morphogenesis in embryonic lung by Rho and cytoskeletal tension.

Local alterations in the mechanical compliance of the basement membrane that alter the level of isometric tension in the cell have been postulated to influence tissue morphogenesis. To explore whether cell tension contributes to tissue pattern formation in vivo, we modulated cytoskeletal force generation in embryonic mouse lung (embryonic days 12-14) rudiments using inhibitors of Rho-associated kinase (ROCK), myosin light chain kinase, myosin ATPase, and microfilament integrity, or a Rho stimulator (cytotoxic necrotizing factor-1). Tension inhibition resulted in loss of normal differentials in basement membrane thickness, inhibition of new terminal bud formation, and disorganization of epithelial growth patterns as well as disruption of capillary blood vessels. In contrast, increasing cell tension through Rho activation, as confirmed by quantitation of myosin light chain phosphorylation and immunohistocytochemical analysis of actin organization, accelerated lung branching and increase capillary elongation. These data suggest that changes in cytoskeletal tension mediated by Rho signaling through ROCK may play an important role in the establishment of the spatial differentials in cell growth and extracellular matrix remodeling that drive embryonic lung development.     

小鼠颌下腺上皮细胞的体外培养及鉴定

目的建立小鼠颌下腺上皮细胞的体外培养方法,探究细胞的最佳分离和培养条件,为干燥综合征等唾液腺相关疾病的研究和药物评价提供体外实验模型。方法用Ⅳ型胶原酶消化分离小鼠的颌下腺细胞;锥虫蓝染色法测定细胞存活率;差速贴壁法纯化细胞。以含有10μg/L表皮生长因子的F-12/DMEM培养液进行培养,光学显微镜观察细胞形态;增殖曲线评估细胞增殖特性;免疫荧光染色法鉴定细胞。结果经胶原酶消化法所获得的细胞存活率为97.5%;镜下观察显示细胞为典型的上皮样,呈多边形,铺路石样排列;根据增殖曲线,细胞增殖能力良好,一般能传3代;免疫荧光结果显示,cytokeratin 8表达阳性,vimentin表达阴性,符合唾液腺细胞的表型特征。结论本实验成功建立了小鼠颌下腺上皮细胞的原代和传代培养方法,方法简便易行,这为进一步实验研究提供了技术基础。     

Neuregulin-1及其受体ErbB在中枢神经系统损伤修复中作用的研究进展

神经调节蛋白-1(NRG-1)是一类包含表皮生长因子样结构域,在神经组织中发挥着多种生物学效应的营养因子。在神经损伤时,NRG-1的作用涉及神经元迁移、轴突生长、髓鞘和突触的形成,以及促进神经再生,近年来受到越来越多的关注,文中将对NRG-1及其Erb B受体的特性及其在中枢神经损伤修复中的作用进行综述。     

Occurrence of endogenous lectins in the mouse submandibular gland: an immunohistochemical approach.

We investigated the occurrence of endogenous lectins in the mouse submandibular gland using anti-lectin antibodies that selectively bound the respective hapten lectins which have formed a complex with the appropriate hapten sugars and endogenous lectins. Findings supported the idea that endogenous lectins could interact with sugar residues and perform several roles including packaging, immobilization and transport of glucidic material. A functional meaning in the defence from some pathogenous agents was also postulated. Animal lectins seem to play roles related to their localization like occurs in the plants.