Molecular profiling of the vestibular lamina highlights a key role for Hedgehog signalling.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

Getting out of a mammalian egg: the egg tooth and caruncle of the echidna.

In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, development of the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence. Unlike therian tooth germs that develop by placode invagination, the echidna egg tooth developed by evagination, similar to the first teeth in some reptiles and fish. The egg tooth ankylosed to the premaxilla, rather than forming a tooth root with ligamentous attachment found in other mammals, with loss of the egg tooth associated with high levels of activity odontoclasts and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium on top of the nose expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with typical reptilian teeth, suggesting that this tooth has been conserved from a common ancestor of mammals and reptiles.

Craniofacial transitions: the role of EMT and MET during head development.

Within the developing head, tissues undergo cell-fate transitions to shape the forming structures. This starts with the neural crest, which undergoes epithelial-to-mesenchymal transition (EMT) to form, amongst other tissues, many of the skeletal tissues of the head. In the eye and ear, these neural crest cells then transform back into an epithelium, via mesenchymal-to-epithelial transition (MET), highlighting the flexibility of this population. Elsewhere in the head, the epithelium loses its integrity and transforms into mesenchyme. Here, we review these craniofacial transitions, looking at why they happen, the factors that trigger them, and the cell and molecular changes they involve. We also discuss the consequences of aberrant EMT and MET in the head.

Evolution and development of the mammalian jaw joint: Making a novel structure.

A jaw joint between the squamosal and dentary is a defining feature of mammals and is referred to as the temporomandibular joint (TMJ) in humans. Driven by changes in dentition and jaw musculature, this new joint evolved early in the mammalian ancestral lineage and permitted the transference of the ancestral jaw joint into the middle ear. The fossil record demonstrates the steps in the cynodont lineage that led to the acquisition of the TMJ, including the expansion of the dentary bone, formation of the coronoid process, and initial contact between the dentary and squamosal. From a developmental perspective, the components of the TMJ form through tissue interactions of muscle and skeletal elements, as well as through interaction between the jaw and the cranial base, with the signals involved in these interactions being both biomechanical and biochemical. In this review, we discuss the development of the TMJ in an evolutionary context. We describe the evolution of the TMJ in the fossil record and the development of the TMJ in embryonic development. We address the formation of key elements of the TMJ and how knowledge from developmental biology can inform our understanding of TMJ evolution.

Epithelial dynamics shed light on the mechanisms underlying ear canal defects.

Defects in ear canal development can cause severe hearing loss as sound waves fail to reach the middle ear. Here, we reveal new mechanisms that control human canal development and highlight for the first time the complex system of canal closure and reopening. These processes can be perturbed in mutant mice and in explant culture, mimicking the defects associated with canal atresia. The more superficial part of the canal forms from an open primary canal that closes and then reopens. In contrast, the deeper part of the canal forms from an extending solid meatal plate that opens later. Closure and fusion of the primary canal was linked to loss of periderm, with failure in periderm formation in Grhl3 mutant mice associated with premature closure of the canal. Conversely, inhibition of cell death in the periderm resulted in an arrest of closure. Once closed, re-opening of the canal occurred in a wave, triggered by terminal differentiation of the epithelium. Understanding these complex processes involved in canal development sheds light on the underlying causes of canal atresia.

Mechanisms driving vestibular lamina formation and opening in the mouse.

The vestibular lamina (VL) forms as an epithelial outgrowth parallel to the dental lamina (DL) in the oral cavity. During late development, it opens to create a furrow that divides the dental tissue from the cheeks and lips and is known as the vestibule. Defects in this process lead to failure in the separation of the teeth from the lips and cheeks, including the presence of multiple frenula. In this paper, the development of the VL is followed in the mouse, from epithelial placode in the embryo to postnatal opening and vestibule formation. During early outgrowth, differential proliferation controls the curvature of the VL as it extends under the forming incisors. Apoptosis plays a role in thinning the deepest part of the lamina, while terminal differentiation of the epithelium, highlighted by the expression of loricrin and flattening of the nuclei, predates the division of the VL into two to create the vestibule. Development in the mouse is compared to the human VL, with respect to the relationship of the VL to the DL, VL morphology and mechanisms of opening. Overall, this paper provides insight into an understudied part of the oral anatomy, shedding light on how defects could form in this region.

Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme.

BACKGROUND: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. RESULTS: Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. CONCLUSIONS: Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.

Revitalising the rudimentary replacement dentition in the mouse.

Most mammals have two sets of teeth (diphyodont) - a deciduous dentition replaced by a permanent dentition; however, the mouse possesses only one tooth generation (monophyodont). In diphyodonts, the replacement tooth forms on the lingual side of the first tooth from the successional dental lamina. This lamina expresses the stem/progenitor marker Sox2 and has activated Wnt/ß-catenin signalling at its tip. Although the mouse does not replace its teeth, a transient rudimentary successional dental lamina (RSDL) still forms during development. The mouse RSDL houses Sox2-positive cells, but no Wnt/ß-catenin signalling. Here, we show that stabilising Wnt/ß-catenin signalling in the RSDL in the mouse leads to proliferation of the RSDL and formation of lingually positioned teeth. Although Sox2 has been shown to repress Wnt activity, overexpression of Wnts leads to a downregulation of Sox2, suggesting a negative-feedback loop in the tooth. In the mouse, the first tooth represses the formation of the replacement, and isolation of the RSDL is sufficient to induce formation of a new tooth germ. Our data highlight key mechanisms that may have influenced the evolution of replacement teeth.This article has an associated 'The people behind the papers' interview.

Cryptophthalmos, dental anomalies, oral vestibule defect, and a novel FREM2 mutation.

FREM2 is a member of the FREM2-FRAS1-FREM1 protein complex which contributes to epithelial-mesenchymal coupling. We report a Thai woman with cryptophthalmos, dental anomalies, and oral vestibule defect. A compound heterozygous mutation (c.6499C>T; p.Arg2167Trp and c.641_642del; p.Glu214GlyfsTer135) in the FREM2 gene was identified. The frameshift variant p.Glu214GlyfsTer135 is de novo and novel. It is predicted to result in the loss of most of the functional domains. The p.Arg2167Trp mutation was predicted to disrupt both Ca2+ binding and conformational change. The Arg2167Trp mutant protein has been shown to cause partial loss of function, decrease its interaction with FREM1 and result in impaired function of the FRAS1-FREM2-FREM1 complex. Frem2 was shown to be expressed in the developing tooth and vestibular lamina. It is hypothesized that these mutations resulted in aberration of the FRAS1-FREM2-FREM1 protein complex, resulting in loss of nephronectin, basement membrane disruption, and abnormal epithelial-mesenchymal interactions leading to dental and oral vestibule malformations.

Expanding genotypic and phenotypic spectrums of LTBP3 variants in dental anomalies and short stature syndrome.

Mutations in LTBP3 are associated with Dental Anomalies and Short Stature syndrome (DASS; MIM 601216), which is characterized by hypoplastic type amelogenesis imperfecta, hypodontia, underdeveloped maxilla, short stature, brachyolmia, aneurysm and dissection of the thoracic aorta. Here we report a novel (p.Arg545ProfsTer22) and a recurrent (c.3107-2A > G) LTBP3 variants, in a Turkish family affected with DASS. The proband, who carried compound heterozygous variant c.3107-2A > G, p.Arg545ProfsTer22, was most severely affected with DASS. The proband's father, who carried the heterozygous variant c.3107-2A > G had short stature and prognathic mandible. The mother and brother of the proband carried the heterozygous variant p.Arg545ProfsTer22, but only the mother showed any DASS characteristics. The c.3107-2A > G and the p.Arg545ProfsTer22 variants are expected to result in abnormal LTPB3 protein, failure of TGFß-LAP-LTBP3 complex formation, and subsequent disruption of TGFß secretion and activation. This is the first report of heterozygous carriers of LTBP3 variants showing phenotypes. The new findings of DASS found in this family include taurodontism, single-rooted molars, abnormal dentin, calcified dental pulp blood vessels, prognathic mandible, failure of mandibular tooth eruption, interatrial septal aneurysm, secundum atrial septal defect, tricuspid valve prolapse, and a recurrent glenohumeral joint dislocation.

Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation.

The middle ear epithelium is derived from neural crest and endoderm, which line distinct regions of the middle ear cavity. Here, we investigate the distribution of putative stem cell markers in the middle ear, combined with an analysis of the location of label-retaining cells (LRCs) to create a map of the middle ear mucosa. We show that proliferating cells and LRCs were associated with specific regions of the ear epithelium, concentrated in the hypotympanum at the base of the auditory bulla and around the ear drum. Sox2 was widely expressed in the endodermally derived ciliated pseudostratified epithelium of the hypotympanum. This part of the middle ear showed high levels of Wnt activity, as indicated by the expression of Axin2, a readout of Wnt signalling. Keratin 5 showed a more restricted expression within the basal cells of this region, with very little overlap between the Sox2- and keratin 5-positive epithelium, indicating that these genes mark distinct populations. Little expression of Sox2 or keratin 5 was observed in the neural crest-derived middle ear epithelium that lined the promontory, except in cases of otitis media when this epithelium underwent hyperplasia. This study lays the foundation for furthering our understanding of homeostasis and repair in the middle ear.

Comparing development and regeneration in the submandibular gland highlights distinct mechanisms.

A common question in organ regeneration is the extent to which regeneration recapitulates embryonic development. To investigate this concept, we compared the expression of two highly interlinked and essential genes for salivary gland development, Sox9 and Fgf10, during submandibular gland development, homeostasis and regeneration. Salivary gland duct ligation/deligation model was used as a regenerative model. Fgf10 and Sox9 expression changed during regeneration compared to homeostasis, suggesting that these key developmental genes play important roles during regeneration, however, significantly both displayed different patterns of expression in the regenerating gland compared to the developing gland. Regenerating glands, which during homeostasis had very few weakly expressing Sox9-positive cells in the striated/granular ducts, displayed elevated expression of Sox9 within these ducts. This pattern is in contrast to embryonic development, where Sox9 expression was absent in the proximally developing ducts. However, similar to the elevated expression at the distal tip of the epithelium in developing salivary glands, regenerating glands displayed elevated expression in a subpopulation of acinar cells, which during homeostasis expressed Sox9 at lower levels. A shift in expression of Fgf10 was observed from a widespread mesenchymal pattern during organogenesis to a more limited and predominantly epithelial pattern during homeostasis in the adult. This restricted expression in epithelial cells was maintained during regeneration, with no clear upregulation in the surrounding mesenchyme, as might be expected if regeneration recapitulated development. As both Fgf10 and Sox9 were upregulated in proximal ducts during regeneration, this suggests that the positive regulation of Sox9 by Fgf10, essential during development, is partially reawakened during regeneration using this model. Together these data suggest that developmentally important genes play a key role in salivary gland regeneration but do not precisely mimic the roles observed during development.

Characteristics of aquaporin 1, 3, and 5 expression during early murine salivary gland development.

Aquaporins (AQPs) are essential to coordinate the transit of water and ions through the cell membrane. In salivary glands (SGs), AQPs have been associated with saliva formation, facilitating water absorption through the epithelium during the formation of hypotonic saliva, which is then secreted into the oral cavity. Different members of the AQP family have been suggested to play distinct roles during embryonic development, highlighted by their specific expression patterns. Here, we have investigated the expression patterns of AQP-1, AQP-3 and AQP-5 by immunofluorescence at key stages of salivary gland development, utilising cultured mouse embryonic submandibular (SMG) and sublingual (SLG) glands. The expression of AQPs was compared to a mitotic marker, phospho-histone 3 (PH3), a myoepithelial marker, smooth muscle actin (SMA), and a vascular marker, CD31. Qualitative analysis revealed that AQP-1 and AQP-3 were primarily expressed during the earlier phases of SG morphogenesis and were associated with cells undergoing mitotic processes (PH3-positive). AQP-5, in contrast, was not associated to mitotic figures, but was predominantly expressed during late stages of SG morphogenesis. Our results highlight that AQPs are expressed from early stages of SG morphogenesis and exhibit complimentary expression patterns that may contribute to the morphogenesis of salivary glands.

Getting out of an egg: Merging of tooth germs to create an egg tooth in the snake.

BACKGROUND: The egg tooth is a vital structure allowing hatchlings to escape from the egg. In squamates (snakes and lizards), the egg tooth is a real tooth that develops within the oral cavity at the top of the upper jaw. Most squamates have a single large midline egg tooth at hatching, but a few families, such as Gekkonidae, have two egg teeth. In snakes the egg tooth is significantly larger than the rest of the dentition and is one of the first teeth to develop. RESULTS: We follow the development of the egg tooth in four snake species and show that the single egg tooth is formed by two tooth germs. These two tooth germs are united at the midline and grow together to produce a single tooth. In culture, this merging can be perturbed to give rise to separate smaller teeth, confirming the potential of the developing egg tooth to form two teeth. CONCLUSIONS: Our data agrees with previous hypotheses that during evolution one potential mechanism to generate a large tooth is through congrescence of multiple tooth germs and suggests that the ancestors of snakes could have had two egg teeth.

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.

Developmental aspects of the tympanic membrane: Shedding light on function and disease.

The ear drum, or tympanic membrane (TM), is a key component in the intricate relay that transmits air-borne sound to our fluid-filled inner ear. Despite early belief that the mammalian ear drum evolved as a transformation of a reptilian drum, newer fossil data suggests a parallel and independent evolution of this structure in mammals. The term "drum" belies what is in fact a complex three-dimensional structure formed from multiple embryonic cell lineages. Intriguingly, disease affects the ear drum differently in its different parts, with the superior and posterior parts being much more frequently affected. This suggests a key role for the developmental details of TM formation in its final form and function, both in homeostasis and regeneration. Here we review recent studies in rodent models and humans that are beginning to address large knowledge gaps in TM cell dynamics from a developmental biologist's point of view. We outline the biological and clinical uncertainties that remain, with a view to guiding the indispensable contribution that developmental biology will be able to make to better understanding the TM.

FGF10 is an essential regulator of tracheal submucosal gland morphogenesis.

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. In the trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with an additional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins and antimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals that regulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development of murine tracheal SMGs, with gland development arresting at the early bud stage in the absence of FGF10 signalling. As Fgf10 knockout mice are lethal at birth, inducible knockdown of Fgf10 at late embryonic stages was used to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygous Fgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. The reduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction in overall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both the number of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMG-dependent respiratory health.

Fgf10 and Sox9 are essential for the establishment of distal progenitor cells during mouse salivary gland development.

Salivary glands are formed by branching morphogenesis with epithelial progenitors forming a network of ducts and acini (secretory cells). During this process, epithelial progenitors specialise into distal (tips of the gland) and proximal (the stalk region) identities that produce the acini and higher order ducts, respectively. Little is known about the factors that regulate progenitor expansion and specialisation in the different parts of the gland. Here, we show that Sox9 is involved in establishing the identity of the distal compartment before the initiation of branching morphogenesis. Sox9 is expressed throughout the gland at the initiation stage before becoming restricted to the distal epithelium from the bud stage and throughout branching morphogenesis. Deletion of Sox9 in the epithelium results in loss of the distal epithelial progenitors, a reduction in proliferation and a subsequent failure in branching. We demonstrate that Sox9 is positively regulated by mesenchymal Fgf10, a process that requires active Erk signalling. These results provide new insights into the factors required for the expansion of salivary gland epithelial progenitors, which can be useful for organ regeneration therapy.

Epithelial stratification and placode invagination are separable functions in early morphogenesis of the molar tooth.

Ectodermal organs, which include teeth, hair follicles, mammary ducts, and glands such as sweat, mucous and sebaceous glands, are initiated in development as placodes, which are epithelial thickenings that invaginate and bud into the underlying mesenchyme. These placodes are stratified into a basal and several suprabasal layers of cells. The mechanisms driving stratification and invagination are poorly understood. Using the mouse molar tooth as a model for ectodermal organ morphogenesis, we show here that vertical, stratifying cell divisions are enriched in the forming placode and that stratification is cell division dependent. Using inhibitor and gain-of-function experiments, we show that FGF signalling is necessary and sufficient for stratification but not invagination as such. We show that, instead, Shh signalling is necessary for, and promotes, invagination once suprabasal tissue is generated. Shh-dependent suprabasal cell shape suggests convergent migration and intercalation, potentially accounting for post-stratification placode invagination to bud stage. We present a model in which FGF generates suprabasal tissue by asymmetric cell division, while Shh triggers cell rearrangement in this tissue to drive invagination all the way to bud formation.

Q&A: Morphological insights into evolution.

In this question and answer article we discuss how evolution shapes morphology (the shape and pattern of our bodies) but also how learning about morphology, and specifically how that morphology arises during development, can shed light on mechanisms that might allow change during evolution. For this we concentrate on recent findings from our lab on how the middle ear has formed in mammals.