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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.

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.

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.

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.

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.

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.

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.

Primary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-D-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system.

Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs) in order to form myelin, which is required for the rapid propagation of action potentials in the vertebrate nervous system. In spite of the considerable clinical importance of myelination, little is known about the basic molecular mechanisms underlying OL differentiation and myelination. Here, we show that cyclin-dependent kinase (Cdk) 5 is activated following the induction of differentiation, and that the Cdk5 inhibitor roscovitine inhibits OL differentiation. The complexity of the OL processes is also diminished after knocking down endogenous Cdk5 using RNAi. We also show that the focal adhesion protein paxillin is directly phosphorylated at Ser244 by Cdk5. Transfection of a paxillin construct harboring a Ser244 to Ala mutation dramatically inhibits its morphological effects. Importantly, phosphorylation of paxillin at Ser244 reduces its interaction with focal adhesion kinase (FAK). Taken together, these results suggest that phosphorylation of paxillin by Cdk5 is a key mechanism in OL differentiation and may ultimately regulate myelination.

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.

Class 3 semaphorin acts as a guidance clue for both cell migration and nerve fiber projection. The signal of class 3 semaphorin travels via a receptor complex consisting of neuropilins and Plexin-A subfamily. Although it has been reported that class 3 semaphorin acts as a repellent for oligodendrocyte precursor cells (OPCs), which migrate actively during brain development, the expression of Plexin-A subfamily has not been reported in OPCs yet. Therefore, it is currently unclear how semaphorin signals can travel in OPCs. In the present study, the expression of Plexin-A4 (PlexA4) was first demonstrated in a newly established OPC line and OPCs in developing brain. In the OPC line, repulsion for process extension was caused by both Sema3A and Sema6A, and the effect of the semaphorins was diminished in cells expressing PlexA4 lacking the cytoplasmic domain. These results strongly suggest that PlexA4 expressed in OPCs acts as a mediator of semaphorin signals.

To bioengineer ectodermal organs such as teeth and whisker follicles, we developed a three-dimensional organ-germ culture method. The bioengineered tooth germ generated a structurally correct tooth, after both in vitro organ culture as well as transplantation under a tooth cavity in vivo, showing penetration of blood vessels and nerve fibers. Our method provides a substantial advance in the development of bioengineered organ replacement strategies and regenerative therapies.

Here we have identified and characterized a novel mitochondrial Ca2+-binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function.

We established clonal cell lines from fetal brains and a cerebellum of p53 deficient mice. In this paper, stem cell lines are selectively described. FBD-103a line generates neurons, astrocytes and oligodendrocytes. 2Y-6fl line generates neurons and astrocytes. 2Y-3t line generates neurons and oligodendrocytes. FBD-104 line generates astrocytes in conventional culture, but formation of spheres in suspension culture gives the line the capability to produce neurons in addition to astrocytes. Analyses of these lines were done mainly by limited dilution cloning, immunostaining and Western blotting. Some factors in culture medium and kidney were shown to regulate differentiation of the stem cell lines.

We previously established cell lines from brains of p53-deficient embryos, and have now characterized one of these lines, FBD-103a, in detail. Recloning FBD-103a yielded 3 types of subclones: 5 generated the neuronal lineage (Type 1), 3 generated the glial lineage (Type 2), and 10 gave rise to both lineages as the parental line (Type 3), indicating that FBD-103a is a multipotent neural progenitor cell line indistinguishable from true neural stem cells. RT-PCR analyses of transcription factor expression indicated that the transition of multipotent Type 3 clones to either neuronally or glially differentiated progeny was marked by down-regulation of Ascl1/Mash1 and Olig1 and up-regulation of Nrsf/Rest. As expected for neural stem cells, FBD-103a and Type 3 clones formed neurospheres when cultured on a non-adhesive substrate in a serum-free medium containing fibroblast growth factor-2 (FGF2). Interestingly, the transition between Type 3 and Type 1 neuronal- or Type 2 glial-specified cells proved to be reversible; Type 1 and Type 2 subclones could also form neurospheres, from which both neuron-generating and glia-generating progenies could be derived. Moreover, when maintained on an adherent substratum that prevented neurosphere formation, but with FGF2 and without serum, Type 2 clones could generate Type 3 multipotent cells. Thus, at least in the absence of p53, specialized cell–cell interactions within neurospheres are not essential for persistence or recapture of the capacity for self-renewal and multipotency by cells differentiating along glial pathways, a result of possible significance to the pathogenesis of malignant brain tumors.

In the present study, a clonal astrocyte progenitor cell line derived from p53-deficient fetal brains, named FBD-104, was characterized in monolayer and suspension culture. In monolayer culture with medium containing 10% serum, FBD-104 cells expressed some markers of astrocytes, such as glial fibrillary acidic protein (GFAP), S100beta, and glutamate aspartate transporter (GLAST). They never expressed any markers of neurons or oligodendrocytes. Thus the cell line appears to be restricted to the astroglial lineage. However, in suspension culture in serum-free medium supplemented with EGF and FGF2, FBD-104 cells proliferated and formed neurospheres expressing mRNAs for Mash1 and Math3, generating cells expressing neuron specific beta-III tubulin. Re-plating the spheres onto an adhesive substrate and withdrawal of the growth factors induced the expression of mRNAs for NeuroD and Olig2 and generated more beta-III tubulin-positive cells. The present study demonstrated that neurosphere culture is an efficient method to induce neurogenesis from the astrocyte progenitor cell line FBD-104. We also determined that pretreatment with FGF2 caused a significant increase in yield of neurospheres. Thus, the FBD-104 line is an interesting in vitro model to study effect of trophic factors and adhesive substrates on lineage determination of neural progenitor cells.

We investigated the effects of both cytokines and extracellular matrices on the proliferation and differentiation of immature adult rat incisor dental pulp cells. These immature cells, which have a high-proliferative potency in vitro and do not express mRNAs for dentin non-collagenous proteins such as dentin sialoprotein (DSP), bone sialoprotein (BSP), and osteocalcin, exist in the root regions of adult rat incisors. Fibroblast growth factor-2 (FGF-2) stimulated the proliferation of these immature cells and the subsequent production of mineralized calcium was induced by beta-glycerophosphate treatment. Additionally, FGF-2 dramatically induced the expression of DSP and BSP mRNAs, but only in collagen type I gel cultures, whereas neither plate-coated collagen type I nor fibronectin, laminin or collagen type IV cultures could produce this effect and generate sufficient physiological levels of these transcripts. Although bone morphogenetic protein-4 could not induce the proliferation of immature dental pulp cells nor upregulate DSP mRNA expression, it had a synergistic effect upon DSP transcript levels in conjunction with FGF-2. These results suggest that both the presence of FGF-2 and the three-dimensional formation of immature dental pulp cells in collagen type I gel cultures are essential for both DSP expression and odontoblast differentiation. These observations provide valuable information concerning the study of the commitment and differentiation of odontoblast lineages, and also provide a basis for the rational design of cytokine and extracellular matrix based compounds for regenerative therapies in new dental treatments.

Clonal cell lines have been established from vaginae of prepubertal female p53-/- mice. Because the mouse vagina has a dual origin (the cranial three-fifths derived from the Mullerian duct and the caudal two-fifths derived from the urogenital sinus), both parts were separately subjected to cloning. Sixteen epithelial and two fibroblastic cell lines established from the cranial three-fifths (Mullerian vagina group), and four epithelial and three fibroblastic cell lines were established from the caudal two-fifths (sinus vagina group). They were maintained in Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 containing 10% fetal calf serum and 17β-estradiol at 10−8 M. Two cell lines (one epithelial and one fibroblastic) were examined using soft agar assay, but no colonies were formed. The doubling time of cell lines was approaximately 24 h, and all of them divided more than 200 times without crisis, suggesting that they were immortalized. All epithelial cell lines expressed cytokeratin 8. However, the epithelial cell lines expressed cytokeratin 14 and cytokeratin 10 when exposed to medium containing different concentrations of Ca2+. Fibroblastic cell lines expressed vimentin. All epithelial and fibroblastic cell lines expressed estrogen receptor-α protein. This is the first successful establishment of clonal cell lines from the normal mouse vagina, and these lines may provide good models in vitro of the vagina for the study of the mechanism of estrogen action.

We attempted to isolate genes involved in neuronal differentiation from a cell line 2Y-3t newly established from a mouse cerebellum. 2Y-3t cells proliferate in serum-containing medium and differentiate into neurons in serum-free medium. We took a subtraction method to isolate genes differerentially expressed in differentiated cells and 17 cDNA clones were isolated. Functions of 6 cDNA clones are unknown. No. 60 cDNA clone has 723 nucleotides encoding 240 amino acid residues. It contains two putative EF-hand motifs and a coiled-coil region at C terminal end. Expression of the clone was undetectable at embryonic stage and was increased in brain during development. In situ hybridization showed that the expression was observed predominantly in neurons, suggesting that the protein may play roles in the neuronal differentiation and function.

Background We demonstrated that p53-deficiency is sufficient for the establishment of clonal cell lines from the uterus and prostate. In the present study, we improved cloning methods to establish androgen-responsive cell lines. Methods In our previous study, a prostatic cell line was established from the ventral prostate of a p53-deficient mouse and was maintained in a medium containing heat-inactivated fetal calf serum at 10% supplemented with insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml) and selenium (10−8 M). In the present study, 5α-dihydrotestosterone (10−8 M) was added to the medium from the beginning of cloning procedures. Results We succeeded in the establishment of an androgen receptor positive prostatic cell line, designated PEA5. PEA5 cells exhibited a typical epithelial morphology in culture and growth was stimulated by androgens in a dose-dependent manner. In addition, they grew and formed three-dimensional structures in collagen gel, in which growth was also stimulated by androgen. Conclusions Although PEA5 lacks p53 gene, it still retains androgen sensitivity. In collagen gel culture, PEA5 cells can grow and form three-dimensional structures similar to those of the primary cultures reported previously. Furthermore, prostates of p53-deficient mice are shown to be useful sources for obtaining androgen-responsive cells lines. Prostate 46:214–225, 2001. © 2001 Wiley-Liss, Inc.

Induction of apoptosis by adenovirus E1A in rodent cells is stimulated by wild type (wt) p53 but completely suppressed by mutated p53. The suppression is overcome by coexpression with Id proteins (Ids). The cells expressing E1A and Ids undergo apoptosis after accumulation in S phase, suggesting that S phase events are perturbed by E1A and Ids. The E1A domains required for induction of apoptosis, analysed by transfection with expression vectors for E1A, Ids and their mutants, followed by flow cytometry, reside in N-terminal (positions 17 - 38), CR1 and CR2 regions. Interaction of E1A with Ids requires the N-terminal and CR1 regions. The cyclin D1 promoter activity in S phase was reduced severely by E1A and this reduction is caused through CR1 and CR2 regions required for interaction with pRB. Analysis of DNA synthesis in G2/M arrested cells indicated that E1A is capable of inducing >4 N cells and this E1A-mediated DNA rereplication is enhanced by coexpression with Id-1H. The E1A domains required for induction of DNA rereplication coincide with those required for apoptosis.

We detected in the brain and then cloned two novel, short forms of human and mouse fibroblast growth factor (FGF)-5 mRNA, which were designated human FGF-5S (hFGF-5S) and mouse FGF-5S (mFGF-5S), respectively. Genomic analysis indicated that mFGF-5S and authentic mFGF-5 mRNAs were transcribed from a single gene; hFGF-5S and mFGF-5S mRNAs were generated by excluding the second exon of the respective FGF-5 genes, and the alternatively spliced mRNAs encoded for 123- and 121-amino acid proteins, respectively. Indeed, a neuron-like cell line expressing mFGF-5S mRNA secreted a protein of the expected size and with FGF-5 antigenicity. In PC12 cells, expression of hFGF-5 or exposure to hFGF-5 protein induced differentiation. Neither expression of hFGF-5S, alone, nor co-expression of hFGF-5S with hFGF-5 induced significant differentiation. At high concentrations, hFGF-5S protein partially antagonized FGF-5 activity, whereas by itself, hFGF-5S exerted very weak neurotrophic activity. hFGF-5S protein binds to FGF receptor (FGFR)-1 on PC12 transfectants and partially inhibits hFGF-5-induced tyrosine phosphorylation of FGFR-1 and an FGFR substrate, but it also induces phosphorylation by itself. These results suggest that FGF-5S is a naturally expressed partial agonist/antagonist of FGF-5 neurotrophic activity in the brain and that its effects are exerted in part at the level of the receptor.

The rat 3Y1 derivative cell lines, EId10 and EId23, established by introducing the adenovirus E1A12S, Id-1H, and Id-2H cDNAs linked to the hormone-inducible promoter, express these proteins upon treatment with dexamethasone and elicit apoptosis, although these cell lines express mutated p53. The E1A mutants containing a deletion in either the N terminus or the conserved region 1 were unable to induce apoptosis in cooperation with Ids. Western blot analysis of the immunoprecipitates prepared from the dexamethasone-treated EId10 cell extract showed that Id-2H preferentially binds to E1A and E2A (E12/E47) helix–loop–helix transcription factorsin vivo, but scarcely to the retinoblastoma protein. After induction of E1A and Ids, EId10 and EId23 cells began to accumulate in S phase and undergo apoptosis before entering G2phase, suggesting that abnormal synthesis of DNA induced by coexpression of E1A, Id-1H, and Id-2H results in the induction of apoptosis. Apoptosis also is induced in mouse A40 (p53−/−) cells by E1A alone or E1A plus Ids after transient transfection of the expression vectors. The induction of apoptosis is stimulated by coexpression with wild-type p53; however, apoptosis induced by E1A alone was suppressed completely by coexpression with mutated p53, whereas apoptosis induced by E1A plus Ids was stimulated by the mutated p53 as done by wild-type p53. These results suggest that the suppressive function of mutated p53 is overcome by Ids.

Brains from 7 p53-deficient mice on fetal day 13 were divided into 4 regions: cerebral cortex, cerebellum, upper spinal cord, and the rest of brain. They were trypsinized and cultured in medium containing 10% fetal calf serum. By dilution culture, 138 clonal lines were established from every regions. The lines were characterized morphologically and immunocytochemically as neuronal, glial, myogenic, or unidentified. They were cultured for more than a year, indicating that the lines are immortalized. p53-deficiency alone is sufficient for establishing clonal cell lines of the central nervous system.

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.

Vertebrate central nervous system develops from a neural tube derived from the embryonic ectoderm. In mouse, the neural tube around embryonic day 10 primarily consists of neural precursor cells (NPCs). During the development of embryonic central nervous system, NPCs proliferate and migrate outward; thus later stages show NPCs toward the lumen of the neural tube and neurofilament-positive differentiated cells toward the periphery. In conventional liquid culture, NPCs isolated from mouse on embryonic day 10 proliferate and differentiate into neurofilament-positive neurons. In the present communication, we show that fragments of neural tubes and aggregates of NPCs, when placed into collagen gel matrix, form three-dimensional structures which resemble the neural tube formed in vivo in the developing embryos. Even dissociated NPCs form the three-dimensional structures in the collagen gel matrix. Our results indicate that individual NPCs or fragments of neural tubes carry morphogenetic information which allows them to reconstruct neural tube-like structures in vitro.

Using a subtraction cloning approach we had previously isolated a series of murine cDNA clones representing the genes predominantly expressed in the embryonic brain and down-regulated during development. We now report that one of these cDNA clones encodes a novel type of GTP-binding protein. The predicted protein of 40.5 kD, named DRG, contains five structural motifs characteristic of the GTP-binding proteins. Consistently, bacterially expressed and cellular DRG proteins are capable of binding GTP in vitro. Sequences closely related to the DRG protein are found in other species including Drosophila and Halobacterium. Based on these observations, we propose that DRG represents an evolutionarily conserved novel class of GTP-binding protein which may play an important role in cell physiology.