Harold C. Slavkin

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.

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.

DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.

Isolation and characterization of a mouse amelogenin expressed in Escherichia coli

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis

A major unsolved problem in developmental biology is to determine when and how time- and position-restricted instructions are signaled and received during secondary embryonic inductions such as branching morphogenesis. The mouse embryonic lung rudiment was used to test the hypothesis that endogenous peptide growth factors, specifically epidermal growth factor (EGF), serve as instructive epigenetic signals for morphogenesis. The presence of EGF precursor mRNA transcripts was detected using the reverse-transcriptase-coupled polymerase chain reaction both in E11-E17-day mouse embryo lung tissues in vivo and in E11-day lung cultured for up to 7 days in vitro under chemically defined, serum-free conditions. Immunolocalization identified a position-restricted distribution of EGF in and around the primitive airways both during in vivo lung morphogenesis and in culture. EGF receptors (EGFR) coimmunolocalized with EGF in the primitive airways. Addition of exogenous EGF to lungs in culture resulted in significant concentration-dependent stimulation of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA. Conversely, the addition of tyrphostin (specific EGF receptor kinase antagonist) to lungs in culture resulted in concentration-dependent inhibition of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA without apparent cytotoxicity. The inhibition of the EGF signal by tyrphostin was confirmed by immunoprecipitation of tyrosine phosphoproteins. We conclude that early mouse embryo lungs express EGF transcripts and corresponding EGF peptides in a specific position-restricted distribution which coimmunolocalizes with EGFR in the primitive airways, while stimulatory and inhibitory studies indicate a functional role for the transduced EGF signal in the epigenetic regulation of lung branching morphogenesis. We speculate that the peptide growth factor EGF serves a function in secondary embryonic morphogenetic inductions, which may be modulated by interaction with other growth factors.

Epithelial-mesenchyme interactions during odontogenesis: IV. Morphological evidence for direct heterotypic cell-cell contacts

Abstract During embryonic and neonatal mouse incisor tooth morphogenesis, direct epithelial-mesenchymal cell contacts were observed by electron microscopy. These direct contacts were evident along the epithelial-mesenchymal interface in the differentiation zone in which inner enamel epithelium was as yet a dividing cell population which had not as yet synthesized and secreted the enamel organic matrix. This region of cell differentiation was also characterized by the appearance of cell processes which extended from the epithelia through the basal lamina. Following the appearance of epithelial cell processes penetrating through the basal lamina, ectomesenchymal cell processes extended across the extracellular matrix and penetrated through the basal lamina and resulted in the formation of contact zones. Following degradation of the basal lamina, the mesenchymal cell processes penetrated into clefts within the preameloblast cells and formed cell contacts. By a combination of tannic acid and uranium acetate staining we observed that the tannic acid stain penetrated through intercellular spaces formed between the apposing mesenchymal and epithelial plasma membrane surfaces. We speculate that direct heterotypic cell contacts, which occur prior to the cessation of preameloblast cell division and precede the secretion of enamel proteins, may be instructive in the induction of enamel protein biosynthesis.

Cleft palate susceptibility linked to histocompatibility-2 (H-2) in the mouse

Data have been obtained indicating that cortisone-induced cleft palate in the mouse is linked to theH-2a complex. Cortisone (2.5 mg) was administered to pregnant females on days 11 through 14 of pregnancy. On day 17 of pregnancy, the fetuses were inspected for cleft palates. Sham experiments were done by injecting sterile saline instead of cortisone. The inbred strains, A/J and C57BL/6, and the congenic strains C57BL/10ScSn and B10.A were tested for susceptibility to cleft palate. The clefting frequency was also observed in hybrids of the congenic strains. The A/J and B10.A strains showed a characteristic high susceptibility to cleft palate (i.e., 99% and 81% incidence of cleft palate, respectively) after teratogenic treatment. The C57BL/6 and C57BL/ 10ScSn demonstrated a significant resistance to the teratogen (i.e., 25% and 21 % incidence of clefting, respectively). The teratogenic treatment of congenic hybrids indicated that maternal influences significantly affected the incidence of cleft palate formation. The maternal influence appeared to depend upon the specificH-2 haplotype of the mother.

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. Dev Den;217:401–414.

Initial enamel crystals are not spatially associated with mineralized dentine

During epithelial-mesenchymal interactions associated with mammalian tooth development, epithelially-derived and mesenchymally-derived extracellular matrix molecules form a discrete dentine-enamel junction. The developmental and molecular processes required to form this junction are not known. To address this problem we designed studies to test the hypothesis that ectodermally-derived epithelial cells synthesize and secrete enamel proteins which function to nucleate and regulate the growth of enamel calcium phosphate crystals. Initial enamel crystals were detected separate from the adiacent dentine. Electron-microprobe analyses revealed that early enamel crystals were octacalciumphosphate or tricalciumphosphate rather than hydroxyapatite. Thereafter, enamel crystals became confluent with the adjacent, albeit significantly smaller hydroxyapatite crystals associated with mineralized dentine. Therefore, we interpret our data to indicate that de novo enamel crystal nucleation and growth are independent from the mineralization processes characterized for dentine. We further argue that gene expression of enamel protein appears to have a constitutive function during early enamel formation and that supramolecular aggregates of amelogenin and enamelin provide the microenvironment for the nucleation and crystal growth of the initial enamel matrix.

Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.