Immunohistochemical localization of LIM mineralization protein 1 during mouse molar development

LIM mineralization protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation, maturation and bone formation. Our previous investigations on the distribution of LMP-1 in mature human teeth indicated that LMP-1 might play a role in the odontoblast differentiation and dentin matrix mineralization. The aim of the present study was to use immunohistochemistry to determine the expression of LMP-1 during tooth development in mouse molars. In embryonic and postnatal Kunming mice, LMP-1 protein was expressed during molar development, but the expression levels and patterns differed at various developmental stages. At embryonic day 13.5 (E13.5), LMP-1 was found in the enamel organ. At E14.5, LMP-1 was detected in the entire enamel organ and in the underlying mesenchyme. At E16.5, LMP-1 was observed in the inner and outer enamel epithelium and the stratum intermedium. The expression also converged at the cusps in the dental papilla. At E18.5 and postnatal day 2.5 (P2.5), LMP-1 was restricted to the stratum intermedium, in differentiating dental papilla cells at cusps, while it disappeared in terminal differentiated ameloblasts and odontoblasts. At P13.5, no positive staining was detected in the odontoblasts or in the dental pulp cells. Therefore, LMP-1 showed spatiotemporal expression patterns during molar development and might participate in molar crown morphogenesis and odontoblast differentiation at late molar development.     

The Kindlins: subcellular localization and expression during murine development

The three Kindlins are a novel family of focal adhesion proteins. The Kindlin-1 (URP1) gene is mutated in Kindler syndrome, the first skin blistering disease affecting actin attachment in basal keratinocytes. Kindlin-2 (Mig-2), the best studied member of this family, binds ILK and Migfilin, which links Kindlin-2 to the actin cytoskeleton. Kindlin-3 is expressed in hematopoietic cells. Here we describe the genomic organization, gene expression and subcellular localization of murine Kindlins-1 to -3. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia, and Kindlin-2 in striated and smooth muscle cells. Kindlins-1 and -2 are both expressed in the epidermis. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in the F-actin surrounding ring structure of podosomes, which are specialized adhesion structures of hematopoietic cells.     

Expression and function of amphiregulin during murine preimplantation development

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-α significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-α, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-α in these embryos. Mol. Reprod. Dev. 47:271–283, 1997. © 1997 Wiley-Liss, Inc.     

Expression of DRG during murine embryonic development.

We had previously characterised a cDNA which encodes a novel GTP-binding protein DRG. The expression of drg gene is down-regulated during the embryonic development of murine central nervous system. Further analysis of drg mRNA and protein in adult mouse tissues and various cell lines of different origins indicated that it is expressed widely, albeit at low and variable levels. In situ hybridisation analysis of mRNA expression in sections of mouse embryos indicated that drg is expressed strongly in various embryonic tissues. The expression of drg mRNA is greatly reduced in newborn animals. At cellular level, DRG protein can be detected in the cytoplasm. These observations suggest that DRG may play multiple roles in development and normal cell metabolism.     

Expression and subcellular localization of X-ATM during early Xenopus development

ATM, the gene mutated in ataxia telangiectasia, is a protein essential for handling DNA strand breaks. We recently isolated the Xenopus homologue of ATM, X-ATM and we report here the detailed expression pattern of the protein and the mRNA during early Xenopus development. During the cleavage stages, ATM protein was concentrated in and around the nuclei of all cells with low levels of expression also detected in the cytoplasm. Following neurulation, increased protein levels were detected in the nuclei of developing somites and in the central nervous system. Areas of high protein expression correlated with areas of increased mRNA expression which was detected in the nuclei of somites and the developing lens. Received: 2 December 1999 / Accepted: 4 February 2000     

Expression of murine Ia antigens during embryonic development.

An immunochemical analysis of the kinetics of appearance of Ia antigens during embryonic development was performed. Ia antigens first appear on the surface of embryonic cells 11 days postconception and their expression between days 11 and 16 of gestation is confined to the fetal liver. Ia antigen synthesis by fetal liver cells is detectable at day 14. Ia seems to precede Ig as a surface marker of embryonic liver cells, since Ig cannot be detected until day 16 of gestation. H-2 antigens may be immunoprecipitated from day 10 whole embryo cells. F9 primitive teratocarcinoma cells are Ia negative and H-2 negative.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Expression analysis of asthma candidate genes during human and murine lung development

Background

Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.

Objective

To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.

Methods

Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.

Results

In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.

Conclusions

Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.     

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.     

Expression of Dlx genes during the development of the murine dentition

The mammalian Dlx homeobox gene family has been shown to play multiple roles in tooth development, but a detailed comparison of the expression pattern of all members throughout tooth development has been lacking. We provide such an analysis for the six known murine Dlx genes. The expression patterns for these genes allow a refinement of previously proposed models for the role of Dlx genes in tooth type specification and raise the possibility of roles for subsets of these genes in tooth initiation, morphogenesis (enamel navel formation, enamel knot induction, cervical loop growth), and enamel formation. The relationship of Dlx gene expression to their genomic organization suggests coordinate regulation of linked genes at early stages but regulatory differences at later stages. Received: 19 July 1999 / Accepted: 3 December 1999     

Pim-1 kinase expression during murine mammary development

Pim-1 kinase phosphorylates substrates whose activities are linked to proliferation, survival, differentiation, and apoptosis. Although pim-1 is induced by hormones and cytokines, the hormonal control and contribution of Pim-1 to mammary gland development have not been evaluated. We examined Pim-1 expression in mammary cell lines, investigated whether Pim-1 levels could be altered in breast epithelia by mammogenic hormones, and evaluated Pim-1 expression during mammary development. We found that Pim-1 was elevated in most mammary carcinoma cell lines and progesterone increased Pim-1 protein to some extent in non-tumorigenic mammary epithelia. Pim-1 expression in situ was consistent with the documented profile of progesterone activity in mouse mammary glands. Pim-1 nuclear localization correlated with cytoplasmic distribution for its substrate, p21(CIP/Waf1), and we found that Pim-1 and p21 associate in vitro. Our results suggest that Pim-1 expression may be regulated by progesterone during mammary development and Pim-1 associates with p21 in mammary epithelial cells.