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M L Snead

Publications and source records attributed to M L Snead.

At least 19 recordsLinked to original sources
PubMed1999

Cloning and characterization of the murine ameloblastin promoter.

The molecular mechanisms directing the highly restricted expression pattern of murine ameloblastin were characterized by cloning and functional analysis of the ameloblastin promoter. The transcription start site, mapped by primer extension, was located 19 base pairs (bp) 5' of the published cDNA. The promoter was analyzed in a mouse ameloblast-like cell line (LS8) and was compared with promoter activity in primary gingival fibroblasts and pulp fibroblasts. Sequential 5'-deletion mutants encompassing DNA sequences from -1616 to -781 bp exhibited high promoter activity in LS8 cells, whereas the promoter activity decreased to 50% of the full-length construct in the -781- and -477-bp regions. The -217-bp promoter region regained promoter activity that approached the activity of the full-length promoter construct, suggesting that both positive and negative cis-acting regions may be involved in ameloblastin transcriptional regulation. Activity of the ameloblastin promoter in gingival and pulp fibroblasts was minimal and ranged from 8 to 30% of the activity in ameloblast-like cells. Several DNA-protein complexes were formed between functionally important promoter fragments and nuclear extracts from LS8 cells. The inactivity of promoter constructs in pulp and gingival fibroblasts as well as the absence of similar DNA-protein complexes from these cells suggest that regulatory regions of the murine ameloblastin promoter may function in a cell-specific manner.

Animals↗
PubMed1999

Msx2 gene dosage influences the number of proliferative osteogenic cells in growth centers of the developing murine skull: a possible mechanism for MSX2-mediated craniosynostosis in humans.

Throughout its complex morphogenesis, the vertebrate skull must at once protect the brain and expand to accommodate its growth. A key structural adaptation that allows this dual role is the separation of the bony plates of the skull with sutures, fibrous joints that serve as growth centers and allow the calvarial bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of one or more calvarial bones with consequent abnormalities in skull shape, is a common developmental anomaly that disrupts this process. We found previously that a single amino acid substitution in the homeodomain of the human MSX2 gene is associated with the autosomal dominant disorder craniosynostosis, Boston type. This mutation enhances the affinity of Msx2 for its target sequence, suggesting that the mutation acts by a dominant positive mechanism. Consistent with this prediction, we showed that general overexpression of Msx2 under the control of the broadly expressed CMV promoter causes the calvarial bones to invade the sagittal suture. Here we use tissue-specific overexpression of Msx2 within the calvarial sutures to address the developmental mechanisms of craniosynostosis and skull morphogenesis. We demonstrate that a segment of the Msx2 promoter directs reporter gene expression to subsets of cells within the sutures. In late embryonic and neonatal stages, this promoter is expressed in undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter activity is reduced in the suture, exhibiting a punctate pattern in undifferentiated osteoblastic cells in the outer margin of the osteogenic front. Overexpression of Msx2 under the control of this promoter is sufficient to enhance parietal bone growth into the sagittal suture by P6. This phenotype is preceded by an increase in both the number and the BrdU labeling of osteoblastic cells in the osteogenic fronts of the calvarial bones. These findings suggest that an important early event in MSX2-mediated craniosynostosis in humans is a transient retardation of osteogenic cell differentiation in the suture and a consequent increase in the pool of osteogenic cells.

Animals↗
PubMed1998

Protein-to-protein interactions: criteria defining the assembly of the enamel organic matrix.

Enamel crystallites form in a protein matrix located proximal to the ameloblast cell layer. This unique organic extracellular matrix is constructed from structural protein components biosynthesized and secreted by ameloblasts. To date, three distinct classes of enamel matrix proteins have been cloned. These are the amelogenins, tuftelin, and ameloblastin, with recent data implicating ameloblastin gene expression during cementogenesis. The organic enamel extracellular matrix undergoes assembly to provide a three-dimensional array of protein domains that carry out the physiologic function of guiding enamel hydroxyapatite crystallite formation. Using the yeast two-hybrid system, we have surveyed these three known enamel gene products for their ability to direct self-assembly. We measured the capacity of the enamel gene products to direct protein-to-protein interactions, a characteristic of enamel proteins predicated to be required for self-assembly. We provide additional evidence for the self-assembly nature of amelogenin and tuftelin. Ameloblastin self-assembly could not be demonstrated, nor were protein-to-protein interactions observed between ameloblastin and either amelogenin or tuftelin. Within the limits of the yeast two-hybrid assay, these findings constrain the emerging model of enamel matrix assembly by helping to define the limits of enamel matrix protein-protein interactions that are believed to guide enamel mineral crystallite formation.

Amelogenesis↗
PubMed1998

Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors.

Mineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and mid- to late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 microm to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. (J Histochem Cytochem 46:911-934, 1998)

Amelogenin↗
PubMed1997

Protein interactions during assembly of the enamel organic extracellular matrix.

Enamel is the outermost covering of teeth and contains the largest hydroxyapatite crystallites formed in the vertebrate body. Enamel forms extracellularly through the ordered assembly of a protein scaffolding that regulates crystallite dimensions. The two most studied proteins of the enamel extracellular matrix (ECM) are amelogenin and tuftelin. The underlying mechanism for assembly of the proteins within the enamel extracellular matrix and the regulatory role of crystallite-protein interactions have proven elusive. We used the two-hybrid system to identify and define minimal protein domains responsible for supra molecular assembly of the enamel ECM. We show that amelogenin proteins self-assemble, and this self-assembly depends on the amino-terminal 42 residues interacting either directly or indirectly with a 17-residue domain in the carboxyl region. Amelogenin and tuftelin fail to interact with each other. Based upon this data, and advances in the field, a model for amelogenin assemblies that direct enamel biomineralization is presented.

Amelogenin↗
PubMed1997

Problem-based learning at the University of Southern California School of Dentistry.

Responding to the recent Institute of Medicine report on dental education, the Center for Craniofacial Molecular Biology (CCMB) of the University of Southern California School of Dentistry has developed a parallel track program in dental education leading to the D.D.S. degree. This program was proposed in May of 1995, and the first class of twelve students was admitted in September of that year. Currently two classes are enrolled and plans to admit a further twelve students (Class of 2001) are in place. The educational strategy for this program is totally problem-based. Students work in groups of six with a faculty facilitator, not necessarily a content expert. Facilitators are largely drawn from the multidisciplinary pool of research faculty at the center. All learning is mediated through biomedical and biodental problem cases. No formal lectures or classes are scheduled. The learning of clinical dental skills is promoted through focussed dental patient simulations in which students review clinical charts, radiographs, medical reports and then explore identified, hands-on learning needs using patient simulators in a clinical context. Early patient exposure is obtained through dental office visits and other special patient clinics. Initial experience with this program suggests that the problem-based learning (PBL) students learn as well (if not better) than their traditional program peers and develop excellent group and cognitive analytical skills. The absence of a pool of dentally related biomedical cases suitable for a PBL program has necessitated the use of innovative approaches to their development and presentation. It is believed that this educational approach will produce dental clinicians equipped with the self-motivated, life-long learning skills required in the ever-changing world of bio-dental sciences in the twenty-first century.

California↗
PubMed1996

The murine amelogenin promoter: developmentally regulated expression in transgenic animals.

We are interested in understanding hierarchical regulation pathways that control gene expression in developing teeth. In pursuit of the molecular basis for the regulated expression of amelogenin by developing ameloblasts during tooth formation, we isolated the murine amelogenin promoter. Analysis of this promoter will provide additional details towards the identification of signals generated through instructive-, dissimilar-germ layer interactions that are for responsible for temporal- and spatial-regulation for amelogenin gene expression. Using transgenic mice we demonstrate that a 2263 nucleotide stretch of the murine amelogenin promoter conveys appropriate temporal- and spatial-regulation for amelogenin gene expression in response to instructive-signals. These transgenic animals are useful reagents to further dissect signaling pathways responsible for regulated gene expression by terminally differentiated ameloblasts.

3T3 Cells↗
PubMed1996

Carboxyl-region of tuftelin mediates self-assembly.

Enamel biomineralization relies on a complex series of protein-protein interactions resulting in the formation of an enamel matrix. This protein matrix is subsequently replaced by a fully mineralized crystallite material. The enamel extracellular matrix is comprised principally by two gene products; the amelogenins and enamelins. The enamelins, including the 389 amino-acid, 44 kDa tuftelin, are a group of acidic proteins found in the enamel extracellular matrix. This study has employed the yeast two-hybrid system to investigate the ability of tuftelin to self-assemble and to define protein regions participating in tuftelin self-assembly. We show that for tuftelin the amino-acid residues 252 through 345 contain structurally relevant determinants for self-assembly.

Amino Acid Sequence↗
PubMed1995

Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.

The coordinate growth of the brain and skull is achieved through a series of interactions between the developing brain, the growing bones of the skull, and the fibrous joints, or sutures, that unite the bones. These interactions couple the expansion of the brain to the growth of the bony plates at the sutures. Craniosynostosis, the premature fusion of the bones of the skull, is a common birth defect (1 in 3000 live births) that disrupts coordinate growth and often results in profoundly abnormal skull shape. Individuals affected with Boston-type craniosynostosis, an autosomal dominant disorder, bear a mutated copy of MSX2, a homeobox gene thought to function in tissue interactions. Here we show that expression of the mouse counterpart of this mutant gene in the developing skulls of transgenic mice causes craniosynostosis and ectopic cranial bone. These mice provide a transgenic model of craniosynostosis as well as a point of entry into the molecular mechanisms that coordinate the growth of the brain and skull.

Animals↗
PubMed1995

Genetically engineered mice: tools to understand craniofacial development.

In this review, we provide a survey of the experimental approaches used to generate genetically engineered mice. Two specific examples are presented that demonstrate the applicability of these approaches to craniofacial development. In the first, a promoter analysis of the Msx2 gene is presented which illustrates the cis regulatory interactions that defined cell-specific gene expression. In the second, a mouse model of the human disease craniosynostosis, Boston type, has been created by misregulation of the Msx2 gene product. Finally. we present a formulary of spontaneously occurring and genetically engineered mice that exhibit defects in developmental processes affecting the craniofacial complex. The purpose of this review is to provide insight into the experimental approaches that are used to create genetically engineered mice and to impress upon the reader that genetically engineered mice are well-suited to address fundamental questions pertaining to the development maintenance, and regeneration of tissues and organs.

Animals↗
PubMed1995

Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line.

During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping.

To probe osteoclast gene expression, we combined the techniques of cell microisolation and RT-PCR to develop a novel and sensitive method for the isolation and mRNA phenotyping of small numbers of authentic osteoclasts and spleen cell polykaryons. Using this method we report (1) direct evidence for the presence of calcitonin receptor mRNA in osteoclasts, (2) confirmation of the recent finding of osteopontin mRNA in osteoclasts, and (3) demonstration that the specific expression of mRNA for tartrate-resistant acid phosphatase, carbonic anhydrase II, calcitonin receptor, and osteopontin enable one to distinguish the osteoclast from the morphologically similar and developmentally related spleen cell polykaryon. We also show that mRNA associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and type I collagen, are absent in osteoclasts. This is the first report in which such an approach has been used successfully to distinguish the mRNA expression pattern of an authentic osteoclast from a macrophage polykaryon, and as such it should provide an important new tool for evaluating the results of various cell culture model systems designed to examine the origin and ontogeny of osteoclasts. Our results also indicate that these procedures can be used as an alternative to in situ hybridization methods for the cell-specific localization of specific mRNA in a mixed cell preparation and for colocalization of multiple mRNA species to a single cell type.

Animals↗
PubMed1994

Early determination and permissive expression of amelogenin transcription during mouse mandibular first molar development.

The expression of tissue-specific enamel matrix genes is believed to require both instructive and permissive interactions of enamel organ epithelium with dental papilla mesenchyme and/or extracellular matrix during a restricted period of development. Biosynthesis of amelogenin gene products has been found to be associated with the terminal differentiation of inner enamel organ epithelium. The developing mouse first mandibular molar was used for a detailed examination of the temporal initiation and developmental pattern of amelogenin transcription. These studies define temporally instructive versus permissive influences on amelogenin transcription. During in vivo development, amelogenin transcripts were detected in late cap (15 days in utero; E15) through bell stage (E16 through E19) mouse molar tooth formation utilizing reverse transcription coupled to polymerase chain reaction amplification. Alternatively spliced amelogenin transcripts were detected in late bell stage (E18) molars. Amelogenin transcripts were also detected in isolated late cap stage (E15) enamel organ epithelium dissected free of dental papilla mesenchyme and cultured within a substitute basement membrane gel, but not in identical cap stage enamel organ epithelium cultured on plastic or a laminin-coated filter. Amelogenin transcripts were also found in early cap stage (E14) isolated enamel organ epithelium cultured within a basement membrane gel, but were not detected in enamel organ epithelium isolated from earlier stages of odontogenesis and cultured within a basement membrane gel. The results of these experiments indicate that a basement membrane gel is a useful extracellular substrate which provides permissive interactions required for the expression of amelogenin transcripts by enamel organ epithelium and that instructive interactions which determine enamel organ epithelium to become committed to amelogenin transcription occur prior to the early cap stage (E14) of odontogenesis. The results also suggest that continued interactions of enamel organ epithelium with dental papilla mesenchyme serve to regulate amelogenin transcription and post-transcriptional amelogenin RNA splicing in a complex manner during odontogenesis.

Amelogenin↗
PubMed1994

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.

Amelogenin↗
PubMed1994

Regulation of the Msx2 homeobox gene during mouse embryogenesis: a transgene with 439 bp of 5' flanking sequence is expressed exclusively in the apical ectodermal ridge of the developing limb.

Msx2, a member of the highly conserved and widely distributed msh homeobox gene family, is expressed in a variety of sites in the vertebrate embryo, including craniofacial structures, heart, limb buds and otic and optic vesicles. In many of these sites, its expression is regulated by tissue interactions. Here we address the cis-trans regulatory interactions that direct Msx2 expression to specific regions of the embryo and enable it to respond to tissue interactions. We created a series of Msx2-lacZ fusion constructs with varying amounts of Msx2 genomic sequences. These were introduced into mouse embryos and their expression monitored by staining for beta-galactosidase activity. A construct bearing 5.2 kb of 5' flanking sequence, the intron, both exons and 3 kb of 3' flanking sequence was expressed in a pattern that closely resembled that of the endogenous Msx2 gene. In the E12.5 embryo, sites of expression included craniofacial mesenchyme, portions of the neural ectoderm, mesoderm in the distal limb bud and the overlying apical ectodermal ridge (AER). Removal of intronic and 3' UTR sequences slightly altered the pattern of Msx2 expression in the neural ectoderm of the E12 embryo. Deletion of 5' flanking sequences to -0.5 kb eliminated Msx2 expression in all sites except the AER. The proximal Msx2 promoter, including sequences required for the AER-specific expression of the -0.5 lacZ transgene, is highly conserved between mouse and human, one stretch exhibiting 100% identity over 72 bp. This conservation suggests that the AER element is under remarkably tight evolutionary constraint.

Amino Acid Sequence↗
PubMed1993

Genomic structure, chromosomal location, and evolution of the mouse Hox 8 gene.

We isolated genomic clones containing the mouse Hox 8 gene, a member of the msh gene family. We show that Hox 8 comprises two exons of approximately 600 and 691 bp separated by a 3.5-kb intron, and that it cosegregates with previously mapped markers in the distal region of mouse chromosome 13. In midgestation embryos, the Hox 8 gene produces transcripts of 1.4 and 2.2 kb. Both transcripts are present in facial tissues of the newborn mouse, though the ratio of the 2.2-kb transcript to the 1.4-kb transcript is reduced relative to the ratio observed for midgestation embryos. An alignment of the homeobox sequences of previously characterized members of the msh family revealed three subclasses: Hox 7-like genes, Hox 8-like genes, and msh-like genes. Both the Hox 7-like genes and Hox 8-like genes are present throughout the vertebrates. Representatives of the third subclass, the msh-like genes, are found in a protostome (Drosophila) and a deuterostome (Ciona) and are thus likely to be phylogenetically widespread. To investigate the distribution of Hox 8-like genes outside the chordates, we used the polymerase chain reaction and degenerate Hox 8 primers to screen genomic DNA of the purple sea urchin (Strongylocentrotus purpuratus, Phylum Echinodermata). We isolated a gene with greater sequence similarity to mouse Hox 8 than to members of the Hox 7 or msh subfamilies, demonstrating that the Hox 8 subfamily has been in existence at least since the echinoderms diverged from the lineage that gave rise to the chordates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗