Developmental mechanisms driving complex tooth shape in reptiles.

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

Regulation of ciliary function by fibroblast growth factor signaling identifies FGFR3-related disorders achondroplasia and thanatophoric dysplasia as ciliopathies.

Cilia project from almost every cell integrating extracellular cues with signaling pathways. Constitutive activation of FGFR3 signaling produces the skeletal disorders achondroplasia (ACH) and thanatophoric dysplasia (TD), but many of the molecular mechanisms underlying these phenotypes remain unresolved. Here, we report in vivo evidence for significantly shortened primary cilia in ACH and TD cartilage growth plates. Using in vivo and in vitro methodologies, our data demonstrate that transient versus sustained activation of FGF signaling correlated with different cilia consequences. Transient FGF pathway activation elongated cilia, while sustained activity shortened cilia. FGF signaling extended primary cilia via ERK MAP kinase and mTORC2 signaling, but not through mTORC1. Employing a GFP-tagged IFT20 construct to measure intraflagellar (IFT) speed in cilia, we showed that FGF signaling affected IFT velocities, as well as modulating cilia-based Hedgehog signaling. Our data integrate primary cilia into canonical FGF signal transduction and uncover a FGF-cilia pathway that needs consideration when elucidating the mechanisms of physiological and pathological FGFR function, or in the development of FGFR therapeutics.

Multikinase activity of fibroblast growth factor receptor (FGFR) inhibitors SU5402, PD173074, AZD1480, AZD4547 and BGJ398 compromises the use of small chemicals targeting FGFR catalytic activity for therapy of short-stature syndromes.

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of human dwarfism, achondroplasia (ACH). Small chemical inhibitors of FGFR tyrosine kinase activity are considered to be viable option for treating ACH, but little experimental evidence supports this claim. We evaluated five FGFR tyrosine kinase inhibitors (TKIs) (SU5402, PD173074, AZD1480, AZD4547 and BGJ398) for their activity against FGFR signaling in chondrocytes. All five TKIs strongly inhibited FGFR activation in cultured chondrocytes and limb rudiment cultures, completely relieving FGFR-mediated inhibition of chondrocyte proliferation and maturation. In contrast, TKI treatment of newborn mice did not improve skeletal growth and had lethal toxic effects on the liver, lungs and kidneys. In cell-free kinase assays as well as in vitro and in vivo cell assays, none of the tested TKIs demonstrated selectivity for FGFR3 over three other FGFR tyrosine kinases. In addition, the TKIs exhibited significant off-target activity when screened against a panel of 14 unrelated tyrosine kinases. This was most extensive in SU5402 and AZD1480, which inhibited DDR2, IGF1R, FLT3, TRKA, FLT4, ABL and JAK3 with efficiencies similar to or greater than those for FGFR. Low target specificity and toxicity of FGFR TKIs thus compromise their use for treatment of ACH. Conceptually, different avenues of therapeutic FGFR3 targeting should be investigated.

Age-related changes in the tooth-bone interface area of acrodont dentition in the chameleon.

Chameleon teeth develop as individual structures at a distance from the developing jaw bone during the pre-hatching period and also partially during the post-hatching period. However, in the adult, all teeth are fused together and tightly attached to the jaw bone by mineralized attachment tissue to form one functional unit. Tooth to bone as well as tooth to tooth attachments are so firm that if injury to the oral cavity occurs, several neighbouring teeth and pieces of jaw can be broken off. We analysed age-related changes in chameleon acrodont dentition, where ankylosis represents a physiological condition, whereas in mammals, ankylosis only occurs in a pathological context. The changes in hard-tissue morphology and mineral composition leading to this fusion were analysed. For this purpose, the lower jaws of chameleons were investigated using X-ray micro-computed tomography, laser-induced breakdown spectroscopy and microprobe analysis. For a long time, the dental pulp cavity remained connected with neighbouring teeth and also to the underlying bone marrow cavity. Then, a progressive filling of the dental pulp cavity by a mineralized matrix occurred, and a complex network of non-mineralized channels remained. The size of these unmineralized channels progressively decreased until they completely disappeared, and the dental pulp cavity was filled by a mineralized matrix over time. Moreover, the distribution of calcium, phosphorus and magnesium showed distinct patterns in the different regions of the tooth-bone interface, with a significant progression of mineralization in dentin as well as in the supporting bone. In conclusion, tooth-bone fusion in chameleons results from an enhanced production of mineralized tissue during post-hatching development. Uncovering the developmental processes underlying these outcomes and performing comparative studies is necessary to better understand physiological ankylosis; for that purpose, the chameleon can serve as a useful model species.

Polarized Sonic Hedgehog Protein Localization and a Shift in the Expression of Region-Specific Molecules Is Associated With the Secondary Palate Development in the Veiled Chameleon.

Secondary palate development is characterized by the formation of two palatal shelves on the maxillary prominences, which fuse in the midline in mammalian embryos. However, in reptilian species, such as turtles, crocodilians, and lizards, the palatal shelves of the secondary palate develop to a variable extent and morphology. While in most Squamates, the palate is widely open, crocodilians develop a fully closed secondary palate. Here, we analyzed developmental processes that underlie secondary palate formation in chameleons, where large palatal shelves extend horizontally toward the midline. The growth of the palatal shelves continued during post-hatching stages and closure of the secondary palate can be observed in several adult animals. The massive proliferation of a multilayered oral epithelium and mesenchymal cells in the dorsal part of the palatal shelves underlined the initiation of their horizontal outgrowth, and was decreased later in development. The polarized cellular localization of primary cilia and Sonic hedgehog protein was associated with horizontal growth of the palatal shelves. Moreover, the development of large palatal shelves, supported by the pterygoid and palatine bones, was coupled with the shift in Meox2, Msx1, and Pax9 gene expression along the rostro-caudal axis. In conclusion, our results revealed distinctive developmental processes that contribute to the expansion and closure of the secondary palate in chameleons and highlighted divergences in palate formation across amniote species.

Angled Growth of the Dental Lamina Is Accompanied by Asymmetrical Expression of the WNT Pathway Receptor Frizzled 6.

Frizzled 6 (FZD6) belongs to a family of proteins that serve as receptors in the WNT signaling pathway. FZD6 plays an important role in the establishment of planar cell polarity in many embryonic processes such as convergent extension during gastrulation, neural tube closure, or hair patterning. Based on its role during hair development, we hypothesized that FZD6 may have similar expression pattern and function in the dental lamina, which is also a distinct epithelial protrusion growing characteristically angled into the mesenchyme. Diphyodont minipig was selected as a model species because its dentition closely resemble human ones with successional generation of teeth initiated from the dental lamina. We revealed asymmetrical expression of FZD6 in the dental lamina of early as well as late stages during its regression with stronger expression located on the labial side of the dental lamina. During lamina regression, FZD6-positive cells were found in its superficial part and the signal coincided with the upregulation of molecules involved in epithelial-mesenchymal transition and increased migratory potential of epithelial cells. FZD6-expression was also turned on during differentiation of cells producing hard tissues, in which mature odontoblasts, ameloblasts, or surrounding osteoblasts were FZD6-positive. On the other hand, the tip of successional lamina and its lingual part, in which progenitor cells are located, exhibited FZD6-negativity. In conclusion, asymmetrical expression of FZD6 correlates with the growth directionality and side-specific morphological differences in the dental lamina of diphyodont species. Based on observed expression pattern, we propose that the dental lamina is other epithelial tissue, where planar cell polarity signaling is involved during its asymmetrical growth.

Fate of the molar dental lamina in the monophyodont mouse.

The successional dental lamina (SDL) plays an essential role in the development of replacement teeth in diphyodont and polyphyodont animals. A morphologically similar structure, the rudimental successional dental lamina (RSDL), has been described in monophyodont (only one tooth generation) lizards on the lingual side of the developing functional tooth. This rudimentary lamina regresses, which has been proposed to play a role in preventing the formation of future generations of teeth. A similar rudimentary lingual structure has been reported associated with the first molar in the monophyodont mouse, and we show that this structure is common to all murine molars. Intriguingly, a lingual lamina is also observed on the non-replacing molars of other diphyodont mammals (pig and hedgehog), initially appearing very similar to the successional dental lamina on the replacing teeth. We have analyzed the morphological as well as ultrastructural changes that occur during the development and loss of this molar lamina in the mouse, from its initiation at late embryonic stages to its disappearance at postnatal stages. We show that loss appears to be driven by a reduction in cell proliferation, down-regulation of the progenitor marker Sox2, with only a small number of cells undergoing programmed cell death. The lingual lamina was associated with the dental stalk, a short epithelial connection between the tooth germ and the oral epithelium. The dental stalk remained in contact with the oral epithelium throughout tooth development up to eruption when connective tissue and numerous capillaries progressively invaded the dental stalk. The buccal side of the dental stalk underwent keratinisation and became part of the gingival epithelium, while most of the lingual cells underwent programmed cell death and the tissue directly above the erupting tooth was shed into the oral cavity.

The development of complex tooth shape in reptiles.

Reptiles have a diverse array of tooth shapes, from simple unicuspid to complex multicuspid teeth, reflecting functional adaptation to a variety of diets and eating styles. In addition to cusps, often complex longitudinal labial and lingual enamel crests are widespread and contribute to the final shape of reptile teeth. The simplest shaped unicuspid teeth have been found in piscivorous or carnivorous ancestors of recent diapsid reptiles and they are also present in some extant carnivores such as crocodiles and snakes. However, the ancestral tooth shape for squamate reptiles is thought to be bicuspid, indicating an insectivorous diet. The development of bicuspid teeth in lizards has recently been published, indicating that the mechanisms used to create cusps and crests are very distinct from those that shape cusps in mammals. Here, we introduce the large variety of tooth shapes found in lizards and compare the morphology and development of bicuspid, tricuspid, and pentacuspid teeth, with the aim of understanding how such tooth shapes are generated. Next, we discuss whether the processes used to form such morphologies are conserved between divergent lizards and whether the underlying mechanisms share similarities with those of mammals. In particular, we will focus on the complex teeth of the chameleon, gecko, varanus, and anole lizards using SEM and histology to compare the tooth crown morphology and embryonic development.