Spatio-temporal expression of Sox genes in murine palatogenesis.

Members of the Sox gene family play critical roles in many biological processes including organogenesis. We carried out comparative in situ hybridisation analysis of seventeen Sox genes (Sox1-14, 17, 18 and 21) during murine palatogenesis from initiation to fusion of the palatal shelves above the dorsal side of the tongue. At palatal shelf initiation (E12.5), the localized expression of six Sox genes (Sox2, 5, 6, 9, 12 and 13) was observed in the shelves, whereas Sox4 and Sox11 showed ubiquitious expression. During the down-growth of palatal shelves (E13.5), Sox4, Sox5, and Sox9 exhibited restricted expression to the interior side of the palatal shelves facing the tongue. Following elevation of the palatal shelves (E14.5), Sox2, Sox11 and Sox21 expression was present in the midline epithelial seam. We thus identify dynamic spatio-temporal expression of Sox gene family during the process of palatogenesis.

Regional regulation of Filiform tongue papillae development by Ikkα/Irf6.

BACKGROUND: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. RESULTS: We found Ikkα and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikkα and Irf6 in filiform papillae development. Ikkα and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. CONCLUSIONS: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikkα and Irf6. Developmental Dynamics 245:937-946, 2016. © 2016 Wiley Periodicals, Inc.

Expression of Sox genes in tooth development.

Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

R-spondins/Lgrs expression in tooth development.

BACKGROUND: Tooth development is highly regulated in mammals and it is regulated by networks of signaling pathways (e. g. Tnf, Wnt, Shh, Fgf and Bmp) whose activities are controlled by the balance between ligands, activators, inhibitors and receptors. The members of the R-spondin family are known as activators of Wnt signaling, and Lgr4, Lgr5, and Lgr6 have been identified as receptors for R-spondins. The role of R-spondin/Lgr signaling in tooth development, however, remains unclear. RESULTS: We first carried out comparative in situ hybridization analysis of R-spondins and Lgrs, and identified their dynamic spatio-temporal expression in murine odontogenesis. R-spondin2 expression was found both in tooth germs and the tooth-less region, the diastema. We further examined tooth development in R-spondin2 mutant mice, and although molars and incisors exhibited no significant abnormalities, supernumerary teeth were observed in the diastema. CONCLUSIONS: R-spondin/Lgr signaling is thus involved in tooth development.

Cytoplasmic plaque formation in hemidesmosome development is dependent on SoxF transcription factor function.

Hemidesmosomes are composed of intricate networks of proteins, that are an essential attachment apparatus for the integrity of epithelial tissue. Disruption leads to blistering diseases such as epidermolysis bullosa. Members of the Sox gene family show dynamic and diverse expression patterns during development and mutation analyses in humans and mice provide evidence that they play a remarkable variety of roles in development and human disease. Previous studies have established that the mouse mutant ragged-opossum (Ra(op)) expresses a dominant-negative form of the SOX18 transcription factor that interferes with the function of wild type SOX18 and of the related SOXF-subgroup proteins SOX7 and -17. Here we show that skin and oral mucosa in homozygous Ra(op) mice display extensive detachment of epithelium from the underlying mesenchymal tissue, caused by tearing of epithelial cells just above the plasma membrane due to hemidesmosome disruption. In addition, several hemidesmosome proteins expression were found to be dysregulated in the Ra(op) mice. Our data suggest that SOXF transcription factors play a role in regulating formation of cytoplasmic plaque protein assembly, and that disrupted SOXF function results in epidermolysis bullosa-like skin phenotypes.

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.

Bmp signalling in filiform tongue papillae development.

OBJECTIVE: Tongue papillae are critical organs in mastication. There are four different types of tongue papillae; fungiform, circumvallate, foliate, and filiform papillae. Unlike the other three taste papillae, non-gustatory papillae, filiform papillae cover the entire dorsal surface of the tongue and are important structures for the mechanical stress of sucking. Filiform papillae are further classified into two subtypes with different morphologies, depending on their location on the dorsum of the tongue. The filiform papillae at the intermolar eminence have pointed tips, whereas filiform papillae with rounded tips are found in other regions (anterior tongue). It remains unknown how the shape of each type of filiform papillae are determined during their development. Bmp signalling pathway has been known to regulate mechanisms that determine the shapes of many ectodermal organs. The aim of this study was to investigate the role of Bmp signalling in filiform papillae development. DESIGN: Comparative in situ hybridization analysis of six Bmps (Bmp2-Bmp7) and two Bmpr genes (Bmpr1a and Bmpr1b) were carried out in filiform papillae development. We further examined tongue papillae in mice over-expressing Noggin under the keratin14 promoter (K14-Noggin). RESULTS: We identified a dynamic temporo-spatial expression of Bmps in filiform papillae development. The K14-Noggin mice showed pointed filiform papillae in regions of the tongue normally occupied by the rounded type. CONCLUSIONS: Bmp signalling thus regulates the shape of filiform papillae.

Expression of Fgf signalling pathway related genes during palatal rugae development in the mouse.

Fgf signalling plays critical roles in the development of many ectodermal organs. Palatal rugae are ectodermal corrugated structures of the hard palate and in common with other ectodermal appendages, their development is initiated as epithelial thickenings that form placodes as the underlying mesenchymal cells condense. The placode regions then bulge towards to oral cavity to form an overall corrugated appearance. We carried out comparative in situ hybridization analysis of 18 Fgf ligands (Fgf1-Fgf10, Fgf15-Fgf18, Fgf20-Fgf23), four Fgf receptors (Fgfr1-Fgfr4) and four other Fgf signalling related molecules (Spry1, Spry2, Spry4 and Etv5) during murine palatal rugae development. Fgfr1 and Etv5 showed restricted expression in the interplacode epithelium whereas Fgf18 expression was localized to mesenchyme underneath the interplacode epithelium. The expression of Fgf9 was restricted to epithelial ruga placodes whereas Spry4 expression was observed in mesenchyme underneath the placodes. The localized expression of Fgf2, Fgf8, Fgf16, Fgfr4 and Spry1 were found in bulge mesenchyme. Fgf3, Fgf6, Fgfr2 and Spry2 showed expression in the entire epithelium whereas Fgf10 was expressed throughout the mesenchyme. Fgf signalling thus shows dynamic temporo-spatial expression in murine palatal rugae development.

A role for suppressed incisor cuspal morphogenesis in the evolution of mammalian heterodont dentition.

Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra "cones" to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4-mediated signaling.

Lrp4 modulates extracellular integration of cell signaling pathways in development.

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.

Targeted discovery of novel human exons by comparative genomics.

A complete and accurate set of human protein-coding gene annotations is perhaps the single most important resource for genomic research after the human-genome sequence itself, yet the major gene catalogs remain incomplete and imperfect. Here we describe a genome-wide effort, carried out as part of the Mammalian Gene Collection (MGC) project, to identify human genes not yet in the gene catalogs. Our approach was to produce gene predictions by algorithms that rely on comparative sequence data but do not require direct cDNA evidence, then to test predicted novel genes by RT-PCR. We have identified 734 novel gene fragments (NGFs) containing 2188 exons with, at most, weak prior cDNA support. These NGFs correspond to an estimated 563 distinct genes, of which >160 are completely absent from the major gene catalogs, while hundreds of others represent significant extensions of known genes. The NGFs appear to be predominantly protein-coding genes rather than noncoding RNAs, unlike novel transcribed sequences identified by technologies such as tiling arrays and CAGE. They tend to be expressed at low levels and in a tissue-specific manner, and they are enriched for roles in motor activity, cell adhesion, connective tissue, and central nervous system development. Our results demonstrate that many important genes and gene fragments have been missed by traditional approaches to gene discovery but can be identified by their evolutionary signatures using comparative sequence data. However, they suggest that hundreds-not thousands-of protein-coding genes are completely missing from the current gene catalogs.