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The mammary gland develops from the placode at ectodermal invagination. The rudimentary parenchyma (mammary bud) develops mammary trees and alveolar structures, suggesting that the mammary bud consists of stem/progenitor cells. Here, we established a clonal stem cell line from a mammary bud of a p53 null female embryo at day 14.5. FP5-3-1 line was a homogeneous cell population with polygonal epithelial morphology and spontaneously became heterogeneous during passages. Recloning gave rise to four sublines; three sublines have basal epithelial property and one subline has luminal epithelial property. The former sublines generate functional mammary glands when injected into cleared fat pads and the latter subline does not. The cell lines also express many stemness-related genes. The clonal cell lines established in the present study are shown to be mammary stem cells and not tumorigenic. They provide useful models for normal and tumor biology of the mammary gland in vivo and in vitro.

Oviduct, uterus, and vagina are derived from Müllerian ducts. But only in the vagina, the epithelium differentiates into stratified layers. Organ-specific secreted factors derived from the stroma of a neonatal mouse induce epithelial differentiation in the female reproductive tracts. However, the effects of the components and mechanical property of extracellular matrix (ECM) on the regulation of gene expression in the mesenchymal cells of neonatal stroma and differentiation of epithelium in the female reproductive tracts have been overlooked. In the present study, we have developed a simple 3D neonatal vaginal model using clonal cell lines to study the effect of ECM’s components and stiffness on the epithelial stratification. Transcriptome analysis was performed by DNA-microarray to identify the components of ECM involved in the differentiation of vaginal epithelial stratification. The knockdown experiment of the candidate genes relating to vaginal epithelial stratification was focused on fibromodulin (Fmod), a collagen cross-linking protein. FMOD was essential for the expression of Bmp4, which encodes secreted factors to induce the epithelial stratification of vaginal mesenchymal cells. Furthermore, stiffer ECM as a scaffold for epithelial cells is necessary for vaginal epithelial stratification. Therefore, the components and stiffness of ECM are both crucial for the epithelial stratification in the neonatal vagina.

Uterine smooth muscle cells differentiate from mesenchymal cells, and gap junctions connect the muscle cells in the myometrium. At the neonatal stage, a uterine smooth muscle layer is situated away from the epithelium when smooth muscle cells are grafted near the epithelium, suggesting that the epithelium plays an important role in differentiation, proliferation, and/or migration of smooth muscle cells. In this study, developmental mechanisms regulating the formation of the smooth muscle layers in the mouse uterus were analyzed using an in vitro culture model. Differentiation of smooth muscle cells occurs at a neonatal stage because ACTA2 gene expression was increased at the outer layer, and GJA1 was not expressed in cellular membranes of uterine smooth muscle cells by postnatal day 15. To analyze the effects of the epithelium on the differentiation of smooth muscle cells, a bulk uterine mesenchymal cell line was established from p53−/− mice at postnatal day 3 (P3US cells). Co-culture with Müllerian ductal epithelial cells (E1 cells) induced repulsive migration of ACTA2-positive cells among bulk P3US cells from E1 cells, but it had no effects on the migration of any of 100% ACTA2-positive or negative smooth muscle cell lines cloned from P3US cells. Thus, uterine epithelial cells indirectly affected the repulsive migration of smooth muscle cells via mesenchymal cells. Conditioned medium by E1 cells inhibited differentiation into smooth muscle cells of clonal cells established from P3US cells. Therefore, the uterine epithelium inhibits the differentiation of stem-like progenitor mesenchymal cells adjacent to the epithelium into smooth muscle cells.

Stratification of the vaginal epithelium is regulated by stromal factors. To analyze the mechanisms of stratification in vitro, 3 dimensional (3D) co-culture models were established with clonal cell lines. In the models, stromal cells were embedded in collagen gel and epithelial cells were seeded on the gel. In the 3D co-culture, stromal SV-6c4a1b cells induced epithelial stratification but stromal MV-1e6g1a cells did not, suggesting that SV-6c4a1b cells secrete molecules to induce stratification. Microarray analyses of these stromal cell lines identified chordin-like 1 (Chrd1) and WNT1 inducible signaling pathway protein 2 (Wisp2) as candidate genes inducing stratification. Chrdl1 variant1 and variant2 mRNAs were expressed not only in stromal SV-6c4a1b and MV-1e6g1a cells but also in epithelial SV-4b6b cells. Wisp2-overexpressing MV-1e6g1a cells, secreting WISP2 as much as SV-6c4a1b cells, induced stratification of epithelial cells. In addition, Wisp2-knockdowned SV-6c4a1b cells were unable to induce epithelial stratification. These results suggest that WISP2 is one of the stromal factors inducing stratification of the mouse vaginal epithelium.

Extracellular vesicles (EVs) have been shown to transport miRNA, mRNA and protein, suggesting that they are new communication mediators. Diffusible mesenchymal factors determine the fate of Műllerian epithelial cells into oviductal ciliated cells. In the present study, we investigated whether EVs mediate the communication in the epithelial-mesenchymal interaction during oviductal ciliogenesis. EVs were isolated from cells of oviductal mesenchymal cell line (S1 cells) and characterized by TEM and expression of exosomal marker CD81. CD81 protein was also detected in oviductal mesenchyme, suggesting that CD81-expressing exosomes may be secreted from oviductal mesenchyme, as well as S1 cells. β-actin, Gapdh and Vimentin mRNAs and miRNAs were detected in the exosomes. mRNA in S1 cells was able to be transported into cells of Műllerian epithelial cell line (E1 cells) via the exosomes. The effects of exosomes derived from S1 cells on ciliogenesis of E1 cells were analyzed by in vitro models. Culture with exosomes increased the number of ciliated cells in E1 cells. These results suggest that exosomes derived from mesenchymal cells modulate the oviductal ciliogenesis and open new avenues for developmental study of EVs.

The mouse vagina consists of stratified squamous epithelium and stroma and is regulated by ovarian hormones. Vaginal epithelial cells do not stratify, but rather form a monolayer and show an inconsistent responsiveness to ovarian hormones when cultured on plastic dish or matrix. To address the discrepancy between in vivo and in vitro observations, three-dimensional (3D) co-culture models are developed with clonal vaginal epithelial and stromal cell lines; stromal cells are embedded in collagen gel and epithelial cells are seeded on the gel. In the 3D models, epithelial cells express Transformation related protein 63 (Trp63) and begin to stratify when they are co-cultured with two out of three stromal cell lines, but not with the other stromal cell line. Stroma may consist of various types of cells with distinct functions.

We previously reported that in rats, chronic exposure to stress inhibits the induction of the common receptor (T1R3) for sweet and umami tastes. Here, we investigated whether endogenous glucocorticoids (GCs) might be responsible for this inhibition. In addition, we used mouse taste-bud cells (TB cells) expressing T1R3 to examine the effect of exogenous GC on T1R3 induction. Both adrenal glands were removed from rats [adrenalectomized (ADX) rats] and T1R3 mRNA expression in fungiform papillae was examined by real-time RT-PCR. T1R3 mRNA expression was significantly reduced in the ADX rats (versus sham-ADX rats). The reduced mRNA expression was restored to the level seen in the sham-ADX rats by administration of dexamethasone (DEX) at the smallest dose tested (0.1ng/kg, i.p.). However, with larger doses of DEX (10 and 1000ng/kg, i.p.) there was no such restoration (i.e., the expression level did not differ from that seen in ADX rats). Expression of the mRNA for the GC receptor-α was detected in mouse TB cells by RT-PCR. Significantly reduced T1R3 mRNA expression, as measured by real-time RT-PCR, was observed in TB cells at 24h after application of DEX (0.1, 1.0, or 10μM). These results suggest that in rodents: (a) a low concentration of endogenous GC is necessary and sufficient for induction of T1R3 expression, and that higher concentrations may actually inhibit such induction, and (b) this inhibitory effect may be due, at least in part, to a direct action of GC on taste cells.

The mouse vagina consists of stratified squamous epithelium and stroma and is regulated by ovarian hormones. Vaginal epithelial cells do not stratify, but rather form a monolayer and show an inconsistent responsiveness to ovarian hormones when cultured on plastic dish or matrix. To address the discrepancy between in vivo and in vitro observations, three-dimensional (3D) co-culture models are developed with clonal vaginal epithelial and stromal cell lines; stromal cells are embedded in collagen gel and epithelial cells are seeded on the gel. In the 3D models, epithelial cells express Transformation related protein 63 (Trp63) and begin to stratify when they are co-cultured with two out of three stromal cell lines, but not with the other stromal cell line. Stroma may consist of various types of cells with distinct functions.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.

An improved organ culture system allowed to observe morphogenesis of mouse lingual papillae and taste buds relatively for longer period, in which fetal tongues were analyzed for 6 d. Taste cells were defined as eosinophobic epithelial cells expressing CK8 and Sox2 within lingual epithelium. Addition of glycogen synthase kinase 3 beta inhibitor CHIR99021 induced many taste cells and buds in non-gustatory and gustatory stratified lingual epithelium. The present study clearly demonstrated induction of taste cells and buds ectopically and without innervation.

The Wnt/β-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated β-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/β-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of β-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that β-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that β-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that β-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of β-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.

Objectives p51/p63 gene, one of the p53 families, is specifically expressed in tooth germ epithelial cells and is essential for tooth development. This study aims to elucidate roles of p51/p63 in ameloblastic cell differentiation. Materials and methods We determined expression pattern of each of p51/p63 isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting using emtg (epithelium of molar tooth germ)-1, -2, -3, -4, and -5 cell lines established from a mandibular molar tooth germ of p53-deficient mice and SF2 cells which differentiates into ameloblasts upon exposure to NT4. Furthermore, we investigated the function of p51/p63 in these cells by Tet system, which enables inducible expression and knock down of the target genes of interest by exposing cells to doxycycline. Results The expression of ΔNp51B/ΔNp63α, an isoform without transactivation domain, was detected at high level in immature cells, while the expression of TAp51/TAp63 isoforms, isoforms of with the transactivation domain, was detected at high level in mature cells. Moreover, induction of TAp51A/TAp63γ expression led to down-regulation of ΔNp51B/ΔNp63α expression and cell proliferation. Interestingly, this also led to up-regulation of ameloblastin expression, a differentiation marker of amelogenesis. Conclusions The results suggested that p51/p63 might regulate the cell proliferation and differentiation of tooth germ epithelial cells.

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

A taste bud is a sensory organ and consists of 50-100 spindle-shaped cells. The cells function as taste acceptors. They have characteristics of both epithelial and neuronal cells. A taste bud contains four types of cells, type I, type II, type III cells, and basal cells. Taste buds were isolated from a tongue of a p53-deficient mouse at day 12, and 11 clonal taste bud (TBD) cell lines were established. In immunochemical analysis, all cell lines expressed cytokeratin 18, gustducin, T1R3, and neural cellular adhesion molecule, but not GLAST. In RT-PCR analysis, shh was not expressed in any of the cell lines. Further analysis with RT-PCR was conducted on four cell lines. They expressed G protein-coupled taste receptors; T1R3, T2R8 for sweet, bitter, umami. And they also expressed α-ENaC for salty taste. While, a candidate for sour receptor HCN4 was expressed in TBD-a1 and TBD-a7 lines. And another candidate for sour receptor PKD1L3 was slightly expressed in TBD-a1 and TBD-c1.

The mouse oviductal epithelium is a simple monolayer until Postnatal Day 7 and subsequently consists of differentiated secretory cells and ciliated cells. In adult oviduct, the two types of epithelial cells are unevenly distributed; ciliated cells are dominant in the ampulla and secretory cells are dominant in the isthmus. Recombinants of enzymatically separated epithelial and mesenchymal tissues of oviducts were grafted under kidney capsule for 4 wk. The recombinants developed structures with a lumen covered with a monolayer of ciliated cells and secretory cells, demonstrating that the recombinant tissues reconstructed oviductal structure. Geographically (ampulla versus isthmus) heterotypic recombinants were prepared from neonatal oviducts at Day 3. The epithelia in reconstructed oviducts took the patterns of cell distribution depending on the origin of the mesenchymal tissues. The results indicate that the mesenchyme geographically has distinct abilities to determine undifferentiated epithelial cells to ciliated cells or secretory cells in the mouse oviduct.

In the past few years, the plasticity of the regional specification of the CNS has been widely debated on the results from in utero transplantation. Two different results are reported with this transplantation method. One is that the distribution of transplanted cells is dependent on the donor origin, and the other is that the distribution is independent on the donor cell origin. The present study attempted to examine closely the plasticity of the regional specification by in utero transplantation method with clonal neural cell lines, 2Y-3t and 2Y-5o2b. These lines were established from a cerebellum of an adult p53 -deficient mouse. Our results showed that transplanted cells migrated into various regions of the CNS and supported the independent distribution. Moreover, different distribution patterns of transplanted cells were observed between host sexes. Labeled cells were localized around the ventricle of neonatal host brains, where they were undifferentiated. In 2–3 weeks after birth, labeled cells were found in the brain parenchyma and some of them took neuronal morphology. In the rostral migratory stream (RMS), cells with unipolar or bipolar morphology were still undifferentiated. In other regions, labeled cells were often associated with blood vessels; the soma were on the surface of vessels, extending processes or neurites into surrounding brain parenchyma. Time-lapse imaging demonstrated that they were migrating with blood vessels.

Interactions between oligodendroglial and astroglial lineages during development are still unclear. In this study, FBD-102b, derived from p53-/- fetal brains, was characterized as an oligodendroglial progenitor cell (OPC) line. In co-culture with 102b cells, cells of an astrocyte progenitor cell line, FBD-104, elongated monopolar processes, like radial glia, and actively proliferated. Conditioned medium of 102b cells also induced the process elongation that was associated with down-regulation of GFAP and up-regulation of vimentin and nestin. These results suggested that OPCs secrete a radial glia-inducing factor that might maintain the radial glial processes to use as scaffolds of migration.

Aim: Term pregnancy complicated by premature rupture of membranes (PROM) is thought to be associated in part with subclinical infection, and places mothers and neonates at an increased risk for several complications. Therefore, perinatal care would be greatly helped if a reliable clinical measure were available for predicting the incidence of PROM. Methods: One hundred and ninety-six pregnant women who consented to enter this study were screened using a method developed to assess active ceruloplasmin in cervicovaginal secretion as a clinical marker for predicting the incidence of PROM. Cervicovaginal secretions were obtained from the cervical canal at about 36 weeks of pregnancy. The active ceruloplasmin level in the cervicovaginal secretion was measured using an original enzyme-linked immunoabsorbent assay. Results: Of 196 women, 27 women (13.8%) developed PROM and 169 women (86.2%) did not develop PROM. Active ceruloplasmin in the cervicovaginal secretion was significantly higher in the PROM group than in the non-PROM group (P

Here we report developmental characteristics of a clonal cell line 2Y-3t established from a multifocal neoplasm that arose in a cerebellum of an adult p53-deficient mouse. The tumorigenicity of the line was not observed in soft agar assay or in nude mouse assay. In serum-containing medium, 2Y-3t cells were epithelial-like in morphology and were mitotic. When they were cultured in serum-free medium, the expressions of neural stem and/or progenitor cell markers were decreased. Concomitantly, the expressions of neuronal and oligodendrocyte markers were increased in concert with morphological differentiation, and DNA synthesis ceased. None of astrocyte markers were detected under these culture conditions. Double-labelling studies revealed that two cell populations coexisted, expressing neuronal or oligodendrocyte markers. Triiodothyronine (T3) increased the oligodendrocyte population when 2Y-3t cells were cultured in serum-free medium. Recloning of the line gave rise to three types of subclones. Sixteen subclones were capable of generating both neurons and oligodendrocytes, four subclones were capable of generating only neurons and one subclone was capable of generating only oligodendrocytes. Thus, 2Y-3t cells have characteristics of bipotent neural progenitor cells capable of generating both neurons and oligodendrocytes. In addition, the line expressed mRNA for Pax-2 and had GAD67-positive cells when cultured in serum-free medium. However, none of the mRNAs for Zic-1, Math1, zebrin or Calbindin-D28k were detected, suggesting that the 2Y-3t line might generate the GABAergic interneuron lineage of the mouse cerebellum.

Here we report developmental characteristics of clonal cell line 2Y6f1, which was established from an adult p53 −/− mouse cerebellum. 2Y6f1 began as a homogeneous population of small polygonal epithelial cells, but during passages it gradually became heterogeneous, containing cells of varying size and shape that expressed either neuron- or astrocyte-specific proteins. Supplements to the culture medium altered the levels of some of the cell type markers. For example, addition of insulin increased expression of neurofilaments, while cholera toxin increased that of glial fibrillary acidic protein. In a colony assay, 2Y6f1 cells gave rise to both homogeneous and heterogeneous colonies, consistent with the idea that they contained multipotent neural progenitor cells. Establishment of subclones that were exclusively neuronal or astroglial in differentiation further supported the conclusion that 2Y6f1 cells have many features that may qualify them as bona fide stem cells and make them a useful new model in neural stem cell biology.

Many molluscs perform sex reversal, and sex hormones may be involved in the process. In adult scallops, Patinopecten yessoensis, gonadotropin releasing hormone and 17β-estradiol (E2) are involved in male sexual maturation, however, little is known about the effects of E2 and testosterone (T) on the gonadal differentiation in young scallops. In the present study, scallop gonadal development was analyzed to determine the sex reversal stage in Funka bay, and effects of E2 and T were examined. In Funka bay, almost all scallops were male at month 12. Scallops equipped with ambiguous gonads were 61.1% at month 16 and disappeared at month 18. Therefore, sex reversal in Funka bay occurs at around month 16. For establishment of organ culture systems for bivalves, Manila clam gonads were cultured in 15% L-15 medium diluted with HBSS containing 10% KSR on agarose gel at 10°C, and the gonads survived for 14 days. Scallop gonads were also able to be cultured in 30% L15 medium diluted with ASW containing 10% KSR on agarose gel for seven days. At mature stage, Foxl2 and Tesk were predominantly expressed in ovary and testis, respectively. When scallop gonads at sex reversal stage were organ-cultured, sex steroid treatment decreased Tesk expression in the majority of scallop gonads at sex reversal stage. However, no obvious change in Foxl2 and Tesk expression was detected in mature gonads in response to either E2 or T in culture, suggesting sex steroid treatment might affect gonadal development at sex reversal stage.

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

The sensitivities of apoptosis induced by E1A, c-Myc, Bax, and Nip3 to wild-type (wt) and mutated p53 and Id proteins were analyzed by transient transfection followed by flow cytometry with p53 null mouse cerebellum cell lines W7 and M13 that express wt and mutated p53 in response to dexamethasone, respectively. Apoptosis induced by c-Myc was stimulated weakly by wt p53, strongly by Ids, but suppressed completely by mutated p53 irrespective of coexpression with Ids, while apoptosis induced by E1A was suppressed by mutated p53 but stimulated when coexpressed with Ids. Apoptosis induced by Bax was little affected by wt and mutated p53, but inhibited by Ids, while apoptosis induced by Nip3 was inhibited by both wt and mutated p53 and inhibition was stimulated by Ids. Caspase-1 was activated only by Bax significantly when coexpressed with mutated p53 but not with wt p53. These results indicate that the apoptotic processes elicited by these inducers are different and differently affected by wt and mutated p53 and by Ids.

p53-Deficient mice are prone to develop spontaneous tumors [L.A. Donehower, M. Harvey, B.L. Slagle, M.J. McArthur, C.A. Montgomery Jr. , J.S. Butel, A. Bradley, Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors, Nature 356 (1992) 215-221], but brain neoplasms are rare and difficult to culture in vitro. Here we describe cloning and long-term culture of heterogeneous neural cell lines from a multifocal cerebellar neoplasm that arose in an adult p53-/- mutant mouse. These lines may be useful for studies of neoplastic transformation and cell lineage in cerebellar development.

UE8 is a uterine epithelial cell line established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture and is strongly positive for cytokeratin, but negative for vimentin in immunocytochemistry. UE8 shows an active growth in a phenol red free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 supplemented with 3% heat-inactivated and dextran-coated charcoal-treated fetal calf serum. Both immunocytochemistry and immunoblot analyses confirmed that UE8 was negative for the estrogen receptor. Diethylstilbestrol added to the medium at concentrations between 10−6 and 10−8 M had no significant effect on the proliferative rate, and estradiol-17β at 10−6 M was slightly inhibitory. Unexpectedly, however, the“pure anti-estrogen”ICI 182,780 at 10−6 M significantly enhanced cell proliferation. This is the first observation that the“pure anti-estrogen”ICI 182,780 stimulates the growth of uterine epithelial cells without estrogen receptors.

BACKGROUND We demonstrated that p53-deficiency is sufficient for immortalization of fetal uterine cells. In the present study, we further extended our previous observations to prostate tissues from a young p53-deficient adult mouse. METHODS Cell lines were established from the ventral prostate of a p53-deficient male mouse and maintained in medium containing 10% heat-inactivated fetal calf serum supplemented with insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and selenium (10−8 M). RESULTS Pro9ad, one of the lines established, exhibits a typical epithelial morphology in culture. Despite the possession of androgen receptors, the growth of Pro9ad was not stimulated by 5α-dihydrotestosterone. Hepatocyte growth factor (HGF) slightly stimulated proliferation, whereas fibroblast growth factor-1 (FGF-1), keratinocyte growth factor (KGF), and platelet-derived growth factor AB (PDGF-AB) had no stimulating effect on growth. However, FGF-2, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) accelerated proliferation in a dose-dependent manner. EGF and IGF-1 additively stimulated growth. CONCLUSIONS These results suggest that Pro9ad shares characteristics in common with primary prostatic epithelial cells despite p53-deficiency, and that p53-deficiency alone allows establishment of clonal cell lines of the prostate epithelium. Furthermore, the prostates of p53-deficient mice are useful sources for obtaining cell lines. Prostate 36:102–109, 1998. © 1998 Wiley-Liss, Inc.

UE8 is one of several uterine cell lines established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture; it is strongly positive for cytokeratin, but negative for vimentin. Immunoblot analysis confirmed that UE8 has glucocorticoid receptors. It grows actively in medium supplemented with 3% heat-inactivated, dextran-coated charcoal-treated fetal calf serum. Dexamethazone (DEX) inhibited its growth dose-dependently at concentrations between 10−6and 10−9 M. The inhibition by DEX was further examined in chemically defined medium (CDM): DMEM/F12 containing transferrin (10 μg/ml), selenium (10−8 M) and 0.1% bovine serum albumin. DEX at 10−6 M inhibited the growth of UE8 in CDM or in CDM supplemented with insulin-like growth factor-1 (IGF-1: 10 ng/ml), but there was no inhibition by DEX(10−6M) in the presence of epidermal growth factor (10 ng/ml). Thus, DEX inhibits the growth of a uterine epithelial cell line derived from p53-deficient mice in vitro, and also ...

Mammary gland involution after cessation of milk production is associated with extensive loss of secretory epithelial cells. In order to study the mechanism of mammary gland involution, litters were removed on day 20 of lactation and morphological and biochemical changes were examined in GR/A mice andlprcg mice lacking functional Fas. DNA fragmentation occurred 24 h after weaning both in GR/A mice andlprcg mice indicating that apoptotic cell death occurs during involution of mammary glands.In situ 3′-end labelling method revealed apoptotic cells in epithelial cells lining alveolar lumens and in cells shed into the alveolar lumen. The number of apoptotic cells plateaued on day 2 of weaning in mammary glands of GR/A mice. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that expression of bcl-2, Fas ligand, keratinocyte growth factor (KGF) and transforming growth factor-β1 (TGF-β1) increased on day 1 of weaning in the process of involution of mammary glands in GR/A mice. In mammary glands oflprcg mice, RT-PCR showed that expression of bcl-2, Fas ligand, KGF, tumour necrosis factor-α(TNF-α) and TGF-βs increased in the process of involution. These results suggest that the Fas ligand, TGF-β1 and TNF-α are involved in the involution of mammary glands after weaning. TNF-α and anti-Fas antibody directly killed more than 80% of mammary cells from p53 knockout micein vitro within 24 h in the presence of actinomycin D, supporting the hypothesis that Fas and/or TNF-α are involved in the induction of apoptosis of mouse mammary glands.

Rat embryos at the head-hold stage (Slc:SD strain; 9.5 days of gestation) were cultured for 48 h in rat serum with the anti-nestin peptide antiserum. The antiserum identified a single band in Western blots of the tissue extracts from rat embryos and stained the cells from the neural tube, migrating neural crest, and somites immunohistochemically. The antiserum-treated embryos appeared to develop normally for the most part. However, histological observation disclosed that the ventral portion of the neural tube was deformed. The cells in the deformed portion did not show the elongated shape but were round. These round cells tended to crowd near the ventricular surface, and a gap was observed between the original pial surface and cells arranged in the most pialward region. The penetration of the anti-nestin peptide antibody into the embryos from the culture medium was confirmed by visualization of the penetrated antibody using biotinylated anti-rabbit IgG antibody raised in goats and Texas red-conjugated streptavidin. These results indicate that the nestin protein plays an important role in the organization or the maintenance of neuroepithelial cells of the elongated shape spanning the neural tube from the luminar to the pial side.

Our previous study has confirmed that the distribution of oligodendrocyte precursor cells (OPCs) is disturbed in the embryonic cerebral cortex of Plexin-A4 knockout mice, and that Sema6A is expressed in OPCs in the region. The present study examined whether Sema6A expressed in OPCs is involved in their own migration, and used a clonal FBD-102b line as OPCs model. In an in vitro migration assay, Sema6A knockdown repressed the migration of FBD-102b cells. Additionally, in co-culture, 3T3 cells ectopically expressing Plexin-A4 were segregated from 3T3 cells ectopically expressing Sema6A. When FBD-102b cells were seeded in a spot and exposed to a gradient of both Sema3A and Sema6A, dispersion of FBD-102b cells was suppressed, and Plexin-A4 knockdown in FBD-102b cells attenuated the suppressive effect of the Semaphorins. These results indicate that Sema6A expressed in OPCs is involved in their autonomous migration through ligand-receptor interaction with Plexin-A4 expressed in surrounding cells.

Molecular mechanisms regulating positions of oligodendrocyte precursor cells (OPCs) remain unclear in developing cerebral cortex. To explore the mechanisms, we investigated how Plexin-A4, receptor of Semaphorin in OPCs, is involved in the positioning. We found that Plexin-A4 knockout mice exhibited (1) an increased number of OPCs in both the upper- and middle-regions of the cortical plate, where both indirect- and direct-ligands of Plexin-A4, Sema3A and Sema6A, respectively, were continuously expressed, and (2) aberrant distributions of OPCs in both the intermediate zone and corpus callosum, where Plexin-A4 was richly expressed in wild-type mice. These results suggest that Plexin-A4 is involved in the precise positioning of OPCs in developing cerebral cortex.

We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.

p51/p63, one of the tumor suppressor p53 family members, is known to maintain proliferative potential and immaturity of epithelial stem cells. p51/p63 is known to be expressed in enamel organ epithelium during tooth development. Nevertheless, its functions in tooth development have remained unclear. Thus, functions of p51/p63 in tooth development were investigated. First, expression patterns of p51/p63 in 5 cell lines established from a lower mandibular molar tooth germ of a fetal mouse were examined by western blot analysis and RT-PCR analysis. ΔNp51B/ΔNp63α expression (one of p51/p63 isoforms), which is known to maintain immaturity of epithelial stem cells, was detected in immature cell lines at high levels and in mature cell lines at low levels. These results suggest that ΔNp51B/ΔNp63α may play significant roles in amelogenesis.

To identify tyrosine kinases which play roles in mammalian early development, the 3' rapid amplification of cDNA ends (RACE) was performed on mouse embryonic stem (ES) cells. Among eight tyrosine kinases thus identified, we report here a novel tyrosine kinase, hyk (adhesion structures linked tyrosine kinase). The sequences of the 4.7 kb cDNA indicated the presence of RGD motif and three epidermal growth factor-like domains put between two immunoglobulin-like domains and three fibronectin type III domains in its extracellular region. It is strongly expressed in ES cells and later stages of embryos, but at low levels in midgestation embryos. It is also expressed at a low level in neural precursor cells from 10-day embryos, but at high levels in embryonic day 15 and neonatal brains. In adult tissues it is expressed ubiquitously.

A full length cDNA whose corresponding mRNA is down-regulated during the mouse embryonic brain development was isolated. The cDNA contains a single long open reading frame which could encode a protein with relative molecular mass of 41 kDa. The predicted gene product contains long stretches of prolines towards the NH2-terminus, followed by a leucine/proline rich region. The cDNA probe detected a number of mRNA species in Northern blot analysis. The reverse transcriptase-polymerase chain reaction analysis of mRNA from adult mouse tissues indicated that heart and testis expressed this gene (named NDPP-1) at relatively high levels, while lower levels of mRNA were detected in a number of other tissues. Expression of NDPP-1 was also detected in embryonic carcinoma and pheochromocytoma cell lines, but not in fibroblasts. The cDNA hybridized to genomic DNA from several vertebrates species in Southern blot analysis indicating interspecies conservation of this gene. The interesting pattern of expression of the NDPP-1 gene during mouse brain development and the structure of its putative protein product indicate that this gene may play an important biological role in the development of mouse central nervous system.

The mouse oviductal epithelium is a simple monolayer until Postnatal Day 7 and subsequently consists of differentiated secretory cells and ciliated cells. In adult oviduct, the two types of epithelial cells are unevenly distributed; ciliated cells are dominant in the ampulla and secretory cells are dominant in the isthmus. Recombinants of enzymatically separated epithelial and mesenchymal tissues of oviducts were grafted under kidney capsule for 4 wk. The recombinants developed structures with a lumen covered with a monolayer of ciliated cells and secretory cells, demonstrating that the recombinant tissues reconstructed oviductal structure. Geographically (ampulla versus isthmus) heterotypic recombinants were prepared from neonatal oviducts at Day 3. The epithelia in reconstructed oviducts took the patterns of cell distribution depending on the origin of the mesenchymal tissues. The results indicate that the mesenchyme geographically has distinct abilities to determine undifferentiated epithelial cells to ciliated cells or secretory cells in the mouse oviduct.