During palatogenesis, fusion of the palatine shelves is a crucial event, the failure of which results in the birth defect, cleft palate. The fate of the midline epithelial seam (MES), which develops transiently upon contact of the two palatine shelves, is still strongly debated. Three major mechanisms underlying the regression of the MES upon palatal fusion have been proposed: (1) apoptosis has been evidenced by morphological and molecular criteria; (2) epithelial-mesenchymal transformation has been suggested based on ultrastructural and lipophilic dye cell labeling observations; and (3) migration of MES cells toward the oral and nasal areas has been proposed following lipophilic dye cell labeling. To verify whether epithelial-mesenchymal transformation of MES cells takes place during murine palatal fusion, we used the Cre/lox system to genetically mark Sonic hedgehog- and Keratin-14-expressing palatal epithelial cells and to identify their fate in vivo. Our analyses provide conclusive evidence that rules out the occurrence of epithelial-mesenchymal transformation of MES cells.

SummaryWe show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.

A total of 102 strains of Actinomyces were isolated from teeth, buccal mucosa and tongue in eight individuals. The isolates were characterized by multivariate statistical analyses of phenotypic characteristics, serotyping and binding to beta-linked galactosamine (N-acetyl-beta-D-galactosamine) and acidic proline-rich protein structures. Based on these characteristics, isolates were classified into three major groups: (i) Isolates of Actinomyces naeslundii genospecies 2 were the dominant species on teeth and buccal mucosa and bound commonly to N-acetyl-beta-D-galactosamine (63 of 63 isolates) and acidic proline-rich proteins (63 of 63 isolates), regardless of tissue origin. They all exhibited a N-acetyl-beta-D-galactosamine binding specificity signified by N-acetyl-beta-D-galactosamine-inhibitable coaggregation with the streptococcal strains LVG1, GVE1, 24892 and MPB1; (ii) Isolates of A. naeslundii genospecies 1 were prevalent on teeth in certain individuals and bound commonly to N-acetyl-beta-D-galactosamine (20 of 20 isolates), but less commonly to acidic proline-rich proteins (5 of 20 isolates). They all possessed another N-acetyl-beta-D-galactosamine specificity, i.e. N-acetyl-beta-D-galactosamine-inhibitable coaggregation with the same streptococcal strains except for strain MPB1; (iii) Isolates of Actinomyces odontolyticus, the dominant species on the tongue (17 of 19 isolates), bound commonly to unknown structures on streptococci (17 of 19 isolates) but rarely to N-acetyl-beta-D-galactosamine (2 of 19 isolates) or acidic proline-rich proteins (3 of 19 isolates). In conclusion, A. naeslundii genospecies 1 and 2 exhibit different patterns of N-acetyl-beta-D-galactosamine and acidic proline-rich protein specificities to colonize dental and buccal mucosa surfaces, whereas A. odontolyticus utilizes another specificity to colonize the tongue.

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.