Distinct roles of MicroRNAs in epithelium and mesenchyme during tooth development

Background: Tooth development is known to be mediated by the cross‐talk between signaling pathways, including Shh, Fgf, Bmp, and Wnt. MicroRNAs (miRNAs) are 19‐ to 25‐nt noncoding small single‐stranded RNAs that negatively regulate gene expression by binding target mRNAs, which is believed to be important for the fine‐tuning signaling pathways in development. To investigate the role of miRNAs in tooth development, we examined mice with either mesenchymal (Wnt1Cre/Dicer^fl/fl) or epithelial (ShhCre/Dicer^fl/fl) conditional deletion of Dicer, which is essential for miRNA processing. Results: By using a CD1 genetic background for Wnt1Cre/Dicer^fl/fl, we were able to examine tooth development, because the mutants retained mandible and maxilla primordia. Wnt1Cre/Dicer^fl/fl mice showed an arrest or absence of teeth development, which varied in frequency between incisors and molars. Extra incisor tooth formation was found in ShhCre/Dicer^fl/fl mice, whereas molars showed no significant anomalies. Microarray and in situ hybridization analysis identified several miRNAs that showed differential expression between incisors and molars. Conclusion: In tooth development, miRNAs thus play different roles in epithelium and mesenchyme, and in incisors and molars. Developmental Dynamics 241:1465–1472, 2012. © 2012 Wiley Periodicals, Inc.     

Postnatal epithelium and mesenchyme stem/progenitor cells in bioengineered amelogenesis and dentinogenesis

Rodent incisors provide a classic model for studying epithelial–mesenchymal interactions in development. However, postnatal stem/progenitor cells in rodent incisors have not been exploited for tooth regeneration. Here, we characterized postnatal rat incisor epithelium and mesenchyme stem/progenitor cells and found that they formed enamel- and dentin-like tissues in vivo. Epithelium and mesenchyme cells were harvested separately from the apical region of postnatal 4–5 day rat incisors. Epithelial and mesenchymal phenotypes were confirmed by immunocytochemistry, CFU assay and/or multi-lineage differentiation. CK14+, Sox2+ and Lgr5+ epithelium stem cells from the cervical loop enhanced amelogenin and ameloblastin expression upon BMP4 or FGF3 stimulation, signifying their differentiation towards ameloblast-like cells, whereas mesenchyme stem/progenitor cells upon BMP4, BMP7 and Wnt3a treatment robustly expressed Dspp, a hallmark of odontoblastic differentiation. We then control-released microencapsulated BMP4, BMP7 and Wnt3a in transplants of epithelium and mesenchyme stem/progenitor cells in the renal capsule of athymic mice in vivo. Enamel and dentin-like tissues were generated in two integrated layers with specific expression of amelogenin and ameloblastin in the newly formed, de novo enamel-like tissue, and DSP in dentin-like tissue. These findings suggest that postnatal epithelium and mesenchyme stem/progenitor cells can be primed towards bioengineered tooth regeneration.     

The dual origin of vaginal epithelium

The absence of the cranial three-fifths of the vaginae of male mice carrying the gene, Testicular Feminization, provides strong support for the view that vaginal epithelium is of dual origin, the cranial three-fifths being of Mullerian in origin and the caudal two-fifths being derived from the urogenital sinus.     

Tissue culture of guinea-pig gall-bladder epithelium

Tissue culture of gall-bladder was attempted in the following media: Dulbecco, Eagle's minimum essential medium, NCTC 135, medium 199 and Ham's F12. Growth occurred in all of them for up to 2 weeks assessed by light microscopy. No enhancement of growth was induced by collagenase trypsin insulin or hydrocortisone. Scanning electron microscopy confirmed that new cells colonised the free surface of the explant. Transmission electron microscopy showed good preservation of the original tall epithelial cells for the period of study. The new migrating cells were flatter, but retained the morphological features of the columnar cells. Secretory granules were absent after 1 day in culture but increased amounts of glycogen and lipid began to appear in the epithelium.     

Detection of differentChlamydia trachomatis serovars of urogenital origin by enzyme-immunoassay

European Journal of Clinical Microbiology &; Infectious Diseases -     

Differentiation of the mammalian retinal pigment epithelium in vitro: Influence of presumptive retinal neuroepithelium and head mesenchyme

The ancestor cells of the pigment epithelium of the mammalian eye are derived from the neuroepithelial cells of the neural plate. They are neurally determined in the process of neurulation but finally decide to follow the pigment cell lineage, whereas the adjacent tissue develops into the neuroretina and the optic stalk. This decision is most probably made in the developmental stage of eye cup formation. The pigment epithelium becomes restricted to the outer leaf of the eye cup and does not encroach on the adjacent neuroepithelial tissues of the internal leaf and the eye stalk. It is therefore supposed to be channelled by a locally confined determinant factor that has not yet been identified. In the present study, development of the mammalian eye and the neural versus pigment cell decision were investigated in mouse embryos. Three approaches were used to discover the source of the putative determinant involved in the process of neuroepithelial decision. First, eye primordia were cultured from stage 11 embryos (0 somites, early neural plate stage, embryonic day 7 1/2–8) to stage 16 embryos (34 somites, neural tube stage, ed 10); this is prior to pigment cell induction. The eye primordia were first cultured in head segments and their natural position. In these experiments, 50% of the ocular neuroepithelia developed along the nerve cell and glial cell lineage. However, the other 50% of the cultured specimens partly developed into pigment epithelia. In these specimens the determinant factors had obviously remained functionally intact in vitro. In the second type of experiment, the eye primordia were also cultured within the head segments, but with the prospective neuroretina selectively removed. This experiment should show whether the inner layer of the eye cup (the prospective neuroretina) is involved in the neuroepithelial lineage decision. In these experiments 90% of the cultured eye primordia failed to develop pigmented cells. The prospective neuroretina was therefore considered as a candidate for the production of an inductive factor. Finally, eye primordia from stage 14–15 embryos (13–29 somites, ed 9–9 1/2) were either transplanted into heterotopic tissues, such as mesenchymal organs, neuroepithelium or heterochronic muscle, or grown as controls in their natural position and tissue environment. In these conditions both transplanted eye primordia and controls bore pigmented epithelium. Hence, the lineage decision, whether to form neural or pigment cell, remained undisturbed in all epitopes tested. On the basis of these experiments, it seemed unlikely that the development of pigment cells was initiated by a mesenchyme-derived factor exclusively produced near the eye vesicle.     

Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme

Nephrogenesis in the mouse kidney begins at embryonic day 11 and ends approximately 10 days postpartum. During this period, new nephrons are continually being generated from a stem-cell population-the nephrogenic mesenchyme-in response to signals emanating from the tips of the branching ureter. Relatively little is known about the mechanism by which the nephrogenic mesenchyme cell population is maintained at the tips of the ureter in the presence of signals promoting tubulogenesis. Previous studies have shown that a loss of Bmp7 function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. The studies presented here demonstrate that BMP7 signaling can prevent apoptosis in explants of metanephric mesenchyme. The surviving mesenchyme cell population, however, is not competent to respond to signals promoting tubulogenesis. In conjunction with FGF2, BMP7 promotes growth and maintains competence of the mesenchyme in vitro. In addition, FGF2 and BMP7 signaling, both independently and in combination, inhibit tubulogenesis. Interestingly, FGF2 and BMP7 also promote expansion of the stromal progenitor cell population in whole kidney culture. Because BMP7 is not produced by stromal progenitor cells, these data suggest a novel interaction between the nephrogenic mesenchyme and stromal progenitor cell populations. A model for the regulation of nephrogenesis by FGF and BMP signaling is presented.     

Coordinated induction of cell proliferation and syndecan expression in dental mesenchyme by epithelium: evidence for diffusible signals.

Epithelial-mesenchymal interactions induce the expression of syndecan, a cell surface proteoglycan, and tenascin, an extracellular matrix glycoprotein in the mesenchymal component of many organ rudiments including the tooth. Experimental recombination cultures of early dental epithelium and mesenchyme were analysed by double immunostaining to compare the distribution of syndecan, tenascin, and proliferating cells (BrdU incorporation) in the induced dental mesenchyme. After 5-9 hr in culture expression of syndecan and tenascin as well as an increase in BrdU incorporation were evident in the mesenchymal cells adjacent to the epithelium and the positive area enlarged with time. Syndecan and tenascin were colocalized only partially in some explants. The expression of syndecan and tenascin in the recombinants correlates with their stage-dependent expression pattern during early tooth development in vivo (Vainio and Thesleff, 1992). The area of increased cell proliferation in the mesenchyme correlated closely with syndecan expression. In none of the explants was increased BrdU incorporation observed in syndecan negative areas. Epithelium induced also condensation of the mesenchymal cells. Induction and spread of the syndecan-positive zone in the dental mesenchyme required close and continuous contact with the epithelium. The mechanism by which the induction of syndecan expression spreads in the mesenchyme was studied in rat-mouse interspecies recombination cultures, using syndecan antibodies that recognize mouse but not rat syndecan. The rat mesenchyme and epithelium were first cultured in contact for 24 hr. Then the epithelium was removed and freshly dissected, "uninduced" mouse mesenchyme was placed in contact with different aspects of the rat mesenchyme. The rat mesenchymal cells that had located next to the epithelial tissue stimulated syndecan expression in adjacent mouse mesenchyme. The induction potential was gradually lost toward the periphery of the rat mesenchyme. Based on these findings we suggest that diffusible signal molecules mediate the spread of syndecan induction in the mesenchyme and that syndecan plays a role in the regulation of cell proliferation.     

Effects of BMP-7 on mouse tooth mesenchyme and chick mandibular mesenchyme.

BMP-7 is a member of the BMP family of signaling molecules that are thought to play key roles in mediating inductive events during embryogenesis. In the present study the possible roles of BMP-7 in mediating inductive events during the initiation phase of odontogenesis and mandibular morphogenesis were investigated. To do so, we have examined the effects of agarose beads soaked in recombinant BMP-7 on E11 mouse molar-forming mesenchyme and stage 23 chick mandibular mesenchyme, and analyzed the patterns of expression of Bmp-7 in developing mouse and chick first branchial arches. Beads releasing BMP-7 induced a translucent zone, cellular proliferation, and expression of Msx-1, Msx-2, and Bmp-4 in molar-forming mesenchyme after 24 hr. The effects of BMP-7 on molar-forming mesenchyme are similar to the effects of BMP-4 and are consistent with their overlapping patterns of expression in the thickened epithelium of the early developing tooth buds, which is suggestive of cooperative and/or redundant roles of BMPs in mediating the inductive interactions during the early stages of odontogenesis. Our studies in the developing chick mandible showed that Bmp-7 is expressed in the mandibular epithelium. In the absence of mandibular epithelium, BMP-7 beads maintained cell proliferation and Msx expression in the medial mandibular mesenchyme and were able to induce cell proliferation, cell death, and Msx expression in the lateral chick mandibular mesenchyme. The effects of BMP-7 on the expression of Msx genes in lateral chick mandibular mesenchyme, although different from the effects of lateral mandibular epithelium, are similar to the effects of epithelium from the medial region where multiple Bmps are expressed. We also showed that laterally placed BMP-7 beads induced ectopic expression of Msx genes and changes in the development of posterior skeletal elements in the maxillary and mandibular arches. However, despite its proliferative effects on mandibular mesenchyme, BMP-7 did not support the directional outgrowth of the mandible. These observations suggest that epithelial-mesenchymal interactions in the medial region of the mandibular arch regulating directional outgrowth of the mandibular mesenchyme are mediated by cooperative interactions between BMPs and other growth factors. Our observations also indicated that EGF, another growth factor implicated in mediating epithelial-mesenchymal interactions in the initiation phase of odontogenesis and morphogenesis of the developing mandible, induces an extensive translucent zone and cellular proliferation in the E11 mouse molar-forming mesenchyme and stage 23 chick mandibular mesenchyme. However, in contrast to BMPs, EGF did not induce Msx-1, Msx-2, and Bmp-4, but modulated the effects of BMPs on the expression of Msx-1 and Msx-2 in these mesenchymes. Our combined data suggest that BMP-7 is a component of the signaling network mediating epithelial-mesenchymal interactions during the initiation phase of odontogenesis and morphogenesis of the mandibular arch.     

Epithelial-mesenchymal interactions in differentiation of stomach epithelium in fetal mice

Summary Epithelial-mesenchymal interactions during development of the forestomach and glandular stomach in fetal mice were investigated by recombination experiments in vitro. Stomach epithelium could not survive when cultivated alone, but its development was supported by the presence of homologous or heterologous mesenchyme.The developmental fate of the epithelium was not affected by recombination with heterologous mesenchyme, but the expression of epithelial differentiation was influenced by the type of mesenchyme. The rate of keratinization of the forestomach epithelium was significantly greater on recombination with homologous mesenchyme than on recombination with heterologous mesenchyme. Moreover, the rate of formation of glandular structures in the glandular stomach epithelium was significantly greater on recombination with 16.5-day stomach mesenchyme than on recombination with 14.5- or 18.5-day stomach mesenchyme.     

The bronchial epithelium: translating gene and environment interactions in asthma

The rising trends in asthma over the past 30 years are likely to be a consequence of changes in the environment acting on a susceptible genotype. Recognising that environmental agents impact on the bronchial epithelium, this structure is in a key position to translate and coordinate these gene-environment interactions. In asthma, the bronchial epithelium is stressed and damaged, with shedding of the columnar cells into the airway lumen. The aim of this review is to consider recent advances in our understanding of why the epithelium is damaged and how the ensuing repair responses orchestrate airway inflammation and remodelling leading to the development and maintenance of the asthmatic state.     

The origin of the basal cell potential in frog corneal epithelium

1. As the micro-electrode penetrated through the epithelial cell membranes of frog cornea, a stepwise increase in the potential reaching a maximum value of - 60 mV (;basal cell potential') was observed. Upon penetrating the stroma, the potential dropped abruptly but remained negative (;stroma potential').2. The basal cell potential was determined as a K(+) or a Cl(-) electrode, but the cell membrane was far more permeable to Cl(-) than to K(+). It was also permeable to Na(+), but to a smaller extent.3. When the product [K](o).[Cl](o) was kept constant, for a tenfold increase in [K](o) the slope of the basal cell potential approached 58 mV at high [K](o). The slope was greater in the excised cornea than in the whole eye.4. The basal cell membrane was more permeable to SCN(-) than to Cl(-). Its selectivity towards cations was in the order K(+) > Rb(+) > Cs(+) > NH(4) (+).5. In Li(+) solution the basal cell membrane in the excised cornea caused a hyperpolarization, followed by a gradual depolarization. In contrast, a slow progressive hyperpolarization occurred in the whole eye.6. Ouabain applied to the epithelial surface of both kinds of preparations did not affect the basal cell potential, but the potential was reduced when applied to the endothelium. MIAA and FDNB also depolarized when applied to both sides of the basal cell.7. With 90 mM-K(+) solution containing Cl(-) or CH(3)SO(4) (-) applied to the epithelial surface, the potential change occurred only in the high K(+) solution with CH(3)SO(4) (-).8. The significance of these results for understanding the role of the epithelial boundary regulating the influence of Na(+), K(+), Cl(-) and drugs is discussed.     

Interactions between rat epididymal epithelium and spermatozoa

We studied in the rat epididymis the presence of membrane-bounded vesicles in the stereociliar areas of the epithelial cells. The intimate contact between principal cell stereocilia and luminal spermatozoa was also explored. The epididymidis of adult male albino rats were fixed with Mollenhauer's fixative via the thoracic aorta; they were removed and the caput and the cauda were separated and fixed for 4 additional hours at 4°C. After fixation, the samples were processed with routine techniques for transmission and scanning electron microscopy. The study showed membrane-bounded vesicles in the lumen of the caput and cauda epididymidis. They are present between stereocilia, in the most peripheral regions of the epididymal lumen, and in a stereocilia-free zone in the apical plasma membrane of the principal cells. The smaller vesicles are located near the apical surface of the latter, and the larger ones are located near the tips of the stereocilia. Their contents are electron lucent in some images and electron dense in others. In several thin sections some of the vesicles are observed to have a stalk. This suggests that the possible mode of production may be an exocytotic process. Some membrane-bounded vesicles were found to be in contact with the head or the tail of maturating spermatozoa. Moreover, an intimate contact was found to exist in the epididymidis between the plasma membranes of the spermatozoa and the stereocilia. These observations seem to suggest two possible mechanisms for sperm-epididymal cell relations: 1) release of a secretion product via the membrane-bounded vesicles and 2) direct contact between stereocilia and spermatozoa.     

Helicobacter pylori chemotaxis modulates inflammation and bacterium-gastric epithelium interactions in infected mice

The ulcer-causing pathogen Helicobacter pylori uses directed motility, or chemotaxis, to both colonize the stomach and promote disease development. Previous work showed that mutants lacking the TlpB chemoreceptor, one of the receptors predicted to drive chemotaxis, led to less inflammation in the gerbil stomach than did the wild type. Here we expanded these findings and examined the effects on inflammation of completely nonchemotactic mutants and mutants lacking each chemoreceptor. Of note, all mutants colonized mice to the same levels as did wild-type H. pylori. Infection by completely nonchemotactic mutants (cheW or cheY) resulted in significantly less inflammation after both 3 and 6 months of infection. Mutants lacking either the TlpA or TlpB H. pylori chemotaxis receptors also had alterations in inflammation severity, while mutants lacking either of the other two chemoreceptors (TlpC and HylB) behaved like the wild type. Fully nonchemotactic and chemoreceptor mutants adhered to cultured gastric epithelial cells and caused cellular release of the chemokine interleukin-8 in vitro similar to the release caused by the wild type. The situation appeared to be different in the stomach. Using silver-stained histological sections, we found that nonchemotactic cheY or cheW mutants were less likely than the wild type to be intimately associated with the cells of the gastric mucosa, although there was not a strict correlation between intimate association and inflammation. Because others have shown that in vivo adherence promotes inflammation, we propose a model in which H. pylori uses chemotaxis to guide it to a productive interaction with the stomach epithelium.     

Accessibility and possibility of elimination of breast epithelium: the theoretical possibility of preventing breast carcinoma through destruction of the epithelium of origin

Two characteristics of breast biology appear to constitute weak points in the fight against breast carcinoma. First, the epithelium from which breast carcinoma arises is of relatively easy accessibility, as it lines cavities which are in close communication with the outside. Second, the elimination of breast epithelium can be tolerated by the organism. Theoretically, breast carcinoma may be prevented by destroying breast epithelium through the injection of an agent into the ductal orifices at the nipple. This strategy appears relatively easy to perform in comparison with conventional strategies, which have not given reliable results. Therefore the possibility presented here should not be neglected.