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 Meckel's cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-alpha (TGF-alpha), 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 Meckel's 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 Meckel's 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.

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling.

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.

Identification of markers of the midface.

Currently, much remains unknown of the genes that mediate the biological events during growth and fusion of the midfacial region, and the possible pathways through which these genes function. We took advantage of high-throughput microarray analysis to search for genes that may play a critical role in the growth and fusion of the midfacial region to become the primary palate. We identified several genes that were potentially expressed at different levels between tail somite (TS) 6-8 (pre-fusion) and TS 12-14 (fusion) in the 3 midfacial processes. Expression of 4 of these genes (Tbx14/15, Dickkopf-1, Fibroblast Growth Factor 8, and Keratin-18) was further verified by reverse-transcription/quantitative PCR and in situ hybridization at the 2 stages of midfacial development. With the identification of these genes, and possibly others, functional analyses can be conducted to improve our understanding of the mechanisms and pathways by which the midface forms.

Desmin expression during early mouse tongue morphogenesis.

Occipital somites provide progenitor cells for craniofacial muscle development including the tongue musculature. Serum-derived factors are assumed to be pre-requisite for myogenesis in vitro. To test these assertions, we designed experiments to determine whether early mouse tongue development in general, and desmin localization in particular, were expressed during the development of embryonic mouse first branchial arch explants cultured in serumless, chemically-defined medium. Immunohistochemical techniques determined the chronology and positions of desmin expression during early craniofacial development. Occipital somites expressed desmin at E9 (9 days +/- 2 h post-fertilization, 18-20 somites). A discrete cell migration pathway initiating in the somites and terminating in the lateral lingual processes of the tongue primordium was defined based upon desmin expression patterns in E9-E11 embryos and computer-assisted three dimensional reconstructions. The in vitro model system was permissive for tongue morphogenesis, allowing development and fusion of the lateral lingual processes with the tuberculum impar. During culture myoblasts were not observed to fuse into myotubes with sarcomeric assembly, even though explant myoblasts produced muscle-specific protein. E10 explants cultured for 9 days demonstrated a five-fold increase in cell number that expressed desmin (P less than 0.05) when compared to the E10 starting material. We interpret these results to indicate that the tongue myogenic cell lineage was determined between E8 and E11, and that this resident population expanded within explants cultured in serumless medium by several explanations: (i) cells other than progenitor myoblasts (e.g., satellite cells) were induced to become myoblasts, and/or (ii) progenitor myoblasts within the original explants expanded by cell division in the absence of serum factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis.

The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.

Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis.

Bone morphogenetic protein 4 (BMP4) induces, whereas epidermal growth factor (EGF) inhibits chondrogenesis. We hypothesize that BMP4 and EGF mediated intracellular signals are both coupled in the regulation of Meckel's cartilage development. Two chondrogenic experimental model systems were employed to test the hypothesis: (1) an ex vivo, serum-free, organ culture system for mouse embryonic mandibular processes, and (2) a micromass culture system for chicken embryonic mandibular processes. Chondrogenesis was assayed by alcian blue staining and expression of Sox9 and type II collagen. Exogenous EGF inhibited and BMP4 induced ectopic cartilage in a dose-dependent manner. When BMP4- and EGF-soaked beads were implanted in juxtaposition within embryonic day 10 mouse mandibular processes, the incidence and amount of ectopic cartilage, and Sox9 and type II collagen expression induced by BMP4, were significantly reduced as the concentration of EGF was increased. Similarly, in chicken serum-free micromass cultures, expression of a constitutively active BMP receptor type IB by replication competent avian retrovirus system promoted the rate and extent of chondrogenesis; however, exogenous EGF attenuated this effect. In micromass cultures, BMP signaling resulted in nuclear translocation and accumulation of the signaling molecule Smad1, whereas the addition of EGF inhibited this event. Our results suggest that BMP4 and EGF function antagonistically, yet are coupled in the regulation of initial chondrogenesis. Smad1 serves as a point of convergence for the integration of two different growth factor signaling pathways during chondrogenesis.

Pharmacokinetics of theophylline in hepatic disease.

The disposition of theophylline was examined in eight male cirrhotic (six proven by biopsy) patients without heart failure. An oral dose of 100 mg of theophylline per square meter of surface area was administered, and samples of serum and saliva were collected from 0 to 60 hours and were assayed by high-pressure liquid chromatographic techniques. Controls were 57 young normal subjects and 25 age-matched patients. The body clearance of theophylline in cirrhotic patients was low, averaging 18.8 +/- 11.3 ml/kg/hr (+/- SD) vs 53.7 +/- 19.3 and 63.0 +/- 28.5 ml/kg/hr in the control patients and the normal subjects, respectively. The half-life of theophylline in cirrhotic patients was prolonged wiht a mean of 28.8 +/- 14.3 hours compared to 6.0 +/- 2.1 hours in normal subjects. Patients with cirrhosis proven by biopsy had significantly lower values for body clearance and longer half-lives than subjects without biopsies. The values for body clearance correlated well with the serum level of bilirubin (r = -0.81) and the serum level of bile acids (r = -0.81). The slow and variable metabolism in cirrhotic patients necessitates a reduction in the maintenance dosage of aminophylline to 0.20 to 0.45 mg/kg/hr and monitoring of the serum level during therapy.

TGF-beta 3 decreases type I collagen and scarring after labioplasty.

Cleft lip is a common congenital malformation, and labioplasty performed on infants to repair such defects often results in severe scar formation. Since TGF-beta 3 has been implicated in wound healing, we therefore hypothesized that TGF-beta 3 functions to reduce scarring after cleft lip repair. In this investigation, we demonstrated that exogenous TGF-beta 3 reduced scar formation in an incised and sutured mouse lip in vivo. During labioplasty, endogenous TGF-beta 3 expression was also elevated. In vitro experiments showed that exogenous TGF-beta 3 reduced type I collagen accumulation. Furthermore, TGF-beta 3 inhibited alpha-smooth-muscle actin expression, a marker for myofibroblasts. In tandem, TGF-beta 3 induced the expression and activity of MMP-9. Analysis of our data suggests that TGF-beta 3 is normally secreted following labioplastic wound healing. An elevated level of TGF-beta 3 reduces type I collagen deposition by restricting myofibroblast differentiation and thereby collagen synthesis, and by promoting collagen degradation by MMP-9. In combination, these events lead to TGF-beta 3-mediated reduced scar formation.

Distribution of type I collagen, type II collagen and PNA binding glycoconjugates during chondrogenesis of three distinct embryonic cartilages.

Previous studies of chondrogenesis have been focused on limb bud cartilage, whereas little is known about chondrogenic processes of other cartilages with different developmental fates. We hypothesize that cartilages with various developmental fates might show identical characteristics of chondrogenesis. The chondrogenic processes in the nasal septum, the mandible, and the limb bud of the mouse were examined by means of PNA-binding glycoconjugate, and types I and II collagen expression. Swiss-Webster mouse embryos of 11 days (E11) to 14 days (E14) gestation were fixed and processed for immuno- and lectin histochemistry. The blastema of mesenchymal cell aggregates stained positively with anti-type I collagen, but very weakly with anti-type II collagen in all three models at E12, whereas PNA bound to the blastema in the limb bud but not in nasal septum or mandible. Types I and II collagens coexisted in cartilages at E13. Type II collagen was predominant in E14; type I collagen was confined to the peripheral region. The synchronized transitional expression of the collagen phenotypes in all three embryonic cartilages may be systemically regulated. The presence or absence of the PNA-binding glycoconjugates may be involved in characterizing the nature of the cartilages.

Induced expression of myoD, myogenin and desmin during myoblast differentiation in embryonic mouse tongue development.

Significant progress has been made in defining mechanisms governing myogenesis at the transcriptional levels, but the extracellular signal-transduction pathways involved in myogenesis are not as yet defined. The developing mouse tongue provides a model for the regulation of myogenesis during precise time periods in embryogenesis. The molecular cues that regulate the close-range autocrine and/or paracrine signalling processes required for the fast-twitch complex tongue musculature are not known. This study was designed to test the hypothesis that transforming growth factor-alpha (TGF alpha) controls myogenesis in embryonic mouse tongue through the induction of myogenic regulatory factors such as myoD, myf5, myogenin and MRF4/myf6/herculin. To test this hypothesis, the effects of exogenous TGF alpha on the transcription of myoD, myf5, myogenin, MRF4 and desmin were examined in tongue samples from embryonic day-10.5 mandibular explants cultured in serum-free, chemically defined medium and then processed for competitive, reverse transcription-polymerase chain reaction. TGF alpha induced myoD, myogenin and desmin expression. Treatment with 20 and 40 ng/ml TGF alpha decreased or downregulated myf5 mRNA. MRF4 was not detected in the explants. TGF alpha apparently induces the early developmental stages of myogenesis through sequential upregulation of myoD and myogenin, downregulation of myf5 and corresponding significant increases in muscle-specific gene expression such as desmin transcription.

Synergistic roles of amelogenin and ameloblastin.

Amelogenin and ameloblastin, the major enamel matrix proteins, are important for enamel mineralization. To identify their synergistic roles in enamel development, we generated Amel X(-/-)/Ambn(-/-) mice. These mice showed additional enamel defects in comparison with Amel X(-/-) or Ambn(-/-) mice. In 7-day-old Amel X(-/-)/Ambn(-/-) mice, not only was the ameloblast layer irregular and detached from the enamel surface, as in Ambn(-/-), but also, the enamel width was significantly reduced in the double-null mice as compared with Amel X(-/-) or Ambn(-/-) mice. Proteomic analysis of the double-null teeth revealed increased levels of RhoGDI (Arhgdia), a Rho-family-specific guanine nucleotide dissociation inhibitor, which is involved in important cellular processes, such as cell attachment. Both Amel X(-/-)/Ambn(-/-) mice and Ambn(-/-) mice displayed positive staining with RhoGDI antibody in the irregularly shaped ameloblasts detached from the matrix. Ameloblastin-regulated expression of RhoGDI suggests that Rho-mediated signaling pathway might play a role in enamel formation.

Progress toward understanding craniofacial malformations.

Significant advances in the study of the human face have revealed the genetic and gene-environment bases of numerous common and rare craniofacial disorders. Classification of craniofacial malformations based on clinical phenotypes is sometimes quite different from the genetic findings of patients. Different mutations in a single gene can cause distinct syndromes, and mutations in different genes can cause the same syndrome. The extracellular signaling molecule SHH, fibroblast growth factor receptors, and transcription factors GLI3, MSX2, and TWIST are discussed as examples of molecules involved in interrelated signal transduction networks regulating craniofacial development. Progress in the understanding of normal and abnormal craniofacial development, through the study of morphoregulatory signaling pathways, has benefited from multifactorial approaches recommended 40 years ago at the National Institute of Dental Research-sponsored landmark Gatlinburg Conference. The utilization of biochemistry, protein structure analyses, tissue culture, and animal model systems for developmental genetics has resulted in remarkable scientific advances. The evolutionary conservation of morphoregulatory pathways has revealed the homology of genes associated with human craniofacial malformations and their counterparts that regulate the morphogenesis of fruit flies. The continued investments in basic, translational, and patient-oriented research regarding normal and abnormal craniofacial development will translate into substantial improvements in the prevention, diagnosis, and treatment of craniofacial diseases and disorders.

Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2.

Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9, and type II collagen demonstrate temporally and spatially restrictive localization patterns suggestive of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants induced ectopic cartilage formation in the proximal position of the explant. However, BMP4-soaked beads implanted at the rostral position did not have an inductive effect. Ectopic chondrogenesis was associated with the up-regulation of Sox9 and Msx2 expression in the immediate vicinity of the BMP4 beads 24 hours after implantation. Control beads had no effect on cartilage induction or Msx2 and Sox9 expression. Sox9 was induced at all sites of BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and suggested that Msx2 expression was inhibitory to chondrogenesis. To test the hypothesis that over-expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery strategy. Microinjection of the Msx2-adenovirus to the proximal position inhibited BMP4-induced chondrogenesis. Over-expression of Msx2 also resulted in the abrogation of endogenous cartilage and the down-regulation of type II collagen expression. Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2. Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear to establish a threshold for Sox9 function and thereby restricts the position of chondrogenesis.

Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells.

During early mouse embryogenesis, cranial neural crest cells (CNCC) emigrate from the posterior midbrain and rhombomeres 1 and 2 of the anterior hindbrain into the first branchial arch-derived maxillary and mandibular processes and there provide cell lineages for several phenotypes, including cartilage, bone, and tooth. Here, we report that Sox9 and Msx2 were coexpressed in a subpopulation of CNCC during their migration. Because Sox9 is a transactivator of chondrogenesis, and Msx genes can act as transcriptional repressors, we hypothesized that Sox9 expression indicates the determination of CNCC-derived chondrogenic cell lineage and that Msx2 represses chondrogenic differentiation until CNCC migration is completed within the mandibular processes. To test whether Msx2 represses chondrogenesis, we designed experiments to inhibit Msx2 function in migratory CNCC in primary cultures through the expression of loss-of-function Msx2 mutants. We showed that infection of migratory CNCC with adenovirus Msx2 mutants accelerated the rate and extent of chondrogenesis, as indicated by the expression level of type II collagen and aggrecan, and the amount of alcian blue staining. Adenovirus infections did not apparently interfere with CNCC proliferation or migration. These findings suggest that an important early event in craniofacial morphogenesis is a transient expression of both Sox9 and Msx2 during emigration into the forming mandibular processes followed by restricted expression of Sox9 within CNCC- derived chondroprogenitor cells. We conclude that Msx2 serves as a repressor of chondrogenic differentiation during CNCC migration.

Transforming growth factor alpha up-regulates desmin expression during embryonic mouse tongue myogenesis.

Myogenesis is determined by a set of myogenic differentiation factors that are, in turn, regulated by a number of peptide growth factors. During embryonic mouse tongue formation, transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), and their cognate receptor (EGFR) are co-expressed spatially and temporally with desmin, a muscle-specific structural protein. This investigation tested the hypothesis that TGF alpha directly regulates the myogenic program in developing tongue myoblasts. Mandibular processes from the first branchial arch of embryonic day 10.5 (E10.5) mouse embryos were microdissected and explanted into an organ culture system using serumless chemically defined medium. Exogenous TGF alpha at 10 and 20 ng/ml specifically increased the amount of desmin expression and the number of desmin-positive cells without affecting the general growth and development of the mandibles. This inductive response was detected as early as 2 days after treatment and sustained up to 9 days in culture. EGFR antisense oligonucleotides (30 microM) as well as tyrphostin (80 microM) were able to negate TGF alpha-induced up-regulation of desmin expression. These data indicate that autocrine and/or paracrine action of TGF alpha promotes tongue myogenesis, and that this action is mediated through functional kinase activity of the EGFR. We speculate that the myogenic program in the developing mouse tongue is dependent upon growth factor mediated cell-cell communication of mesenchymal cells originating from the occipital somites and ectomesenchymal cells originating from the cranial neural crest.

EGF abrogation-induced fusilli-form dysmorphogenesis of Meckel's cartilage during embryonic mouse mandibular morphogenesis in vitro.

Mutations associated with genes of the EGF superfamily are implicated in facial malformations arising from abnormal development of the first branchial arch. EGF and EGF receptor (EGFr) transcripts are expressed in the mouse embryonic first branchial arch and derivatives from E9 through E15. EGF transcripts are localized to ectomesenchymal cells associated with precartilage, cartilage, bone and tooth-forming cells. EGF and EGFr proteins co-localize to the same cells suggesting an autocrine regulation. To test whether EGF effects the timing and positional information required for Meckel's cartilage (MC) and tooth development, we cultured E10 mandibular explants in serumless, chemically defined medium with either antisense or sense EGF oligodeoxynucleotides. Antisense inhibition of EGF expression produces bilaterally symmetrical Fusilli-form dysmorphogenesis of MC and decreases tooth bud size; these effects are reversed by the addition of exogenous EGF to the culture medium. Tyrphostin RG 50864, which inhibits EGF receptor kinase activity, inhibits EGF stimulation of tyrosine phosphorylation in a concentration-dependent manner and severely retards mandibular development yet increases tooth size. These findings support the hypothesis that endogenous EGF and EGF-like proteins provide signalling to regulate the size and shape both of cartilage and tooth formation during craniofacial morphogenesis.

Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants.

Posterior midbrain and anterior hindbrain neuroectoderm trans-differentiate into cranial neural crest cells (CNCC), emigrate from the neural folds, and become crest-derived ectomesenchyme within the mandibular and maxillary processes. To investigate the growth factor requirement specific for the initiation of tooth morphogenesis, we designed studies to test whether nerve growth factor (NGF) can support odontogenesis in a first branchial arch (FBA) explant culture system. FBA explants containing neural-fold tissues before CNCC emigration and the anlagen of the FBA were microdissected from embryonic day 8 (E8) mouse embryos, and cultured for 8 days in medium supplemented with 10% fetal calf serum only, or serum-containing medium further supplemented with either NGF or epidermal growth factor (EGF) at three different concentrations: 50, 100, or 200 ng/ml. Morphological, morphometric, and total protein analyses indicated that growth and development in all groups were comparable. Meckel's cartilage and tongue formation were also observed in all groups. However, odontogenesis was only detected in explants cultured in the presence of exogenous NGF. NGF-supplemented cultures were permissive for bud stage (50 ng/ml) as well as cap stage of tooth morphogenesis (100 and 200 ng/ml). Morphometric analyses of the volume of tooth organs showed a significant dose-dependent increase in tooth volume as the concentration of NGF increased. Whole-mount in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction for Pax9, a molecular marker of dental mesenchyme, further supported and confirmed the morphological data of the specificity and dose dependency of NGF on odontogenesis. We conclude that (1) E8 FBA explants contain premigratory CNCC that are capable of emigration, proliferation, and differentiation in vitro; (2) serum-supplemented medium is permissive for CNCC differentiation into tongue myoblasts and chondrocytes in FBA explants; and (3) NGF controls CNCC cell fate specification and differentiation into tooth organs.