Päivi Kettunen

Expression and function of FGFs‐4, ‐8, and ‐9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis

To elucidate the roles of fibroblast growth factors (FGF) in the regulation of tooth morphogenesis we have analyzed the expression patterns of Fgf‐4, ‐8, and ‐9 in the developing mouse molar and incisor tooth germs from initiation to completion of morphogenesis by in situ hybridization analysis. The expression of these Fgfs was confined to dental epithelial cells at stages when epithelial‐mesenchymal signaling regulates critical steps of tooth morphogenesis. Fgf‐8 and Fgf‐9 mRNAs were present in the oral epithelium of the first branchial arch at E10 and 1 day later expression became more restricted to the area of presumptive dental epithelium and persisted there until the start of epithelial budding. Fgf‐8 mRNAs were not detected later in the developing tooth. Fgf‐4 and Fgf‐9 expression was upregulated in the primary enamel knot, which is a putative signaling center regulating tooth shape. Subsequently, Fgf‐4 and Fgf‐9 were expressed in the secondary enamel knots at the sites of tooth cusps. Fgf‐9 expression spread from the primary enamel knot within the inner enamel epithelium where it remained until E18. In the continuously growing incisors Fgf‐9 expression persisted in the epithelium of the cervical loops. The effects of FGFs were analyzed on the expression of the homeobox‐containing transcription factors Msx‐1 and Msx‐2, which are associated with tissue interactions and regulated by the dental epithelium. Locally applied FGF‐4, ‐8, and ‐9 stimulated intensely the expression of Msx‐1 but not Msx‐2 in the isolated dental mesenchyme. We suggest that the three FGFs act as epithelial signals mediating inductive interactions between dental epithelium and mesenchyme during several successive stages of tooth formation. This data suggest roles for FGF‐8 and FGF‐9 during initiation of tooth development, and for FGF‐4 and FGF‐9 during regulation of tooth shape. FGF‐9 may also be involved in differentiation of odontoblasts. The coexpression of Fgfs with other signaling molecules including Shh and several Bmps and their partly similar effects suggest that the FGFs participate in the signaling networks during odontogenesis. Dev. Dyn. 1998;211:256–268.

Associations of FGF‐3 and FGF‐10 with signaling networks regulating tooth morphogenesis

The morphogenesis and cell differentiation in developing teeth is governed by interactions between the oral epithelium and neural crest‐derived ectomesenchyme. The fibroblast growth factors FGF‐4, ‐8, and ‐9 have been implicated as epithelial signals regulating mesenchymal gene expression and cell proliferation during tooth initiation and later during epithelial folding morphogenesis and the establishment of tooth shape. To further evaluate the roles of FGFs in tooth development, we analyzed the roles of FGF‐3, FGF‐7, and FGF‐10 in developing mouse teeth. In situ hybridization analysis showed developmentally regulated expression during tooth formation for Fgf‐3 and Fgf‐10 that was mainly restricted to the dental papilla mesenchymal cells. Fgf‐7 transcripts were restricted to the developing bone surrounding the developing tooth germ. Fgf‐10 expression was observed in the presumptive dental epithelium and mesenchyme during tooth initiation, whereas Fgf‐3 expression appeared in the dental mesenchyme at the late bud stage. During the cap and bell stage, both Fgf‐3 and Fgf‐10 were intensely expressed in the dental papilla mesenchymal cells both in incisors and molars. It is of interest that Fgf‐3 expression was also observed in the primary enamel knot, a putative signaling center of the tooth, whereas no transcripts were seen in the secondary enamel knots that appear in the tips of future cusps of the bell stage tooth germs. Down‐regulation of Fgf‐3 and Fgf‐10 expression in postmitotic odontoblasts correlated with the terminal differentiation of the odontoblasts and the neighboring ameloblasts. In the incisors, mesenchymal cells of the cervical loop area showed partially overlapping expression patterns for all studied Fgfs. In vitro analyses showed that expression of Fgf‐3 and Fgf‐10 in the dental mesenchyme was dependent on dental epithelium and that epithelially expressed FGFs, FGF‐4 and ‐8 induced Fgf‐3 but not Fgf‐10 expression in the isolated dental mesenchyme. Beads soaked in Shh, BMP‐2, and TGF‐β1 protein did not induce either Fgf‐3 or Fgf‐10 expression. Cells expressing Wnt‐6 did not induce Fgf‐10 expression. Furthermore, FGF‐10 protein stimulated cell proliferation in the dental epithelium but not in the mesenchyme. These results suggest that FGF‐3 and FGF‐10 have redundant functions as mesenchymal signals regulating epithelial morphogenesis of the tooth and that their expressions appear to be differentially regulated. In addition, FGF‐3 may participate in signaling functions of the primary enamel knot. The dynamic expression patterns of different Fgfs in dental epithelium and mesenchyme and their interactions suggest existence of regulatory signaling cascades between epithelial and mesenchymal FGFs during tooth development.

Association of developmental regulatory genes with the development of different molar tooth shapes in two species of rodents

Abstract While the evolutionary history of mammalian tooth shapes is well documented in the fossil record, the developmental basis of their tooth shape evolution is unknown. We investigated the expression patterns of eight developmental regulatory genes in two species of rodents with different molar morphologies (mouse, Mus musculus and sibling vole, Microtus rossiaemeridionalis). The genes Bmp-2, Bmp-4, Fgf-4 and Shh encode signal molecules, Lef-1, Msx-1 and Msx-2, are transcription factors and p21CIP1/WAF1 participates in the regulation of cell cycle. These genes are all known to be associated with developmental regulation in mouse molars. In this paper we show that the antisense mRNA probes made from mouse cDNA cross-hybridized with vole tissue. The comparisons of gene expression patterns and morphologies suggest that similar molecular cascades are used in the early budding of tooth germs, in the initiation of tooth crown base formation, and in the initiation of each cusp’s development. Furthermore, the co-localization of several genes indicate that epithelial signalling centres function at the three stages of morphogenesis. The earliest signalling centre in the early budding epithelium has not been reported before, but the latter signalling centres, the primary and the secondary enamel knots, have been studied in mouse. The appearance of species-specific tooth shapes was manifested by the regulatory molecules expressed in the secondary enamel knots at the areas of future cusp tips, whilst the mesenchymal gene expression patterns had a buccal bias without similar species-specific associations.

Gene expression patterns associated with suppression of odontogenesis in mouse and vole diastema regions

Abstract Rodents have a toothless diastema region between the incisor and molar teeth which may contain rudimentary tooth germs. We found in upper diastema region of the mouse (Musmusculus) three small tooth germs which developed into early bud stage before their apoptotic removal, while the sibling vole (Microtusrossiaemeridionalis) had only a single but larger tooth germ in this region, and this developed into late bud stage before regressing apoptotically. To analyze the genetic mechanisms of the developmental arrest of the rudimentary tooth germs we compared the expression patterns of several developmental regulatory genes (Bmp2, Bmp4, Fgf4, Fgf8, Lef1, Msx1, Msx2, p21, Pitx2, Pax9 and Shh) between molars and diastema buds of mice and voles. In diastema tooth buds the expression of all the genes differed from that of molars. The gene expression patterns suggest that the odontogenic program consists of partially independent signaling cascades which define the exact location of the tooth germ, initiate epithelial budding, and transfer the odontogenic potential from the epithelium to the underlying mesenchyma. Although the diastema regions of the two species differed, in both species the earliest difference that we found was weaker expression of mesenchymal Pax9 in the diastema region than in molar and incisor regions at the dental lamina stage. However, based on earlier tissue recombination experiments it is conceivable that the developmental arrest is determined by the early oral epithelium.

Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial-mesenchymal interactions, and epithelial Wnt4 and Tgfβ1 regulate semaphorin 3a expression in the dental mesenchyme

During development, trigeminal nerve fibers navigate and establish their axonal projections to the developing tooth in a highly spatiotemporally controlled manner. By analyzing Sema3a and its receptor Npn1 knockout mouse embryos, we found that Sema3a regulates dental trigeminal axon navigation and patterning, as well as the timing of the first mandibular molar innervation, and that the effects of Sema3a appear to be mediated by Npn1 present in the axons. By performing tissue recombinant experiments and analyzing the effects of signaling molecules, we found that early oral and dental epithelia, which instruct tooth formation, and epithelial Wnt4 induce Sema3a expression in the presumptive dental mesenchyme before the arrival of the first dental nerve fibers. Later, at the bud stage, epithelial Wnt4 and Tgfβ1 regulate Sema3a expression in the dental mesenchyme. In addition, Wnt4 stimulates mesenchymal expression of Msx1 transcription factor, which is essential for tooth formation, and Tgfβ1 proliferation of the dental mesenchymal cells. Thus, epithelial-mesenchymal interactions control Sema3a expression and may coordinate axon navigation and patterning with tooth formation. Moreover, our results suggest that the odontogenic epithelium possesses the instructive information to control the formation of tooth nerve supply.

Dynamic expression of Wnt signaling-related Dickkopf1, -2, and -3 mRNAs in the developing mouse tooth.

Wnt signaling is essential for tooth formation. Members of the Dickkopf (Dkk) family modulate the Wnt signaling pathway by binding to the Wnt receptor complex. Comparison of Dkk1, ‐2, and ‐3 mRNA expression during mouse tooth formation revealed that all three genes showed distinct spatiotemporally regulated expression patterns. Dkk1 was prominently expressed in the distal, incisor‐bearing mesenchyme area of the mandibular process during the initial stages of tooth formation. During molar morphogenesis Dkk1 was detected in the dental mesenchyme, including the preodontoblasts. Dkk2 was seen in the dental papilla, whereas Dkk3 was specifically expressed in the putative epithelial signaling centers, the primary and secondary enamel knots. Postnatally, Dkk1 was prominently expressed in the preodonto‐ and odontoblasts, while Dkk3 mRNAs were transiently seen in the preameloblasts before the onset of enamel matrix secretion. These results suggest that modulation of Wnt‐signaling by Dkks may serve important functions in patterning of dentition as well as in crown morphogenesis and dental hard‐tissue formation. Developmental Dynamics 233:161–166, 2005.

Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth.

The semaphorins are a large family of secreted or cell-bound signals needed for the development of the nervous system. We compared mRNA expression of class 3 semaphorins (Sema3A, 3B, 3C and 3F) and their two receptors (Neuropilin-1 and -2) in the embryonic mouse first molar tooth germ (E10-18) by radioactive in situ hybridization. All genes showed distinct developmentally regulated expression patterns during tooth organogenesis. Interestingly, Sema3A and 3C were first detected in the early dental epithelium, and later both genes were present in the epithelial primary enamel knot, a putative signaling center of the embryonic tooth regulating tooth morphogenesis. Prior to birth, Sema3A was also observed in tooth-specific cells, preodontoblasts, which later differentiate into odontoblasts secreting dentin, and in the mesenchymal dental follicle cells surrounding the tooth germ. Sema3B appeared transiently in the dental mesenchyme in the bud and cap stage tooth while Sema3F was expressed in both epithelial and mesenchymal components of the tooth. Of note, Npn-1 expression pattern was largely complementary to that of Sema3A, and transcripts were restricted to the dental mesenchymal cells. Npn-1 expression was first seen in the developing dental follicle, and later transcripts also appeared in the dental papilla mesenchyme. In contrast, Npn-2 signal was seen in both epithelial and mesenchymal tissues such as in the primary enamel knot and preodontoblasts.

Tissue interactions in the regulation of axon pathfinding during tooth morphogenesis

Like many other organs, the tooth develops as a result of the epithelial‐mesenchymal interactions. In addition, the tooth is a well‐defined peripheral target organ for sensory trigeminal nerves, which are required for the function and protection of the teeth. Dental trigeminal axon growth and patterning are tightly linked with advancing tooth morphogenesis and cell differentiation. This review summarizes recent findings on the regulation of dental axon pathfinding, which have provided evidence that the development of tooth trigeminal innervation is controlled by epithelial‐mesenchymal interactions. The early dental epithelium possesses the information to instruct tooth nerve supply, and signals mediating these interactions are part of the signaling networks regulating tooth morphogenesis. Tissue interactions, thus, appear to provide a central mechanism of spatiotemporally orchestrating tooth formation and dental axon navigation and patterning. Developmental Dynamics 234:482–488, 2005.

Fgfr2b mediated epithelial-mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning

Dental trigeminal nerve fiber growth and patterning are strictly integrated with tooth morphogenesis, but it is still unknown, how these two developmental processes are coordinated. Here we show that targeted inactivation of the dental epithelium expressed Fgfr2b results in cessation of the mouse mandibular first molar development at the degenerated cap stage and the failure of the trigeminal molar nerve to establish the lingual branch at E13.5 stage while the buccal branch develops properly. This axon patterning defect correlates to the histological absence of the mesenchymal dental follicle and adjacent Semaphorin3A-free dental follicle target field as well as appearance of ectopic Sema3A expression domain in the lingual side of the epithelial bud. Although the mesenchymal ligands for Fgfr2b, Fgf3 and -10 were present in the Fgfr2b(-/)(-) dental mesenchyme, mutant dental epithelium showed dramatically reduced proliferation and the lack of Fgf3. Tgfbeta1, which controls Sema3A was absent from the Fgfr2b(-/-) tooth germ, and Sema3A was specifically downregulated in the dental mesenchyme at the bud and cap stage. In addition, the epithelial primary enamel knot signaling center although being molecularly present neither was histologically detectable nor expressed Bmp4 and Fgf3 as well as Fgf4, which is essential for tooth morphogenesis and stimulates mesenchymal Fgf3 and Tgfbeta1. Fgf4 beads rescued Tgfbeta1 in the Fgfr2b(-/-) dental mesenchyme explants and Tgfbeta1 induced de novo Sema3A expression in the dental mesenchyme. Collectively these results demonstrate that epithelial Fgfr2b controls tooth morphogenesis and dental axon patterning, and suggests that Fgfr2b, by mediating local epithelial-mesenchymal interactions, integrates these two distinct developmental processes during odontogenesis.

RNA deep sequencing of the Atlantic cod transcriptome

The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.