Abigail S. Tucker

The cutting-edge of mammalian development; how the embryo makes teeth

A wealth of information has recently become available on the molecular signals that are required to form and pattern the dentition in the mouse, shedding light on how important decisions about tooth shape, tooth number and cusp (cone-shaped prominence) number are generated. This information, which has been gleaned principally from knockout mice and manipulation of organ cultures, has been used to identify the genes and developmental processes that underlie the many human disorders in which tooth development is defective. Mouse models of several of these syndromes have also indicated ways in which such conditions could be treated.

Gene defect in ectodermal dysplasia implicates a death domain adapter in development

Members of the tumour-necrosis factor receptor (TNFR) family that contain an intracellular death domain initiate signalling by recruiting cytoplasmic death domain adapter proteins. Edar is a death domain protein of the TNFR family that is required for the development of hair, teeth and other ectodermal derivatives. Mutations in Edar—or its ligand, Eda—cause hypohidrotic ectodermal dysplasia in humans and mice. This disorder is characterized by sparse hair, a lack of sweat glands and malformation of teeth. Here we report the identification of a death domain adapter encoded by the mouse crinkled locus. The crinkled mutant has an hypohidrotic ectodermal dysplasia phenotype identical to that of the edar (downless) and eda (Tabby) mutants. This adapter, which we have called Edaradd (for Edar-associated death domain), interacts with the death domain of Edar and links the receptor to downstream signalling pathways. We also identify a missense mutation in its human orthologue, EDARADD, that is present in a family affected with hypohidrotic ectodermal dysplasia. Our findings show that the death receptor/adapter signalling mechanism is conserved in developmental, as well as apoptotic, signalling.

Molecular genetics of tooth morphogenesis and patterning: the right shape in the right place.

Development of the mammalian tooth has for many years served as a useful model system for the study of cell-cell interactions in organogenesis. Early development of teeth (tooth buds) shows many morphological and molecular similarities with early development of other organs such as the lung, hair, kidney, etc. There has been much progress toward understanding epithelial/mesenchymal cell signaling in tooth germ formation. Advances in understanding the formation of different shapes of teeth (morphogenesis) at their correct positions in the jaws (patterning) has, until recently, been less forthcoming. We review here the latest ideas on the control of odontogenic patterning and morphogenesis. The stages of early tooth development are well-defined histologically and have been described in numerous textbooks. The progression from localized thickenings of oral epithelium to bud, cap, and bell stages provides an adequate description of the gross morphological changes seen in the epithelial cells of early developing tooth germs. Less obvious are the concomitant changes taking place in the dental (ecto)mesenchymal cells which originate from the cranial neural crest and which condense around the tooth bud epithelium. However, it is very clear that these mesenchymal cells are equal partners with epithelium during the early stages of tooth germ formation and undergo complex changes which, although not obvious histologically, are revealed with molecular (gene) probes. Genes identified as being important for the early communication between the epithelial and ectomesenchymal cells mainly comprise those which code for proteins which act as secreted signals between the cells (ligands) and those that code for nuclear proteins that act to control gene expression in response to the signals. Little is presently known about the changes in structural proteins such as cell adhesion molecules which are involved in mediating the physical interactions between cells and generating the morphological changes.

Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme.

Tooth development is regulated by a reciprocal series of epithelial‐mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45–58). A target gene for Bmp4 signalling is the homeobox gene Msx‐1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx‐1 expression. Msx‐1 and Bmp‐4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp‐4 expression precede changes in Msx‐1 expression. At embryonic day (E)10.5–E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx‐1 in underlying mesenchyme. At E11.5, Bmp‐4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up‐regulation of Msx‐1 expression at the sites of Bmp‐4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx‐1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx‐1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp‐4 from the epithelium is responsible for regulating the early spatial expression of Msx‐1. We also show that the mesenchymal shift in Bmp‐4 is responsible for up‐regulating Msx‐1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future sites of tooth formation, creating a positive feedback loop that maintains expression of both genes in tooth mesenchymal cells. Dev. Dyn. 1998;212:533–539.

Tooth Agenesis: from Molecular Genetics to Molecular Dentistry

Tooth agenesis may originate from either genetic or environmental factors. Genetically determined hypodontic disorders appear as isolated features or as part of a syndrome. Msx1, Pax9, and Axin2 are involved in non-syndromic hypodontia, while genes such as Shh, Pitx2, Irf6, and p63 are considered to participate in syndromic genetic disorders, which include tooth agenesis. In dentistry, artificial tooth implants represent a common solution to tooth loss problems; however, molecular dentistry offers promising solutions for the future. In this paper, the genetic and molecular bases of non-syndromic and syndromic hypodontia are reviewed, and the advantages and disadvantages of tissue engineering in the clinical treatment of tooth agenesis are discussed.

Current knowledge of tooth development: patterning and mineralization of the murine dentition

The integument forms a number of different types of mineralized element, including dermal denticles, scutes, ganoid scales, elasmoid scales, fin rays and osteoderms found in certain fish, reptiles, amphibians and xenarthran mammals. To this list can be added teeth, which are far more widely represented and studied than any of the other mineralized elements mentioned above, and as such can be thought of as a model mineralized system. In recent years the focus for studies on tooth development has been the mouse, with a wealth of genetic information accrued and the availability of cutting edge techniques. It is the mouse dentition that this review will concentrate on. The development of the tooth will be followed, looking at what controls the shape of the tooth and how signals from the mesenchyme and epithelium interact to lead to formation of a molar or incisor. The number of teeth generated will then be investigated, looking at how tooth germ number can be reduced or increased by apoptosis, fusion of tooth germs, creation of new tooth germs, and the generation of additional teeth from existing tooth germs. The development of mineralized tissue will then be detailed, looking at how the asymmetrical deposition of enamel is controlled in the mouse incisor. The continued importance of epithelial–mesenchymal interactions at these later stages of tooth development will also be discussed. Tooth anomalies and human disorders have been well covered by recent reviews, therefore in this paper we wish to present a classical review of current knowledge of tooth development, fitting together data from a large number of recent research papers to draw general conclusions about tooth development.

Ror2 Knockout Mouse as a Model for the Developmental Pathology of Autosomal Recessive Robinow Syndrome

Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. We have analyzed Ror2‐/‐ mice as a model for the developmental pathology of RS. Our results demonstrate that vertebral malformations in Ror2‐/‐ mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2‐/‐ mice is a consequence of perturbed chondrocyte differentiation. Moreover, we show that the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2‐/‐ mice makes it likely that Ror2 is involved in genital development. In conclusion, our findings suggest that Ror2 is essential at multiple sites during development. The Ror2‐/‐ mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome. Developmental Dynamics 229:400–410, 2004,

Bapx1 regulates patterning in the middle ear: altered regulatory role in the transition from the proximal jaw during vertebrate evolution

The middle ear apparatus is composed of three endochondrial ossicles (the stapes, incus and malleus) and two membranous bones, the tympanic ring and the gonium, which act as structural components to anchor the ossicles to the skull. Except for the stapes, these skeletal elements are unique to mammals and are derived from the first and second branchial arches. We show that, in combination with goosecoid (Gsc), the Bapx1 gene defines the structural components of the murine middle ear. During embryogenesis, Bapx1 is expressed in a discrete domain within the mandibular component of the first branchial arch and later in the primordia of middle ear-associated bones, the gonium and tympanic ring. Consistent with the expression pattern of Bapx1, mouse embryos deficient for Bapx1 lack a gonium and display hypoplasia of the anterior end of the tympanic ring. At E10.5, expression of Bapx1 partially overlaps that of Gsc and although Gsc is required for development of the entire tympanic ring, the role of Bapx1 is restricted to the specification of the gonium and the anterior tympanic ring. Thus, simple overlapping expression of these two genes appears to account for the patterning of the elements that compose the structural components of the middle ear and suggests that they act in concert. In addition, Bapx1 is expressed both within and surrounding the incus and the malleus. Examination of the malleus shows that the width, but not the length, of this ossicle is decreased in the mutant mice. In non-mammalian jawed vertebrates, the bones homologous to the mammalian middle ear ossicles compose the proximal jaw bones that form the jaw articulation (primary jaw joint). In fish, Bapx1 is responsible for the formation of the joint between the quadrate and articular (homologues of the malleus and incus, respectively) enabling an evolutionary comparison of the role of a regulatory gene in the transition of the proximal jawbones to middle ear ossicles. Contrary to expectations, murine Bapx1 does not affect the articulation of the malleus and incus. We show that this change in role of Bapx1 following the transition to the mammalian ossicle configuration is not due to a change in expression pattern but results from an inability to regulate Gdf5 and Gdf6, two genes predicted to be essential in joint formation.

Neural crest cells provide species-specific patterning information in the developing branchial skeleton

Summary The skeletal elements of the branchial region are made by neural crest cells, following tissue interactions with the pharyngeal endoderm. Previous transplantation experiments have claimed that the cranial neural crest is morphogenetically prespecified in respect of its branchial skeletal derivatives, that is, that information for the number, size, shape, and position of its individual elements is already determined in these cells when they are still in the neural folds. This positional information would somehow be preserved during delamination from the neural tube and migration into the branchial arches, before being read out as a spatial pattern of chondrogenesis and osteogenesis. However, it now appears that signals from the endoderm are able to specify not only the histogenic differentiation state of neural crest cells but also the identity and orientation of the branchial skeletal elements. It is therefore important to ask whether fine details of branchial skeletal pattern such as those that exist between different species are also governed by extrinsic factors, such as the endoderm, or by the neural crest itself. We have grafted neural crest between duck and quail embryos and show that the shape and size of the resulting skeletal elements is donor derived. The ability to form species‐specific patterns of craniofacial skeletal tissue thus appears to be an inherent property of the neural crest, expressed as species‐specific responses to endodermal signals.

Initiation and patterning of the snake dentition are dependent on Sonic Hedgehog signaling

Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation.