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In the short-beaked echidna, Tachyglossus aculeatus, after an initial period of in utero development, 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. However, there are only a few historical references that describe the development of the monotreme egg tooth. Using unprecedented access to echidna pre- and post-hatching tissues, the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence, to map the changes at the morphological and molecular level. Unlike mammalian tooth germs that develop by invagination of a placode, the echidna egg tooth developed by evagination, similar to that of the first teeth in some reptiles. The egg tooth ankylosed to the premaxilla, rather than forming a mammalian thecodont attachment, with loss of the egg tooth post-hatching associated with high levels of 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 expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with reptilian teeth, suggesting that this tooth is conserved from a common ancestor of mammals and reptiles.

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., 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. Our data suggest that developmentally important genes play a key role in salivary gland regeneration but do not precisely mimic the roles observed during development.

Objective. Caspases, cysteine proteases traditionally associated with apoptosis and inflammation, have recently been identified as important regulators of autophagy and reported within the growth plate, a cartilaginous part of the developing bone. The aim of this research was to identify novel autophagy-related molecules affected by inhibition of pro-apoptotic caspases in chondrocytes. Design. Chondrocyte micromasses derived from mouse limb buds were treated with pharmacological inhibitors of caspases. Autophagy-related gene expression was examined and possible novel molecules were confirmed by real-time polymerase chain reaction and immunocytofluorescence. Individual caspases inhibitors were used to identify the effect of specific caspases. Results. Chondrogenesis accompanied by caspase activation and autophagy progression was confirmed in micromass cultures. Expression of several autophagy-associated genes was significantly altered in the caspases inhibitors treated groups with the most prominent decrease for Pik3cg and increase of Tnfsf10. The results showed the specific pro-apoptotic caspases that play a role in these effects. Importantly, use of caspase inhibitors mimicked changes triggered by an autophagy stimulator, rapamycin, linking loss of caspase activity to an increase in autophagy. Conclusion. Caspase inhibition significantly affects regulation of autophagy-related genes in chondrocytes cultures. Detected markers are of importance in diagnostics and thus the data presented here open new perspectives in the field of cartilage development and degradation.

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.

Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and 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. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. 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.

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.

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

Phenotypic variation across mammals is extensive and reflects their ecological diversification into a remarkable range of habitats on every continent and in every ocean. The skull performs many functions to enable each species to thrive within its unique ecological niche, from prey acquisition, feeding, sensory capture (supporting vision and hearing) to brain protection. Diversity of skull function is reflected by its complex and highly variable morphology. Cranial morphology can be quantified using geometric morphometric techniques to offer invaluable insights into evolutionary patterns, ecomorphology, development, taxonomy, and phylogenetics. Therefore, the skull is one of the best suited skeletal elements for developmental and evolutionary analyses. In contrast, less attention is dedicated to the fibrous sutural joints separating the cranial bones. Throughout postnatal craniofacial development, sutures function as sites of bone growth, accommodating expansion of a growing brain. As growth frontiers, cranial sutures are actively responsible for the size and shape of the cranial bones, with overall skull shape being altered by changes to both the level and time period of activity of a given cranial suture. In keeping with this, pathological premature closure of sutures postnatally causes profound misshaping of the skull (craniosynostosis). Beyond this crucial role, sutures also function postnatally to provide locomotive shock absorption, allow joint mobility during feeding, and, in later postnatal stages, suture fusion acts to protect the developed brain. All these sutural functions have a clear impact on overall cranial function, development and morphology, and highlight the importance that patterns of suture development have in shaping the diversity of cranial morphology across taxa. Here we focus on the mammalian cranial system and review the intrinsic relationship between suture development and morphology and cranial shape from an evolutionary developmental biology perspective, with a view to understanding the influence of sutures on evolutionary diversity. Future work integrating suture development into a comparative evolutionary framework will be instrumental to understanding how developmental mechanisms shaping sutures ultimately influence evolutionary diversity.

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.

Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-βgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

Organs throughout the body develop both asymmetrically and symmetrically. Here, we assess how symmetrical teeth in reptiles can be created from asymmetrical tooth germs. Teeth of lepidosaurian reptiles are mostly anchored to the jaw bones by pleurodont ankylosis, where the tooth is held in place on the labial side only. Pleurodont teeth are characterized by significantly asymmetrical development of the labial and lingual sides of the cervical loop, which later leads to uneven deposition of hard tissue. On the other hand, acrodont teeth found in lizards of the Acrodonta clade (i.e. agamas, chameleons) are symmetrically ankylosed to the jaw bone. Here, we have focused on the formation of the symmetrical acrodont dentition of the veiled chameleon (Chamaeleo calyptratus). Intriguingly, our results revealed distinct asymmetries in morphology of the labial and lingual sides of the cervical loop during early developmental stages, both at the gross and ultrastructural level, with specific patterns of cell proliferation and stem cell marker expression. Asymmetrical expression of ST14 was also observed, with a positive domain on the lingual side of the cervical loop overlapping with the SOX2 domain. In contrast, micro-CT analysis of hard tissues revealed that deposition of dentin and enamel was largely symmetrical at the mineralization stage, highlighting the difference between cervical loop morphology during early development and differentiation of odontoblasts throughout later odontogenesis. In conclusion, the early asymmetrical development of the enamel organ seems to be a plesiomorphic character for all squamate reptiles, while symmetrical and precisely orchestrated deposition of hard tissue during tooth formation in acrodont dentitions probably represents a novelty in the Acrodonta clade.

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., Highlighted Article: We reveal new mechanisms that control development of the ear canal and highlight for the first time the complex system of canal closure and reopening.

The pinna (or auricle) is part of the external ear, acting to capture and funnel sound toward the middle ear. The pinna is defective in a number of craniofacial syndromes, including Lacrimo-auriculo-dento-digital (LADD) syndrome, which is caused by mutations inFGF10or its receptorFGFR2b. Here we study pinna defects in theFgf10knockout mouse. We show that Fgf10 is expressed in both the muscles and forming cartilage of the developing external ear, with loss of signaling leading to a failure in the normal extension of the pinna over the ear canal. Conditional knockout ofFgf10in the neural crest fails to recapitulate this phenotype, suggesting that the defect is due to loss ofFgf10from the muscles, or that this source ofFgf10can compensate for loss in the forming cartilage. The defect in theFgf10null mouse is driven by a reduction in proliferation, rather than an increase in cell death, which can be partially phenocopied by inhibiting cell proliferation in explant culture. Overall, we highlight the mechanisms that could lead to the phenotype observed in LADD syndrome patients and potentially explain the formation of similar low-set and cup shaped ears observed in other syndromes.

Meckel’s cartilage was first described by the German anatomist Johann Friedrich Meckel the Younger in 1820 from his analysis of human embryos. Two hundred years after its discovery this paper follows the development and largely transient nature of the mammalian Meckel’s cartilage, and its role in jaw development. Meckel’s cartilage acts as a jaw support during early development, and a template for the later forming jaw bones. In mammals, its anterior domain links the two arms of the dentary together at the symphysis while the posterior domain ossifies to form two of the three ear ossicles of the middle ear. In between, Meckel’s cartilage transforms to a ligament or disappears, subsumed by the growing dentary bone. Several human syndromes have been linked, directly or indirectly, to abnormal Meckel’s cartilage formation. Herein, the evolution, development and fate of the cartilage and its impact on jaw development is mapped. The review focuses on developmental and cellular processes that shed light on the mechanisms behind the different fates of this cartilage, examining the control of Meckel’s cartilage patterning, initiation and maturation. Importantly, human disorders and mouse models with disrupted Meckel’s cartilage development are highlighted, in order to understand how changes in this cartilage impact on later development of the dentary and the craniofacial complex as a whole. Finally, the relative roles of tissue interactions, apoptosis, autophagy, macrophages and clast cells in the removal process are discussed. Meckel’s cartilage is a unique and enigmatic structure, the development and function of which is starting to be understood but many interesting questions still remain.

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.

The vestibular lamina (VL) is a transient developmental structure that forms the lip furrow, creating a gap between the lips/cheeks and teeth (oral vestibule). Surprisingly, little is known about the development of the VL and its relationship to the adjacent dental lamina (DL), which forms the teeth. In some congenital disorders, such as Ellis-van Creveld (EVC) syndrome, development of the VL is disrupted and multiple supernumerary frenula form, physically linking the lips and teeth. Here, we assess the normal development of the VL in human embryos from 6.5 (CS19) to 13 weeks of development, showing the close relationship between the VL and DL, from initiation to differentiation. In the anterior lower region, the two structures arise from the same epithelial thickening. The VL then undergoes complex morphogenetic changes during development, forming a branched structure that separates to create the vestibule. Changing expression of keratins highlight the differentiation patterns in the VL, with fissure formation linked to the onset of filaggrin. Apoptosis is involved in removal of the central portion of the VL to create a broad furrow between the future cheek and gum. This research forms an essential base to further explore developmental defects in this part of the oral cavity.

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need to rely on other mechanisms to allow them to feed. Here, we show that this vital function is carried out by the earlier developing, cartilaginous incus of the middle ear, abutting the cranial base to form a cranio-mandibular articulation. The nature of this articulation varies between monotremes and marsupials, with juvenile monotremes retaining a double articulation, similar to that of the fossil mammaliaform Morganucodon, while marsupials use a versican-rich matrix to stabilise the jaw against the cranial base. These findings provide novel insight into the evolution of mammals and the changing relationship between the jaw and ear., eLife digest The defining feature of all mammals is how the jaw works. Fish, reptiles and other animals with backbones have a lower jaw made of many bones fused together, one of which connects to the upper jaw. The lower jaw in mammals, however, is made of a single bone that connects with the upper jaw using a completely unique jaw joint. This new joint emerged as the ancestors of all mammals split from the reptiles around 200 million years ago. The bones that formed the original jaw joint ended up in the middle ear in mammals and switched to a role in hearing. Nowadays, there are three types of mammals: the placentals, marsupials and monotremes (the egg laying mammals). In mice, humans and other placental mammals, the skeleton of the adult jaw joint forms in the embryo before birth. However, marsupials (such as kangaroos and opossums) and monotremes (platypuses and echidnas) are born at a much earlier embryonic stage, before the adult jaw joint has formed. It is therefore unclear how newborn marsupials and monotremes are able to move their jaws to feed on milk from their mother. Anthwal et al. compared how the jaw develops in mice, opossums, platypuses and echidnas before and after the adult jaw joint becomes functional. The experiments showed that young echidnas, platypuses and opossums use their middle ear bones to articulate the lower jaw with the head before the adult jaw joint forms. In young opossums, the ear bones form a cushion to support the jaw. In juvenile platypuses a double joint is evident, with the ear bones forming a joint at the same time as the newly formed adult jaw joint, similar to the situation observed in fossils of mammal ancestors. The experiments also indicated that mice and other placental mammals may potentially use their ear bones to support the jaw before birth. These findings shed light on why the ear and jaw have such a close connection in mammals. In humans, the ear and jaw bones are still connected by ligaments, explaining why trauma to the jaw joint can cause dislocation of the ear bones. Similarly, defects in the development of the jaw can impact the ear, such as in Treacher Collins Syndrome, where in some cases the jaw joint fails to form and the ear bones appear to try and take this role. Understanding how the ear and jaw evolved will help us understand why they look like they do and why a defect in one can have knock-on consequences for the other.

The novel mammalian jaw joint, known in humans as the temporomandibular joint or TMJ, is cushioned by a fibrocartilage disc. This disc is secondarily absent in therian mammals that have lost their dentition, such as giant anteaters and some baleen whales. The disc is also absent in all monotremes. However, it is not known if the absence in monotremes is secondary to the loss of dentition, or if it is an ancestral absence. We use museum held platypus and echidna histological sections to demonstrate that the developing monotreme jaw joint forms a disc primordium that fails to mature and become separated from the mandibular condyle. We then show that monotreme developmental anatomy is similar to that observed in transgenic mouse mutants with reduced musculature. We therefore suggest that the absence of the disc on monotremes is a consequence of the changes in jaw musculature associated with the loss of adult teeth. Taken together, these data indicate that the ancestors of extant monotremes likely had a jaw joint disc, and that the disc evolved in the last common ancestor or all mammals.

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need to rely on other mechanisms to allow them to feed. Here we show that this vital function is carried out by the earlier developing, cartilaginous incus of the middle ear, abutting the cranial base to form a cranio-mandibular articulation. The nature of this articulation varies between monotremes and marsupials, with monotremes retaining a double articulation, similar to that described in the fossil mammaliaform,Morganucodon, while marsupials use a versican rich matrix to stabilise the jaw against the cranial base. These findings provide novel insight into the evolution of mammals and the relationship between the jaw and ear.

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/β-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/β-catenin signalling. Here, we show that stabilising Wnt/β-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.

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. Inthe trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with anadditional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins andantimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cysticfibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals thatregulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development ofmurine 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 wasused to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygousFgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. Thereduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction inoverall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both thenumber of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMGdependentrespiratory health.

Eilenodontines are one of the oldest radiation of herbivorous lepidosaurs (snakes, lizards and tuatara) characterized by batteries of wide teeth with thick enamel that bear mammal-like wear facets. Unlike most reptiles, eilenodontines have limited tooth replacement, making dental longevity particularly important to them. We use both X-ray and neutron computed tomography to examine a fossil tooth from the eilenodontine Eilenodon (Late Jurassic, USA). Of the two approaches, neutron tomography was more successful and facilitated measurements of enamel thickness and distribution. We find the enamel thickness to be regionally variable, thin near the cusp tip (0.10 mm) but thicker around the base (0.15–0.30 mm) and notably greater than that of other rhynchocephalians such as the extant Sphenodon (0.08–0.14 mm). The thick enamel in Eilenodon would permit greater loading, extend tooth lifespan and facilitate the establishment of wear facets that have sharp edges for orally processing plant material such as horsetails ( Equisetum ). The shape of the enamel dentine junction indicates that tooth development in Eilenodon and Sphenodon involved similar folding of the epithelium but different ameloblast activity.

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.

Background: The submucosal glands (SMGs) of the respiratory system are specialized structures essential for maintaining airway homeostasis. The significance of SMGs is highlighted by their involvement in respiratory diseases such as cystic fibrosis, asthma and chronic bronchitis, where their phenotype and function are severely altered. Uncovering the normal development of the airway SMGs is essential to elucidate their role in these disorders, however, very little is known about the cellular mechanisms and intracellular signals involved in their morphogenesis. Results: This review describes in detail the embryonic developmental journey of the nasal SMGs and the postnatal development of the tracheal SMGs in the mouse. Current knowledge of the genes and signalling molecules involved in SMG organogenesis is also explored. Conclusion: Here we review the temporal localisation and development of the murine respiratory glands in the hope of stimulating further research into the mechanisms required for successful SMG patterning and function. Developmental Dynamics 244:525–539, 2015. © 2015 Wiley Periodicals, Inc.

A key transformation in mammalian ear evolution was incorporation of the primary jaw joint of premammalian synapsids into the definitive mammalian middle ear of living mammals. This evolutionary transition occurred in two steps, starting with a partial or ‘transitional’ mammalian middle ear in which the ectotympanic and malleus were still connected to the mandible by an ossified Meckel’s cartilage (MC), as observed in many Mesozoic mammals. This was followed by MC breakdown, freeing the ectotym- panic and the malleus from the mandible and creating the definitive mammalian middle ear. Here we report new findings on the role of chondroclasts in MC breakdown, shedding light on how therian mammals lost the part of the MC connecting the ear to the jaw. Genetic or pharmacological loss of clast cells in mice and opossums leads to persistence of embryonic MC beyond juvenile stages, with MC ossification in mutant mice. The persistent MC causes a distinctive groove on the postnatal mouse dentary. This morphology phenocopies the ossified MC and Meckelian groove observed in Mesozoic mammals. Clast cell recruitment to MC is not observed in reptiles, where MC persists as a cartilaginous structure. We hypothesize that ossification of MC is an ancestral feature of mammaliaforms, and that a shift in the timing of clast cell recruitment to MC prior to its ossification is a key developmental mechanism for the evolution of the definitive mammalian middle ear in extant therians.

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes – carotid body glomus cells, and ‘pulmonary neuroendocrine cells’ (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive ‘neuroepithelial cells’ (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.

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.

In this issue of Developmental Cell, new findings from the Crump lab (Barske et al., 2018) highlight a role for Nr2f nuclear receptors in controlling maxilla versus mandible fate, playing an important role in shaping the upper jaw in jawed vertebrates.

Summary Salivary glands provide an excellent model for the study of epithelial–mesenchymal interactions. We have looked at the interactions involved in the early initiation and development of murine salivary glands using classic recombination experiments and knockout mice. We show that salivary gland epithelium, at thickening and initial bud stages, is able to direct salivary gland development in non-gland pharyngeal arch mesenchyme at early stages. The early salivary gland epithelium is therefore able to induce gland development in non-gland tissue. This ability later shifts to the mesenchyme, with non-gland epithelium, such as from the limb bud, able to form a branching gland when combined with pseudoglandular stage gland mesenchyme. This shift appears to involve Fgf signalling, with signals from the epithelium inducing Fgf10 in the mesenchyme. Fgf10 then signals back to the epithelium to direct gland down-growth and bud development. These experiments highlight the importance of epithelial–mesenchymal signalling in gland initiation, controlling where, when and how many salivary glands form.

The cranial base exerts a supportive role for the brain and includes the occipital, sphenoid and ethmoid bones that arise from cartilaginous precursors in the early embryo. As the occipital bone and the posterior part of the sphenoid are mesoderm derivatives that arise in close proximity to the notochord and floor plate, it has been assumed that their development, like the axial skeleton, is dependent on Sonic hedgehog (Shh) and modulation of bone morphogenetic protein (Bmp) signalling. Here we examined the development of the cranial base in chick and mouse embryos to compare the molecular signals that are required for chondrogenic induction in the trunk and head. We found that Shh signalling is required but the molecular network controlling cranial base development is distinct from that in the trunk. In the absence of Shh, the presumptive cranial base did not undergo chondrogenic commitment as determined by the loss of Sox9 expression and there was a decrease in cell survival. In contrast, induction of the otic capsule occurred normally demonstrating that induction of the cranial base is uncoupled from formation of the sensory capsules. Lastly, we found that the early cranial mesoderm is refractory to Shh signalling, likely accounting for why development of the cranial base occurs after the axial skeleton. Our data reveal that cranial and axial skeletal induction is controlled by conserved, yet spatiotemporally distinct mechanisms that co-ordinate development of the cranial base with that of the cranial musculature and the pharyngeal arches.

How teeth are replaced during normal growth and development has long been an important question for comparative and developmental anatomy. Non-standard model animals have become increasingly popular in this field due to the fact that the canonical model laboratory mammal, the mouse, develops only one generation of teeth (monophyodonty), whereas the majority of mammals possess two generations of teeth (diphyodonty). Here we used the straw-coloured fruit bat (Eidolon helvum), an Old World megabat, which has two generations of teeth, in order to observe the development and replacement of tooth germs from initiation up to mineralization stages. Our morphological study uses 3D reconstruction of histological sections to uncover differing arrangements of the first and second-generation tooth germs during the process of tooth replacement. We show that both tooth germ generations develop as part of the dental lamina, with the first generation detaching from the lamina, leaving the free edge to give rise to a second generation. This separation was particularly marked at the third premolar locus, where the primary and replacement teeth become positioned side by side, unconnected by a lamina. The position of the replacement tooth, with respect to the primary tooth, varied within the mouth, with replacements forming posterior to or directly lingual to the primary tooth. Development of replacement teeth was arrested at some tooth positions and this appeared to be linked to the timing of tooth initiation and the subsequent rate of development. This study adds an additional species to the growing body of non-model species used in the study of tooth replacement, and offers a new insight into the development of the diphyodont condition.

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., Summary: During tooth development in mice, FGF-dependent vertical cell divisions thicken the tooth placode while Shh drives cell rearrangements that cause invagination.

The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.

Background: The stapes, an ossicle found within the middle ear, is involved in transmitting sound waves to the inner ear by means of the oval window. There are several developmental problems associated with this ossicle and the oval window, which cause hearing loss. The developmental origin of these tissues has not been fully elucidated. Results: Using transgenic reporter mice, we have shown that the stapes is of dual origin with the stapedial footplate being composed of cells of both neural crest and mesodermal origin. Wnt1cre/Dicer mice fail to develop neural crest-derived cartilages, therefore, have no middle ear ossicles. We have shown in these mice the mesodermal stapedial footplate fails to form and the oval window is induced but underdeveloped. Conclusions: If the neural crest part of the stapes fails to form the mesodermal part does not develop, indicating that the two parts are interdependent. The stapes develops tightly associated with the otic capsule, however, it is not essential for the positioning of the oval window, suggesting that other tissues, perhaps within the inner ear are needed for oval window placement. Developmental Dynamics 241:1396-1404, 2012. (c) 2012 Wiley Periodicals, Inc.

Interdigital tissue regression is one of the most well-known examples of embryonic programmed cell death, providing the mechanism behind separation of developing digits. Caspases have been shown to play a key part in this process, with activated caspase-3 localized between the developing digits. In caspase-3 knock-out adult mice, however, the digits are completely separated with no webbing. In other mutants with defects in the apoptotic machinery, such as Apaf1 deficient mice, interdigital tissue regression is initially inhibited but the webbing eventually disappears as alternative/additional cell death mechanisms step in. In order to investigate whether a similar temporal effect occurs after loss of caspase-3, we have used an in vitro approach to inhibit caspase-3 at specific times during digit separation. Previous limb explant culture approaches have encountered problems with proper limb development in culture, and thus a modified technique was used. The new approach enables detailed observation of the effects of caspase-3 inhibition on interdigital regression. Using these methods, we show that caspase-3 inhibition caused a delay in the loss of interdigital tissue compared with control explants, similar to that observed in Apaf1 mutant mice. Along with immunohistochemistry, active caspase-3 positive cells of the interdigital vs. digital regions were measured by flow cytometry. Notably, activated caspase-3 in vivo was found not only in the interdigital mesenchyme but also in the TUNEL negative digit region, supporting a role for caspase-3 in nonapoptotic events.

This paper describes tooth development in a basal squamate, Paroedura picta. Due to its reproductive strategy, mode of development and position within the reptiles, this gecko represents an excellent model organism for the study of reptile development. Here we document the dental pattern and development of non-functional (null generation) and functional generations of teeth during embryonic development. Tooth development is followed from initiation to cytodifferentiation and ankylosis, as the tooth germs develop from bud, through cap to bell stages. The fate of the single generation of non-functional (null generation) teeth is shown to be variable, with some teeth being expelled from the oral cavity, while others are incorporated into the functional bone and teeth, or are absorbed. Fate appears to depend on the initiation site within the oral cavity, with the first null generation teeth forming before formation of the dental lamina. We show evidence for a stratum intermedium layer in the enamel epithelium of functional teeth and show that the bicuspid shape of the teeth is created by asymmetrical deposition of enamel, and not by folding of the inner dental epithelium as observed in mammals.

Having three ossicles in the middle ear is one of the defining features of mammals. All reptiles and birds have only one middle ear ossicle, the stapes or columella. How these two additional ossicles came to reside and function in the middle ear of mammals has been studied for the last 200 years and represents one of the classic example of how structures can change during evolution to function in new and novel ways. From fossil data, comparative anatomy and developmental biology it is now clear that the two new bones in the mammalian middle ear, the malleus and incus, are homologous to the quadrate and articular, which form the articulation for the upper and lower jaws in non-mammalian jawed vertebrates. The incorporation of the primary jaw joint into the mammalian middle ear was only possible due to the evolution of a new way to articulate the upper and lower jaws, with the formation of the dentary-squamosal joint, or TMJ in humans. The evolution of the three-ossicle ear in mammals is thus intricately connected with the evolution of a novel jaw joint, the two structures evolving together to create the distinctive mammalian skull.

Background: The contribution of the endoderm to the oral tissues of the head has been debated for many years. With the arrival of Cre/LoxP technology endoderm progenitor cells can now be genetically labeled and tissues derived from the endoderm traced. Using Sox17-2A-iCre/Rosa26 reporter mice we have followed the fate of the endoderm in the teeth, glands, and taste papillae of the oral cavity. Results: No contribution of the endoderm was observed at any stage of tooth development, or in development of the major salivary glands, in the reporter mouse during development. In contrast, the minor mucous glands of the tongue were found to be of endodermal origin, along with the circumvallate papilla and foliate papillae. The mucous minor salivary glands of the palate, however, were of mixed ectodermal and endodermal origin. Conclusions: In contrast to urodele studies, the epithelium of murine teeth is derived solely from the ectoderm. The border between the ectoderm- and endoderm-derived epithelium may play a role in determining the position of the lingual glands and taste buds, and may explain differences observed between taste buds in the anterior and posterior part of the tongue. Developmental Dynamics 241:1183–1191, 2012. © 2012 Wiley Periodicals, Inc.

Functional tooth germs in mammals, reptiles, and chondrichthyans are initiated from a dental lamina. The longevity of the lamina plays a role in governing the number of tooth generations. Monophyodont species have no replacement dental lamina, while polyphyodont species have a permanent continuous lamina. In diphyodont species, the dental lamina fragments and regresses after initiation of the second tooth generation. Regression of the lamina seems to be an important mechanism in preventing the further development of replacement teeth. Defects in the complete removal of the lamina lead to cyst formation and has been linked to ameloblastomas. Here, we show the previously unknown mechanisms behind the disappearance of the dental lamina, involving a combination of cell migration, cell-fate transformation, and apoptosis. Lamina regression starts with the loss of the basement membrane, allowing the epithelial cells to break away from the lamina and migrate into the surrounding mesenchyme. Cells deactivate epithelial markers (E-cadherin, cytokeratin), up-regulate Slug and MMP2, and activate mesenchymal markers (vimentin), while residual lamina cells are removed by apoptosis. The uncovering of the processes behind lamina degradation allows us to clarify the evolution of diphyodonty, and provides a mechanism for future manipulation of the number of tooth generations.

Apoptosis is an important morphogenetic event in embryogenesis as well as during postnatal life. In the last 2 decades, apoptosis in tooth development (odontogenesis) has been investigated with gradually increasing focus on the mechanisms and signaling pathways involved. The molecular machinery responsible for apoptosis exhibits a high degree of conservation but also organ and tissue specific patterns. This review aims to discuss recent knowledge about apoptotic signaling networks during odontogenesis, concentrating on the mouse, which is often used as a model organism for human dentistry. Apoptosis accompanies the entire development of the tooth and corresponding remodeling of the surrounding bony tissue. It is most evident in its role in the elimination of signaling centers within developing teeth, removal of vestigal tooth germs, and in odontoblast and ameloblast organization during tooth mineralization. Dental apoptosis is caspase dependent and proceeds via mitochondrial mediated cell death with possible amplification by Fas-FasL signaling modulated by Bcl-2 family members.

Objectives The primary enamel knot (PEK) is a population of cells that shows spatio-temporal restricted apoptosis during tooth development. It has been shown that caspase-9 and Apaf-1 are essential for apoptosis in the PEK as well as the central caspase-3. Caspase-7, as another executioner member in the caspase machinery, is considered to have caspase-3 like properties. Design The aim of this study was to detect caspase-7 activation during molar tooth development with a special focus on the cells of the PEK and to correlate the expression with the pattern of apoptosis and caspase-3 activation. Apoptosis in the PEK was investigated in caspase-7 deficient mice to examine the functional consequence of loss of this specific caspase. In addition, odontoblasts and ameloblasts, which are known to undergo cell death during their secretory and maturation stages, were investigated. Results Cleaved caspase-7 was found in the apoptotic region of the PEK, however, caspase-7-deficient mice still possessed apoptotic cells in the PEK in a similar distribution to the wild type. Caspase-7 is therefore not essential for apoptosis in the PEK. Notably, cleaved caspase-7-positive cells were found at later stages in odontoblasts and ameloblasts, but expression did not correlate with apoptosis in these tissues. Conclusions The results indicate a non-essential apoptotic role of caspase-7 in the PEK apoptosis but suggest also possible non-apoptotic functions for caspase-7 in tooth development.

Describing the stages of normal development ofVaranus indicus, the present paper provides the first developmental data on Varanidae. The incubation period is relatively long (180 days at 28°C) and without any diapause. The development is rather slow during the first 50 days, after which a considerable acceleration can be observed. The stage of accelerated growth terminates at app. 100 days when all essential specificities of adult organisation (prolonged narial region with vomeronasal organ, eyes, claws, large heart and robust body and limbs) are established. The remaining period of the embryonic development is characterized by continuation of the respective trends, i.e., enlarging body, prolongation of rostrum, enlarging teeth and claws, keratinisation of claws and scales etc. In short, the second half of the embryonic development ofVaranusis devoted to refining the structures supporting its adaptations for active predation.

The transcription factor c-Myb is involved in the control of cell proliferation, survival and differentiation. As these processes accompany the morphogenesis of developing teeth, this work investigates the possible role of c-Myb during odontogenesis. Analysis of the expression of c-Myb in the monophyodont mouse was followed by similar analysis in a diphyodont species, the pig, which has a dentition more closely resembling that of the human. The distribution of c-Myb was correlated with the pattern of proliferation and apoptosis and the tooth phenotype of c-Myb mutant mice was also assessed. In the mouse, c-Myb expression was detected throughout prenatal development of the first molar tooth. Negative temporospatial correlation was found between c-Myb expression and apoptosis, while c-Myb expression positively correlated with proliferation. c-Myb-positive cells, however, were more abundant than the proliferating cell nuclear antigen positive cells, suggesting other roles of c-Myb in odontogenesis. In the minipig, in contrast to the mouse, there was an asymmetrical arrangement of c-Myb positive cells, with a higher presence on the labial side of the tooth germ and dental lamina. A cluster of negative cells was situated in the mesenchyme close to the tooth bud. At later stages, the number of positive cells decreased and these cells were situated in the upper part of the dental papilla in the areas of future cusp formation. The expression of c-Myb in both species was strong in the odontoblasts and ameloblasts at the stage of dentin and enamel production suggesting a possible novel role of c-Myb during tooth mineralization.

The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.

The parathyroid glands originate from the endoderm of the caudal pharyngeal pouches. How these parathyroids are restricted to developing in the caudal pouches is unclear. In this paper we investigate the role of Shh signalling in patterning the vertebrate pharyngeal pouches, and show that Hh signalling may be involved in restricting the expression of the parathyroid marker Gcm2 in the pharyngeal epithelium. In the chick and mouse, Shh signalling is excluded or highly reduced in the posterior/caudal pouches, where the parathyroid marker Gcm2 is expressed, while remaining at high levels in the more anterior pouches. Moreover, though the block of Shh signalling at early developmental stages results in the loss of chick Gcm2 expression, at later stages, it induces ectopic Gcm2 expression domains in the second and first pharyngeal epithelium, suggesting that HH signalling prevents Gcm2 in those tissues. These ectopic domains go on to express other parathyroid markers but do not migrate and develop into ectopic parathyroids. Differences in the expression of Gcm2 in the chick, mouse and zebrafish, correlate with changing patterns of Shh signalling, indicating a conserved regulatory mechanism that acts to define pouch derivatives.

The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0-2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex.

Teeth develop from epithelium and neural crest-derived mesenchyme via a series of reciprocal epithelial-mesenchymal interactions. The majority of the dental papilla of the tooth has been demonstrated to be of neural crest origin. However, non-neural crest cells have also been observed in this region from the bud stage of tooth development onwards. The number of these non-neural crest-derived cells rises as the dental papilla develops. However, their origin is unknown. We have followed migration of cells into the tooth in vitro using DiI to fate map regions surrounding the developing tooth. To identify the contribution of mesodermally-derived cells, we have utilised Mesp1cre/R26R transgenic reporter mice. We document that cells outside the early tooth primordium migrate into the developing dental papilla from the late cap stage of development. Here, we show that migrating cells are mesodermally-derived and create a network of endothelial cells, forming the blood vessels of the tooth. No cells of mesodermal origin were present in the condensed mesenchyme surrounding the dental epithelium until the cap stage of tooth development. Mesodermally-derived cells start invading the dental papilla at the late cap stage, providing the blood supply to the dental pulp. Endothelial cells are able to invade the developing dental papilla in vitro using the slice culture method. Understanding the origin and timing of migration of the mesodermally-derived cells is an important advance in our understanding of how a tooth develops and is particularly relevant to studies which aim to create bioengineered teeth.

Laser capture microdissection (LCM) uniquely allows the selection of specific cell populations from histological sections. These selected cells are then catapulted into a test tube without any contamination from surrounding tissues. During the last ten years, many significant results have been achieved, particularly at the level of DNA and RNA where amplification techniques are available. However, where amplification procedures are difficult, the benefits of LCM diminish. To overcome such difficulties, a novel approach, combining laser capture microdissection and flow cytometry, has been tested here for detection of apoptosis and proliferation in tissue bound cell populations without any amplification steps. The mouse cap stage molar tooth germ was used as a model. At the centre of the inner enamel epithelium, the primary enamel knot is a clearly defined apoptotic population with minimal proliferation, flanked by the highly proliferative cervical loops on each side. Thus within the tooth germ epithelium at this stage, two distinct populations of cells are found side by side. These populations were selected by laser capture microdissection and then analysed by flow cytometry for apoptosis and proliferation. Flow cytometric results correlated well with immunohistochemical findings, demonstrating the success and sensitivity of this combined procedure.