Mark Tummers

p63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation

Heterozygous germline mutations in p63, a transcription factor of the p53 family, result in abnormal morphogenesis of the skin and its associated structures, including hair follicles and teeth. In mice lacking p63, all ectodermal organs fail to develop, and stratification of the epidermis is absent. We show that the ectodermal placodes that mark early tooth and hair follicle morphogenesis do not form in p63-deficient embryos, although the multilayered dental lamina that precedes tooth placode formation develops normally. The N-terminally truncated isoform of p63 (ΔNp63) was expressed at high levels in embryonic ectoderm at all stages of tooth and hair development, and it was already dominant over the transactivating TAp63 isoform prior to epidermal stratification. Bmp7, Fgfr2b, Jag1 and Notch1 transcripts were co-expressed withΔ Np63 in wild-type embryos, but were not detectable in the ectoderm of p63 mutants. In addition, β-catenin and Edar transcripts were significantly reduced in skin ectoderm. We also demonstrate that BMP2, BMP7 and FGF10 are potent inducers of p63 in cultured tissue explants. Hence, we suggest that p63 regulates the morphogenesis of surface ectoderm and its derivatives via multiple signalling pathways.

The importance of signal pathway modulation in all aspects of tooth development

Most characteristics of tooth shape and pattern can be altered by modulating the signal pathways mediating epithelial-mesenchymal interactions in developing teeth. These regulatory signals function in complex networks, characterized by an abundance of activators or inhibitors. In addition, multiple specific inhibitors of all conserved signal pathways have been identified as modulators in tooth development. The number of teeth as well as molar cusp patterns can be modified by tinkering with several different signal pathways. The inhibition of any of the major conserved signal pathways in knockout mice leads to arrested tooth formation. On the other hand, the stimulation of the Wnt pathway in the oral epithelium in transgenic mice leads to abundant de novo tooth formation. The modulation of some of the signal pathways can rescue the development of vestigial tooth rudiments in the incisor and molar regions resulting in extra premolar-like teeth. The size and the degree of asymmetry of the continuously growing mouse incisor can be modulated by modifying the complex network of FGF, bone morphogenetic protein, and Activin signals, which regulate the proliferation and differentiation of epithelial stem cells. Follistatin, Sprouty, and Sostdc1 are important endogenous inhibitors antagonizing these pathways and they are also involved in regulation of enamel formation, and patterning of teeth in crown and root domains. All these findings support the hypothesis that the diversity of tooth types and dental patterns may have resulted from tinkering with the conserved signal pathways, organized into complex networks, during evolution.

Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species

The rodent incisor grows continuously throughout its lifetime. The epithelial stem cell niche is located at the apical end of the tooth and its progeny gives rise to the ameloblasts that form the hard enamel. Previously, mesenchymal FGF10 was shown to support the niche, in conjunction with epithelial Notch signaling. Here we show that in a different continuously growing tooth type, the molar of the sibling vole, a similar regulatory system is in place. Moreover, the identical expression pattern of Bmp4 compared to Fgf10 suggests that BMP4 could also be involved in the regulation of the epithelial stem cell niche. Notch and FGF10 signaling is mainly absent in the mouse molar, which stops growing and develops roots. The regulation of the epithelial stem cell niche seems to be flexible allowing for the existence of different tooth types, such as continuously growing teeth, and high and low crowned molars.

Expression of Bone Morphogenetic Proteins and Msx Genes during Root Formation

Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

The role of the dental lamina in mammalian tooth replacement.

We have applied the ferret, Mustela putorius furo, as a model for tooth replacement. Ferret has a heterodont dentition, which includes all tooth families, and all antemolar teeth are replaced. Compared with mouse, the ferret therefore has a less derived mammalian dentition resembling that of humans. We have studied tooth replacement in serial histological sections in embryonic and young postnatal ferrets. Our observations indicate that the replacement teeth form from the dental lamina that is intimately connected to the lingual aspect of the deciduous tooth enamel organ. It grows as an offshoot from the enamel organ, elongates in cervical direction and later buds to give rise to the replacement tooth. The extent of the dental lamina growth, preceding replacement tooth budding, varied between different teeth. The dynamic gene expression patterns of Sostdc1, Shh and Axin2 brought new insight into the signal networks regulating the tooth replacement process. The distinct expression of Sostdc1 at the interface between the dental lamina and the deciduous tooth is the first indication of a specific tissue identity of the dental lamina. We suggest that the reactivation of a competent dental lamina is pivotal for the replacement tooth formation.

Tooth morphogenesis and ameloblast differentiation are regulated by micro-RNAs

Teeth form as appendages of the ectoderm and their morphogenesis is regulated by tissue interactions mediated by networks of conserved signal pathways. Micro-RNA (miRNA) pathway has emerged as important regulator of various aspects of embryonic development, but its function in odontogenesis has not been elucidated. We show that the expression of RNAi pathway effectors is dynamic during tooth morphogenesis and differentiation of dental cells. Based on microarray profiling we selected 8 miRNAs expressed during morphogenesis and 7 miRNAs in the incisor cervical loop containing the stem cell niche. These miRNAs were mainly expressed in the dental epithelium. Conditional deletion of Dicer-1 in the epithelium (Dcr(K14)(-)(/)(-)) resulted in rather mild but significant aberrations in tooth shape and enamel formation. The cusp patterns of the Dcr(K14)(-)(/)(-) molar crowns resembled the patterns of both ancestral muroid rodents and mouse mutants with modulated signal pathways. In the Dcr(K14)(-)(/)(-) incisors, longitudinal grooves formed on the labial surface and these were shown to result from ectopic budding of the progenitor epithelium in the cervical loop. In addition, ameloblast differentiation was impaired and resulted in deficient enamel formation in molars and incisors. To help the identification of candidate target genes of the selected tooth enriched miRNAs, we constructed a new ectodermal organ oriented database, miRTooth. The predicted targets of the selected miRNAs included several components of the main morphogenetic signal pathways regulating tooth development. Based on our findings we suggest that miRNAs modulate tooth morphogenesis largely by fine tuning conserved signaling networks and that miRNAs may have played important roles during tooth evolution.

Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role of dental mesenchyme in limiting tooth induction

Like epithelial organs in general, tooth development involves inductive crosstalk between the epithelium and the mesenchyme. Classically, the inductive potential for tooth formation is considered to reside in the mesenchyme during the visible morphogenesis of teeth, and dental mesenchyme can induce tooth formation even when combined with non-dental epithelium. Here, we have investigated induction of mouse incisors using Sostdc1 (ectodin), a putative antagonist of BMP signaling in the mesenchymal induction of teeth. Deletion of Sostdc1 leads to the full development of single extra incisors adjacent to the main incisors. We show that initially, Sostdc1 expression is limited to the mesenchyme, suggesting that dental mesenchyme may limit supernumerary tooth induction. We test this in wild-type incisors by minimizing the amount of mesenchymal tissue surrounding the incisor tooth germs prior to culture in vitro. The cultured teeth phenocopy the extra incisors phenotype of the Sostdc1-deficient mice. Furthermore, we show that minimizing the amount of dental mesenchyme in cultured Sostdc1-deficient incisors causes the formation of additional de novo incisors that resemble the successional incisor development that results from activated Wnt signaling. Finally, Noggin and Dkk1 prevent individually the formation of extra incisors, and we therefore suggest that inhibition of both BMP and Wnt signaling contributes to the inhibitory role of the dental mesenchyme. Considering the role of mesenchyme in tooth induction and the design of tissue engineering protocols, our work may have uncovered how delicate control of tissue quantities alone influences the outcome between induction and inhibition.

Stem Cells and Tissue Engineering: Prospects for Regenerating Tissues in Dental Practice

In general, human tissues have a very limited potential to regenerate. However, recent progress in stem cell research and in tissue engineering promises novel prospects for tissue regeneration in dental practice in the future. Stem cells have been discovered in many adult tissues, including teeth, and stem cells from embryos have the potential to form all adult tissues. Embryonic stem cells can now be cultured and even produced from adult cells by the nuclear transfer method. Due to the rapid progress of research in molecular biology, particularly in the field of developmental biology, we are now starting to understand at the level of genes and molecules how the development of dental tissues is regulated. For instance, specific signal molecules have been identified which regulate the development of teeth and bones from progenitor cells. This information is already being used for the generation of dentoalveolar tissues in vitro and in vivo. Could we perhaps grow new enamel, dentine, periodontal ligament, bone, or even whole new teeth for our patients in the future?

Obesity Is Associated With Low NAD+/SIRT Pathway Expression in Adipose Tissue of BMI-Discordant Monozygotic Twins

CONTEXT Sirtuins (SIRTs) regulate cellular metabolism and mitochondrial function according to the energy state of the cell reflected by NAD(+) levels. OBJECTIVE Our aim was to determine whether expressions of SIRTs and NAD(+) biosynthesis genes are affected by acquired obesity and how possible alterations are connected with metabolic dysfunction while controlling for genetic and familial factors. DESIGN AND PARTICIPANTS We studied a cross-sectional sample of 40 healthy pairs of monozygotic twins, including 26 pairs who were discordant for body mass index (within-pair difference > 3 kg/m(2)), from the FinnTwin12 and FinnTwin16 cohorts. MAIN OUTCOME MEASURES Subcutaneous adipose tissue (SAT) transcriptomics was analyzed by using Affymetrix U133 Plus 2.0 chips, total SAT (poly-ADP) ribose polymerase (PARP) activity by an ELISA kit, body composition by dual-energy x-ray absorptiometry and magnetic resonance imaging/spectroscopy, and insulin sensitivity by an oral glucose tolerance test. RESULTS SIRT1, SIRT3, SIRT5, NAMPT, NMNAT2, NMNAT3, and NRK1 expressions were significantly down-regulated and the activity of main cellular NAD(+) consumers, PARPs, trended to be higher in the SAT of heavier co-twins of body mass index-discordant pairs. Controlling for twin-shared factors, SIRT1, SIRT3, NAMPT, NMNAT3, and NRK1 were significantly negatively correlated with adiposity, SIRT1, SIRT5, NMNAT2, NMNAT3, and NRK1 were negatively correlated with inflammation, and SIRT1 and SIRT5 were positively correlated with insulin sensitivity. Expressions of genes involved in mitochondrial unfolded protein response were also significantly down-regulated in the heavier co-twins. CONCLUSIONS Our data highlight a strong relationship of reduced NAD(+)/SIRT pathway expression with acquired obesity, inflammation, insulin resistance, and impaired mitochondrial protein homeostasis in SAT.

Modulation of Epithelial Cell Fate of the Root in vitro

Mouse molars are normally not capable of continuous growth. We hypothesized that the mouse molar has intrinsic potential to maintain the epithelial stem cell niche and assessed this potential by growth in vitro. Although the tooth germs flattened considerably, they developed a mineralized crown and a root. However, histologically, the root surface was composed of 3 structurally different regions affecting the fate of the dental epithelium. The anterior and posterior aspects maintained the morphological and molecular characteristics of the cervical loop of a continuously growing incisor, with a continuous layer of ameloblasts. The epithelium making contact with the supporting filter resembled Hertwig’ss epithelial root sheath. The top of the cultured molar exposed to air lacked epithelium altogether. We conclude that the fate of the epithelium is regulated by external cues influenced by culture conditions, and that the molar has the intrinsic capacity to grow continuously.