Charles F. Shuler

Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects.

Cleft palate and skull malformations represent some of the most frequent congenital birth defects in the human population. Previous studies have shown that TGFβ signaling regulates the fate of the medial edge epithelium during palatal fusion and postnatal cranial suture closure during skull development. It is not understood, however, what the functional significance of TGFβ signaling is in regulating the fate of cranial neural crest (CNC) cells during craniofacial development. We show that mice with Tgfbr2 conditional gene ablation in the CNC have complete cleft secondary palate, calvaria agenesis, and other skull defects with complete phenotype penetrance. Significantly, disruption of the TGFβ signaling does not adversely affect CNC migration. Cleft palate in Tgfbr2 mutant mice results from a cell proliferation defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Our data suggests that TGFβ IIR plays a crucial, cell-autonomous role in regulating the fate of CNC cells during palatogenesis. During skull development, disruption of TGFβ signaling in the CNC severely impairs cell proliferation in the dura mater, consequently resulting in calvaria agenesis. We provide in vivo evidence that TGFβ signaling within the CNC-derived dura mater provides essential inductive instruction for both the CNC- and mesoderm-derived calvarial bone development. This study demonstrates that TGFβ IIR plays an essential role in the development of the CNC and provides a model for the study of abnormal CNC development.

Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure.

Craniofacial malformations are among the most frequent congenital birth defects in humans; cleft palate, that is inadequate fusion of the palatal shelves, occurs with an annual incidence of 1 in 700 to 1 in 1,000 live births among individuals of European descent. The secondary palate arises as bilateral outgrowths from the maxillary processes, and its formation depends on the coordinated development of craniofacial structures including the Meckels cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-α (TGF-α), an epidermal growth factor receptor (EGFR) ligand made by most epithelia. Here we have characterized craniofacial development in Egfr -deficient (Egfr-/-) mice. Newborn Egfr -/- mice have facial mediolateral defects including narrow, elongated snouts, underdeveloped lower jaw and a high incidence of cleft palate. Palatal shelf explants from Egfr-/- mice fused, but frequently had residual epithelium in the midline. In addition, morphogenesis of Meckels cartilage was deficient in cultured mandibular processes from Egfr -/- embryos. The secretion of matrix metalloproteinases (MMPs) was diminished in Egfr-/- explants, consistent with the ability of EGF to increase MMP secretion and with the decreased MMP expression caused by inhibition of Egfr signalling in wild-type explants. Accordingly, inactivation of MMPs in wild-type explants phenocopied the defective morphology of Meckels cartilage seen in Egfr-/- explants. Our results indicate that EGFR signalling is necessary for normal craniofacial development and that its role is mediated in part by its downstream targets, the MMPs, and may explain the genetic correlation of human cleft palate with polymorphisms in TGFA.

Identification of Microbial Biofilms in Osteonecrosis of the Jaws Secondary to Bisphosphonate Therapy

PURPOSE Biofilm theory has emerged to explain the etiology of the chronic infections that have come to constitute between 65% to 80% of the microbial diseases treated by physicians in the developed world. The purpose of this article is to report for the first time the observation of multispecies microbial biofilms on affected bone in patients with osteonecrosis of the jaws (ONJ) secondary to bisphosphonate therapy. PATIENTS AND METHODS A program has been established at the University of Southern California to monitor and evaluate patients with ONJ as a multidisciplinary collaboration between the School of Dentistry, Center for Biofilms, Center for Craniofacial Molecular Biology and the Keck School of Medicine. From this cohort, 4 patients with active ONJ who were scheduled for necessary treatment in the form of sequestrectomy gave informed consent for this study. Bone samples were evaluated using conventional histopathologic techniques and scanning electron microscopy, a technique applicable to biofilm characterization. RESULTS Bone specimens from affected sites in all patients showed large areas occluded with biofilms comprising mainly bacteria, and occasionally yeast, embedded in extracellular polymeric substance. The number of bacterial morphotypes in the biofilms ranged from 2 to 15, and they included species from the genus Fusobacterium, bacillus, actinomyces, staphylococcus, streptococcus, Selenomonas, and 3 different types of treponemes. The yeast identified was consistent with Candida species. Co-aggregation was observed between different species within the biofilms. CONCLUSION These findings have important clinical and therapeutic implications and may suggest a role for microbial biofilms in the disease process of ONJ.

Transforming growth factor‐β3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane

Studies on transforming growth factor β3 (TGF‐β3) deficient mice have shown that TGF‐β3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF‐β3 in palatogenesis, we cultured TGF‐β3 null mutant and wild‐type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF‐β3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF‐β3 (−/−) samples is not impaired adhesion. Our data define a specific role for TGF‐β3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane. Dev. Dyn. 209:255–260, 1997.

Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo☆

Vital cell labeling techniques were used to trace the fate of the medial edge epithelial (MEE) cells during palatal fusion in vivo. Mouse palatal tissues were labeled in utero with DiI. The fetuses continued to develop in utero and tissues of the secondary palate were examined at several later stages of palatal ontogeny. The presence and distribution of DiI was correlated with the presence of cell phenotype-specific markers. During the initial stages of palatal fusion the DiI-labeled MEE were present in the midline position. These cells were attached to an intact laminin-containing basement membrane and contained keratin intermediate filaments. At later stages of palatogenesis the DiI-labeled MEE were not separated from the mesenchyme by an intact basement membrane and did not contain keratin. In late fetal development, DiI-labeled cells without an epithelial morphology were present in the mesenchyme. The transition of the DiI-labeled cells from an epithelial phenotype to a mesenchymal phenotype is consistent with a fate of epithelial-mesenchymal transformation rather than programmed cell death.

Programmed Cell Death and Cell Transformation in Craniofacial Development

Fusion of branchial arch derivatives is an essential component in the development of craniofacial structures. Bilaterally symmetric branchial arch processes fuse in the midline to form the mandible, lips, and palate. The mechanism for fusion requires several different morphologic and molecular events prior to the completion of the mesenchymal continuity between opposing tissue processes. The ectodermal covering of the branchial arches is one of the cell types that has an important role during craniofacial development. The surface epithelia provide the initial adherence between the processes; however, this population of cells is ultimately absent from the fusion zone. The medial edge epithelium of the secondary palatal shelves is one example of such an epithelium that must disappear from the fusion zone of the secondary palate during development in order to complete palatal fusion. The mechanisms for removal of the epithelial cells from the fusion zone could include either programmed cell death, epithelial-mesenchymal transformation, or migration to adjacent epithelia. All three of these fates have been hypothesized as a mechanism for the removal of the palatal medial edge epithelia. The processes of programmed cell death, epithelial-mesenchymal transformation, and epithelial migration are reviewed with respect to both palatal fusion and results reported in other model systems.

Expression of myogenic regulatory factors during the development of mouse tongue striated muscle

While the role of myogenic regulatory factors (MRFs) in skeletal myogenesis has been well evaluated in limb and trunk muscles, very little is known about their role in tongue myogenesis. Here the expression of MRF mRNA in mouse tongue muscle was examined during development from embryonic day (E)11 to birth and compared them with that in hind-limb muscle. Desmin, muscle creatine kinase and troponin C mRNAs were used as markers for myoblast determination, myotubule formation and myofibre maturation, respectively. The mRNA quantities were determined by competitive reverse transcriptase-polymerase chain reaction. The expression profile of desmin mRNA indicated that myoblast determination occurred before E11 in both the tongue and hind-limb muscles; the profile of muscle creatine kinase and troponin C mRNAs indicated that myotubule formation and myofibre maturation began between E11 and 13 in both tongue and hind-limb muscles, but ended 2 days earlier in the tongue than in the hind limb. Expression of myoD and myogenin mRNAs began at E11, increased, and showed peak values earlier in the tongue muscle (E13) than in the hind-limb muscle (E15). Expression of MRF4 mRNA appeared earlier in the tongue (E13) than in the hind-limb muscle (E15) and increased in both muscles after that. These results suggest that myotubule formation and myofibre maturation in the tongue muscle progress faster than in the hind-limb muscle, a result of earlier expression of myoD, myogenin, and MRF4 in response to earlier functional demands such as suckling immediately after birth.

Cell-Nonautonomous Induction of Ovarian and Uterine Serous Cystadenomas in Mice Lacking a Functional Brca1 in Ovarian Granulosa Cells

Women with germline mutations in BRCA1 have a 40% risk of developing ovarian cancer by age 70 and are also predisposed to cancers of the fallopian tubes. Given that ovulatory activity is a strong risk factor for sporadic ovarian cancer, we hypothesized that reduced BRCA1 expression might predispose to gynecological cancers indirectly, by influencing ovarian granulosa cells. These cells secrete sex steroids that control the ovulatory cycle and influence the growth of ovarian epithelial tumors. Granulosa cells also secrete mullerian inhibiting substance (MIS), a hormone that inhibits both the formation of female reproductive organs in male embryos and the proliferation of ovarian epithelial tumor cells. We tested this hypothesis by using the Cre-lox system to inactivate the Brca1 gene in mouse ovarian granulosa cells. A truncated form of the Fsh receptor promoter served as the Cre driver. Here, we show that indeed, inactivation of the Brca1 gene in granulosa cells led to the development of cystic tumors in the ovaries and uterine horns. These tumors carried normal Brca1 alleles, supporting the view that Brca1 may influence tumor development indirectly, possibly through an effector secreted by granulosa cells.

TGF-β3–dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion

Transforming growth factor (TGF) ‐β3 is known to selectively regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Previous studies suggested that the selective function of TGF‐β3 in MEE was conducted by TGF‐β receptors. Further studies were needed to demonstrate that the TGF‐β signaling mediators were indeed expressed and phosphorylated in the MEE cells. SMAD2 and SMAD3 were both present in the MEE, whereas SMAD2 was the only one phosphorylated during palatal fusion. SMAD2 phosphorylation was temporospatially restricted to the MEE and correlated with the disappearance of the MEE. No phosphorylated SMAD2 was found in MEE in TGF‐β3−/− mice, although nonphosphorylated SMAD2 was present. The results suggest that TGF‐β3 is required for initiating and maintaining SMAD2 phosphorylation in MEE. Phospho‐SMAD3 was not detectable in palate during normal palatal fusion. Previous results suggested TGF‐β–induced cessation of DNA synthesis in MEE cells during palatal fusion in vitro. The present results provide evidence that inhibition of MEE proliferation in vivo was controlled by endogenous TGF‐β3. The number of 5‐bromo‐2′‐deoxyuridine (BrdU) ‐labeled MEE cells was significantly reduced in TGF‐β3+/+ compared with TGF‐β3−/− mice when the MEE seam formed (t‐test, P < 0.05). This finding suggests that TGF‐β3 is required for inhibiting MEE proliferation during palatal fusion. The inhibition of MEE proliferation may be mediated by TGF‐β3–dependent phosphorylation of SMAD2. Developmental Dynamics 227:387–394, 2003.

Human developing enamel proteins exhibit a sex-linked dimorphism.

SummaryThe amelogenin protein of developing dental enamel is generally accepted to mediate the regulation of the form and size of the hydroxyapatite crystallites during enamel biomineralization (1). A genetic disorder of enamel development (amelogenesis imperfecta) has been linked to theamelogenin geneAMEL (2–3), and loci regulating enamel thickness and tooth size have been mapped to the human sex chromosomes (4). In the human genome there are twoAMEL loci with one copy of the gene on each of the sex chromosomes (AMELX andAMELY), whereas in the mouse only anAMELX locus is present (5). It is presently unknown if humanAMELY is transcriptionally active. These observations prompted us to examine specimens of human developing enamel for sexual dimorphism at the protein level. We report here, for the first time, a diagnosis of differences in human enamel proteins which permits the distinction of specimens according to the sex of the individual.