Faculty Bibliography

Studies of facial ontogeny in immature hominins have contributed significantly to understanding the evolution of human growth and development. The recently discovered hominin species Autralopithecus sediba is represented by a well-preserved and nearly complete facial skeleton of a juvenile (MH1) which shows a derived facial anatomy. We examined MH1 using high radiation synchrotron to interpret features of the oronasal complex pertinent to facial growth. We also analyzed bone surface microanatomy to identify and map fields of bone deposition and bone resorption, which affect the development of the facial skeleton. The oronasal anatomy (premaxilla-palate-vomer architecture) is similar to other Australopithecus species. However surface growth remodeling of the midface (nasomaxillary complex) differs markedly from Australopithecus, Paranthropus, early Homo and from KNM-WT 15000 (H. erectus/ergaster) showing a distinct distribution of vertically disposed alternating depository and resorptive fields in relation to anterior dental roots and the subnasal region. The ontogeny of the MH1 midface superficially resembles some H. sapiens in the distribution of remodeling fields. The facial growth of MH1 appears unique among early hominins representing an evolutionary modification in facial ontogeny at 1.9 my, or to changes in masticatory system loading associated with diet.

Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+-EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+-handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs.

ClC-7 is a 2Cl- /1H+ -exchanger expressed at late endosomes and lysosomes, as well as the ruffled border of osteoclasts. ClC-7 deficiencies in mice and humans lead to impaired osteoclast function and therefore osteopetrosis. Failure of tooth eruption is also apparent in ClC-7 mutant animals, and this has been attributed to the osteoclast dysfunction and the subsequent defect in alveolar bone resorptive activity surrounding tooth roots. Ameloblasts also express ClC-7, and this study aims to determine the significance of ClC-7 in enamel formation by examining the dentitions of ClC-7 mutant mice. Micro-CT analysis revealed that the molar teeth of 3-week old ClC-7 mutant mice had no roots, and the incisors were smaller than their age-matched controls. Despite these notable developmental differences, the enamel and dentin densities of the mutant mice were comparable to those of the wild type littermates. Scanning electron microscopy (SEM) showed normal enamel crystallite and prismatic organization in the ClC-7 mutant mice, although the enamel was thinner (hypoplastic) than in controls. These results suggested that ClC-7 was not critical to enamel and dentin formation, and the observed tooth defects may be related more to a resulting alveolar bone phenotype. Micro-CT analysis also revealed abnormal features in the calvarial bones of the mutant mice. The cranial sutures in ClC-7 mutant mice remained open compared to the closed sutures seen in the control mice at 3 weeks. These data demonstrate that ClC-7 deficiency impacts the development of the dentition and calvaria, but does not significantly disrupt amelogenesis

The bicarbonate transport activities of Slc26a1, Slc26a6 and Slc26a7 are essential to physiological processes in multiple organs. Although mutations of Slc26a1, Slc26a6 and Slc26a7 have not been linked to any human diseases, disruption of Slc26a1, Slc26a6 or Slc26a7 expression in animals causes severe dysregulation of acid-base balance and disorder of anion homeostasis. Amelogenesis, especially the enamel formation during maturation stage, requires complex pH regulation mechanisms based on ion transport. The disruption of stage-specific ion transporters frequently results in enamel pathosis in animals. Here we present evidence that Slc26a1, Slc26a6 and Slc26a7 are highly expressed in rodent incisor ameloblasts during maturation-stage tooth development. In maturation-stage ameloblasts, Slc26a1, Slc26a6 and Slc26a7 show a similar cellular distribution as the cystic fibrosis transmembrane conductance regulator (Cftr) to the apical region of cytoplasmic membrane, and the distribution of Slc26a7 is also seen in the cytoplasmic/subapical region, presumably on the lysosomal membrane. We have also examined Slc26a1 and Slc26a7 null mice, and although no overt abnormal enamel phenotypes were observed in Slc26a1-/- or Slc26a7-/- animals, absence of Slc26a1 or Slc26a7 results in up-regulation of Cftr, Ca2, Slc4a4, Slc4a9 and Slc26a9, all of which are involved in pH homeostasis, indicating that this might be a compensatory mechanism used by ameloblasts cells in the absence of Slc26 genes. Together, our data show that Slc26a1, Slc26a6 and Slc26a7 are novel participants in the extracellular transport of bicarbonate during enamel maturation, and that their functional roles may be achieved by forming interaction units with Cftr.

The sodium pump Na(+)/K(+)-ATPase, expressed in virtually all cells of higher organisms, is involved in establishing a resting membrane potential and in creating a sodium gradient to facilitate a number of membrane-associated transport activities. Na(+)/K(+)-ATPase is an oligomer of alpha, beta, and gamma subunits. Four unique genes encode each of the alpha and beta subunits. In dental enamel cells, the spatiotemporal expression of Na(+)/K(+)-ATPase is poorly characterized. Using the rat incisor as a model, this study provides a comprehensive expression profile of all four alpha and all four beta Na(+)/K(+)-ATPase subunits throughout all stages of amelogenesis. Real-time PCR, western blot analysis, and immunolocalization revealed that alpha1, beta1, and beta3 are expressed in the enamel organ and that all three are most highly expressed during late-maturation-stage amelogenesis. Expression of beta3 was significantly higher than expression of beta1, suggesting that the dominant Na(+)/K(+)-ATPase consists of an alpha1beta3 dimer. Localization of alpha1, beta1, and beta3 subunits in ameloblasts was primarily to the cytoplasm and occasionally along the basolateral membranes. Weaker expression was also noted in papillary layer cells during early maturation. Our data support that Na(+)/K(+)-ATPase is functional in maturation-stage ameloblasts.

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

The modern human face differs from that of our early ancestors in that the facial profile is relatively retracted (orthognathic). This change in facial profile is associated with a characteristic spatial distribution of bone deposition and resorption: growth remodeling. For humans, surface resorption commonly dominates on anteriorly-facing areas of the subnasal region of the maxilla and mandible during development. We mapped the distribution of facial growth remodeling activities on the 900-800 ky maxilla ATD6-69 assigned to H. antecessor, and on the 1.5 My cranium KNM-WT 15000, part of an associated skeleton assigned to African H. erectus. We show that, as in H. sapiens, H. antecessor shows bone resorption over most of the subnasal region. This pattern contrasts with that seen in KNM-WT 15000 where evidence of bone deposition, not resorption, was identified. KNM-WT 15000 is similar to Australopithecus and the extant African apes in this localized area of bone deposition. These new data point to diversity of patterns of facial growth in fossil Homo. The similarities in facial growth in H. antecessor and H. sapiens suggest that one key developmental change responsible for the characteristic facial morphology of modern humans can be traced back at least to H. antecessor.

Recent studies suggest that the hypodigms representing the two earliest Australopithecus (Au. anamensis and Au. afarensis) form an ancestor-descendant lineage. Understanding the details of this possible transition is important comparative evidence for assessing the likelihood of other examples of ancestor-descendant lineages within the hominin clade. To this end we have analyzed crown and cusp base areas of high resolution replicas of the mandibular molars of Au. anamensis (Allia Bay and Kanapoi sites) and those of Au. afarensis (Hadar, Laetoli, and Maka). We found no statistically significant differences in crown areas between these hypodigms although the mean of M(1) crowns was smaller in Au. anamensis, being the smallest of any Australopithecus species sampled to date. Intraspecies comparison of the areas of mesial cusps for each molar type using Wilcoxon signed rank test showed no differences for Au. anamensis. Significant differences were found between the protoconid and metaconid of Au. afarensis M(2)s and M(3)s. Furthermore, the area formed by the posterior cusps as a whole relative to the anterior cusps showed significant differences in Au. afarensis M(1)s and in Au. anamensis M(2)s but no differences were noted for M(3)s of either taxon. Developmental information derived from microstructural details in enamel shows that M(1) crown formation in Au. anamensis is similar to Pan and shorter than in H. sapiens. Taken together, these data suggests that the overall trend in the Au. anamensis-Au. afarensis transition may have involved a moderate increase in M(1) crown areas with relative expansion of distal cusps.