Murine MLP: cloning and expression in the embryonic head.

Differential display reverse transcription polymerase chain reaction (DD RT-PRC) was used to isolate cDNA clones of mRNA expressed in particular compartments of the developing murine head. One clone has been characterized and found to be the murine homologue of the MLP genes. In other species the LIM-domain containing MLPs are expressed specifically in muscle. In situ hybridization and RT-PCR have confirmed that the murine gene is expressed during both cardiac and skeletal myogenesis. Our data show that DD RT-PCR is a valuable technique to clone cDNA clones from minute tissue samples by virtue of the gene's expression pattern rather than by a process of library screening by homology.

Sialylation of terminal saccharides of glycoconjugates expressed by murine molar tooth germs developing in vitro and in vivo.

During development of the mammalian tooth germ the pattern of terminal saccharides of glycoconjugates changes, with many structures losing lectin reactivity in a consistent pattern. This study investigated whether the epitopes are lost or become masked by terminal sialylation, using a combination of neuraminidase treatment of sections and sialic acid-reactive lectins. The results suggested that most of the terminal galactosamine and fucose sites in the epithelial enamel organ were removed during morphogenesis. Conversely, during condensation of the dental mesenchyme, masked peanut agglutinin (PNA)-reactive galactose epitopes appeared. During differentiation and organisation of the mesenchyme into odontoblasts and a subodontoblastic layer the PNA-reactive sites became masked again. These regions also specifically expressed sialylated glucosamine. However, at the proliferating epithelial cervical loop galactose sites appeared to be masked. This was more pronounced during in vitro development when abnormal expression of PNA-reactive sites was found at the cervical loop. Additionally, fucosylated sites persisted in the enamel organ, further indicating that the expression of terminal saccharides was disrupted during development in the organ culture system. These data suggest that loss of terminal galactose and galactosamine is related to differentiation of the cells. However, whether this loss occurs by removal or sialic acid masking is not dependent either on the origin of the cells or the epitope being lost.

Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth.

Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.

Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth.

Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be D-galactose-(beta 1----3)DGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia--I (BSL-I) staining was lost from postmitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express beta-D-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.

Morphometric analysis of the developing murine molar tooth in vivo and in vitro.

A morphometric technique is described which has been used to measure morphogenesis and matrix synthesis of developing murine teeth in vivo and in vitro. The growth of bud and cap stage tooth germs in vitro was reduced considerably compared with in vivo. There was no growth of tooth germs explanted and cultured in vitro at the bell stage. The onset of dentine and enamel synthesis were however only slightly delayed in vitro compared with in vivo. The technique had been designed to be insensitive to orientation or artefactual folding of the cultured tissue.

The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro.

Hox 7.1 is a murine homeobox-containing gene expressed in a range of neural-crest-derived tissues and areas of putative epithelial-mesenchymal interactions during embryogenesis. We have examined the expression of Hox 7.1 during craniofacial development in the mouse embryo between days 8 and 16 of development. Whereas facial expression at day 10 of gestation is broadly localised in the neural-crest-derived mesenchyme of the medial nasal, lateral nasal, maxillary and mandibular processes, by day 12 expression is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Hox 7.1 expression in the mesenchyme of the dental papilla and follicle is maximal at the cap stage of development and progressively declines in the bell stage prior to differentiation of odontoblasts and ameloblasts. Hox 7.1 expression in tooth germs is independent of overall embryonic stage of development but is dependent on stage of development of the individual tooth. Similar patterns of transient Hox 7.1 expression can also be detected in tooth germs in vitro in organ cultures of day 11 first branchial arch explants cultured for up to 7 days. Hox 7.1 is also expressed early in development (days 10/11) in the epithelium of the developing anterior pituitary (Rathke's pouch), the connective tissue capsule and meninges of the developing brain, and specific regions of neuroepithelium in the developing brain.

Biocompatibility testing of a posterior composite and dental cements using a new organ culture model.

Mandibular first molars from day 14 and 17 mouse embryos were cultured in vitro for 7 days in chemically defined Pratt's medium in a submerged culture system. Ameloblast and odontoblast polarization and morphogenesis occurred in the control tooth germs. Discs of Occlusin, Silicate, ChemFil, IRM and Dycal were exposed to the culture system as either fresh material, leached discs (by pre-incubation in media) or the leachate from the incubated discs. Their effects on dental differentiation were assessed histologically. Day 17 tooth germs were slightly more sensitive to the effects of the exogenous agents than day 14 tooth germs. In general, silicate and ChemFil were toxic, IRM was slightly less toxic and the major effect was from the leachate. Dycal was toxic but most of this effect resulted from pH changes in the leachate. Occlusin was the most biocompatible material tested. Only a very mild adverse effect was detected, and this appeared to be caused by an agent (not a pH change) released into the leachate.