Biomedical subjects
M W Ferguson
Publications and source records attributed to M W Ferguson.
Differentiation of isolated murine embryonic palatal epithelium in culture: exogenous transforming growth factor alpha modulates matrix biosynthesis in defined experimental conditions.
A novel culture technique, which supports the growth and differentiation of mouse embryonic palatal epithelial cells in the absence of either an extracellular matrix substratum or feeder layers, has been developed. Using this technique we have investigated the effects of exogenous transforming growth factor alpha (TGF alpha) and serum on extracellular matrix biosynthesis by primary cultures of mouse embryonic epithelial sheets under defined experimental conditions. In all culture treatments (chemically defined medium with and without TGF alpha or serum) the palatal epithelial sheets differentiated into three regionally distinct cell phenotypes after 36 h. Nasal and oral cells differentiated into pseudostratified, ciliated columnar, and stratified squamous keratinizing epithelium, respectively. In addition, basal medial edge epithelial (MEE) cells at the oral/nasal regional interface assumed an elongated cobblestoned phenotype. In serum-free medium, collagen types IV and V, laminin, fibronectin, and heparan sulphate proteoglycan were detected immunocytochemically throughout the entire epithelial sheet. Tenascin and collagen IX were present almost exclusively in MEE cells. Types I, II, and III collagen were completely absent. Addition of TGF alpha or serum universally increased the intensity of staining, most notably that for tenascin and collagen IX in MEE cells. These results indicate that mouse embryonic palatal epithelial sheets can be maintained under defined culture conditions during which they exhibit patterns of differentiation similar to those observed in vivo. TGF alpha, known to localize to the MEE in vivo, can modulate palatal extracellular matrix biosynthesis, particularly by the MEE, suggesting a regulatory role for this factor. The culture system is suitable for further investigating the effects of exogenous factors on mouse embryonic palatal epithelial cell bioactivity and differentiation.
The esterification of cholesterol in the yolk sac membrane of the alligator embryo.
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Chemoreception in crocodilians: anatomy, natural history, and empirical results.
Anatomical and behavioral aspects of chemoreception in crocodilians are reviewed. Extant crocodilians possess valvular nostrils regulated by smooth muscles; paired nasal cavities, each with three conchae; and flaps closing the rear of the buccopharyngeal passage to channel inspired air through the nasopharyngeal duct. Olfaction is enhanced by gular pumping, whereby the floor of the pharynx is alternately lowered and raised to pulse air through the nasal cavity. Taste buds occur on the tongue, palate, and pharyngeal walls. Crocodilians in nature or in semi-natural enclosures locate distant carrion or concealed meat, implying chemoattraction to food. The American alligator exhibits underwater head-waving and mouth-opening in response to aqueous and chloroform extracts of meat. Alligators increase gular pumping in response to the scents of various meats, indicating that they detect food chemicals by olfaction. Paired gular and paracloacal glands on the integument of crocodilians are thought to produce pheromones. Juvenile crocodilians exhibit heightened gular pumping in response to airborne skin gland secretions. Observations of adults suggest that gular and paracloacal glands produce chemical signals used in mating and/or nesting activities, but behavioral responses to skin gland exudates are too poorly documented to ascribe pheromonal properties.
Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth.
Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.
Monoclonal antibodies recognising stage and region specific epitopes in embryonic mouse palatal epithelial cells.
Four monoclonal antibodies raised against murine embryonic palatal epithelium were used to stain frontal cryosections of embryonic mouse heads (d 11-15). One antibody (6A11) recognised the nasal epithelium at the angle of the palate prior to shelf elevation (embryonic d 11-d 13) and the epithelia at the tips of the approximated shelves (embryonic d 14.5). Two further antibodies (2H6 and 3H2) recognised epitopes on the entire palatal epithelium until shelf elevation (embryonic d 14.5) when their expression ceased. The epitope recognised by antibody 6D3 was absent from the palatal epithelium until embryonic d 13 when it displayed region specific expression, being present on the nasal aspect in the midpalate and the tip of the palate posteriorly. On embryonic d 14.5 staining of the medial edge epithelia was present in all regions. Whilst the nasal palatal epithelium stained in the anterior and mid regions, only patchy staining of the oral epithelium was detected. By embryonic d 15 staining was present throughout the palatal epithelia. These antibodies are directed against intracellular epitopes, possibly keratins. Indeed, immunoblotting assays revealed that antibody 2H6 recognised keratin 18. The developmental regulation of the epitopes recognised by the antibodies corresponds with differentiative events occurring during murine palatogenesis.
Novel articular cartilage structure in the South American opossum, Monodelphis domestica.
Articular cartilage provides smooth surfaces for low-friction, unrestricted movement of opposing skeletal elements. The surface topography of articular cartilage has been the subject of numerous studies and, with few exceptions, is considered to be smooth (at least at the light microscopic level). Some studies have reported 'humps' on the articular surface which have been related to underlying chondrocytes residing very close to the surface. Here we report on a highly nodular form of articular cartilage in the distal limb joints of the South American opossum, Monodelphis domestica. Unlike previous reports, these articular 'humps' are visible under a dissecting microscope. Each 'hump' or 'nodule' represents the surrounding matrix of single or sometimes paired rounded chondrocytes. Flattened chondrocytes normally associated with mammalian articular cartilage were absent from these joints. Interestingly, the articular cartilage of the more proximal limb joints such as the knee showed more typical features of articular cartilage including flattened superficial chondrocytes.
The distribution of epidermal growth factor binding sites in the developing mouse palate.
The distribution of epidermal growth factor (EGF) receptors in the developing mouse palate was mapped using 125I-EGF labeling of paired palate organ cultures. 125I-EGF binding sites were localized throughout the palate mesenchyme except in a region immediately adjacent to the midline seam. The EGF receptor was detected in all palatal epithelia at the beginning of culture, but as seam formation and subsequent degeneration took place it was down-regulated in the medial edge epithelia. Using submerged culture the mechanism of this down-regulation was investigated by treating with various growth factors such as EGF, basic fibroblast growth factor (bFGF), insulin-like growth factor-II (IGF-II) and transforming growth factors alpha and beta (TGF-alpha, TGF-beta). Conventional Trowell organ culture was not used because it was observed that the Millipore filter blocked growth factor uptake. All three TGF-beta isoforms accelerated palate fusion and TGF-beta 1 reduced 125I-EGF binding throughout the palate, suggesting a potential level of regulation during palatogenesis. Contrary to previous reports, EGF treatment in the absence of serum prevented palatal shelf fusion, and also down-regulated subsequent 125I-EGF binding.
Transgenic mouse model of X-linked cleft palate.
A transgenic mouse line, PyLMP.5, exhibited a sex-linked lethality not observed in any other lines expressing the transgene. In this unique line, the transgene integrated into the X chromosome, yielding a simple tandem duplication of the insert sequences with minimal, if any, additional rearrangement of the cellular sequences. The predominant phenotype was a cleft secondary palate and neonatal lethality in males. Survival of females was dependent on the mouse strain background. The disrupted cellular sequences have been mapped to the proximal region of the mouse X chromosome. The disrupted locus may represent the mouse counterpart to a human locus mutated in an X-linked cleft secondary palate syndrome.
Cloning and expression analysis of two ZFY-related zinc finger genes from Alligator mississippiensis, a species with temperature-dependent sex determination.
In order to investigate the molecular mechanism of temperature-dependent sex determination, a human zinc finger gene (ZFY), known to be highly conserved amongst other species, was used to isolate homologues from the genome of the American alligator, Alligator mississippiensis. ZFY was originally a candidate for the primary testis-determining gene in man, but is now thought to function further down the sex-determining cascade. Two alligator genes are described, Zfc and Znc6. Both code for zinc finger proteins and exhibit amino acid (aa) homologies to ZFY of 91% and 73%, respectively. Znc6 shows aa homology of 88% to the protein encoded by the zinc finger exon of the human ZFY-related gene, ZNF6, recently found on the X chromosome. Analysis of Zfc and Znc6 expression during embryonic development identified two major transcripts of 5.9 kb and 2.7 kb coding for Zfc, whilst only one transcript of 4.8 kb was detected for Znc6. Both genes are transcribed at all stages tested, from day 3 (post egg laying) throughout gestation. The expression level of all transcripts appears to decline towards the time of hatching (65-72 days). No sex-specific differences in the expression were observed. The extensive sequence conservation of the genes between reptiles and humans suggests major functional constraints. The expression patterns indicate that these genes do not play a primary role in temperature-dependent sex determination.
Control of scarring in adult wounds by neutralising antibody to transforming growth factor beta.
Adult wounds heal with scar-tissue formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. We injected the margins of healing dermal wounds in adult rats with neutralising antibody (NA) to transforming growth factor-beta (TGF-beta). All control wounds (irrelevant antibody, or TGF-beta, or no injection) healed with scarring, whereas the NA-treated wounds healed without scar-tissue formation; NA-treated wounds had fewer macrophages and blood vessels, lower collagen and fibronectin contents, but identical tensile strength and more normal dermal architecture than the other wounds. Early manipulation of the concentrations of selected cytokines may be a new approach to the control of scarring.
Changes in the extracellular matrix of the normal human breast during the menstrual cycle.
The normal human mammary gland undergoes a well defined sequence of histological changes in both epithelial and stromal compartments during the menstrual cycle. Studies in vitro have suggested that the extracellular matrix surrounding the individual cells plays a central role in modulating a wide variety of cellular events, including proliferation, differentiation and gene expression. We therefore investigated the distribution of a number of extracellular matrix molecules in the normal breast during the menstrual cycle. By use of indirect immunofluorescence, with specific antibodies, we demonstrated that laminin, heparan sulphate proteoglycan, type IV collagen, type V collagen, chondroitin sulphate and fibronectin undergo changes in distribution during the menstrual cycle, whereas collagen types I, III, VI and VII remain unchanged. These changes were most marked in the basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts surrounding the ductules where basement membrane markers such as laminin, heparan sulphate proteoglycan, and type IV and V collagens appear greatly reduced during the mid-cycle period (days 8 to 22). These results suggest that some extracellular matrix molecules may act as mediators in the hormonal control of the mammary gland, whereas others may have a predominantly structural role.
The effects of epidermal growth factor, transforming growth factors alpha and beta and platelet-derived growth factor on murine palatal shelves in organ culture.
Palatal shelves isolated from day-13 embryonic mice were explanted on to the surfaces of collagen gels either singly or in pairs with their medial edges in contact, and cultured submerged in a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F12 medium. The medium was supplemented with either 10 ng/ml epidermal growth factor (EGF), 10 ng/ml transforming growth factor alpha (TGF alpha), 1 ng/ml transforming growth factor beta (TGF beta 1) or 2 ng/ml platelet-derived growth factor (PDGF) all in the presence or absence of 2.5% donor calf serum (DCS). Cultures were terminated after 0, 24, 48 or 72 h and processed for histological and immunocytochemical examination. In serum-free medium and medium supplemented with 2.5% DCS the palatal epithelia differentiated in a manner similar to that seen in vivo (oral, keratinization; nasal, pseudostratified, ciliated columnar cells and medial edge, epithelial degeneration). A similar pattern was obtained in serum-free medium supplemented with either EGF or TGF alpha. However in cultures with either EGF or TGF alpha plus 2.5% DCS present in the medium, medial-edge epithelial degeneration was inhibited and the oral epithelia were more heavily keratinized. The mesenchyme of such cultures stained more intensely for various extracellular matrix molecules. In TGF beta 1-supplemented cultures (with, but especially without, serum supplementation) the epithelia were thin, medial-edge epithelial degeneration was marked, and the fibronectin content of the mesenchyme was increased. PDGF prevented medial-edge epithelial degeneration in the presence, but not in the absence, of serum; mesenchymal extracellular molecules were not as prevalent as with the EGF treatment. These results indicate that exogenous growth factors (including those present in serum) exert effects on organ-cultured mouse palatal shelves in a fashion similar to their effects in cell culture and that controlled physiological levels of such factors may be important in mouse palatal development.
Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth.
Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be D-galactose-(beta 1----3)DGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia--I (BSL-I) staining was lost from postmitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express beta-D-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.
The effects of transforming growth factor-beta 1 on protein production by mouse embryonic palate mesenchymal cells in the presence or absence of serum.
Mouse embryonic palatal mesenchyme cells were cultured on a variety of substrata (plastic, on a collagen gel or within a collagen gel). On each substratum TGF-beta 1 (1 ng/ml) inhibited cell proliferation. Cells cultured within a collagen gel had the lowest rate of proliferation, but were metabolically the most active in terms of incorporation of [3H]-proline into both collagenous and non-collagenous proteins. TGF-beta 1, in the presence of 2.5% donor calf serum stimulated the production of fibronectin and the major collagen types I, III and V. However, in serum-free medium, TGF-beta 1 induced a large reduction in total collagen production, mainly due to an effect on type I collagen, whilst stimulating production of some non-collagenous proteins. Experiments involving combinations of TGF-beta 1 with other growth factors suggested that the different effects of TGF-beta 1 on collagen production, in the presence and absence of serum, may be due to an interaction with platelet-derived growth factor.
Serum parathyroid hormone and calcitonin levels following ovariectomy in the adult rat.
The ovariectomized rat is a reasonably good and often utilized model of human post-menopausal osteoporosis (Devlin and Ferguson, 1989). Mature rats were either ovariectomized or underwent a sham operation, killed at intervals following operation and the serum concentration of parathyroid hormone and calcitonin at death related to the histomorphometry of the cancellous bone of the proximal ilium. The mean serum calcitonin concentration for all ovariectomized rats was 66.4 pg/ml (SD = 18) and for the control rats was 76.42 pg/ml (SD = 20). The serum calcitonin concentration in the ovariectomized rats was significantly negatively correlated with the extent of erosion cavities of the cancellous bone (r = -0.598, t = -2.36, p = 0.039). No significant correlation was observed for the control rats (r = -0.107, t = -0.357, p = 0.727). The mean extent of erosion cavities in the ovariectomized group was 6.81% (SD = 1.94) and in the control group was 6.47% (SD = 2.21), with no significant differences between the two groups (t = 0.41, p = 0.69). No significant correlations were observed in serum parathyroid hormone (PTH) concentration with the bone resorption parameters. From this study it was concluded that calcitonin may be of aetiological importance in bone loss in the ovariectomized rat.
The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy.
Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.
Expression patterns of the homeobox gene, Hox-8, in the mouse embryo suggest a role in specifying tooth initiation and shape.
We have studied the expression patterns of the newly isolated homeobox gene, Hox-8 by in situ hybridisation to sections of the developing heads of mouse embryos between E9 and E17.5, and compared them to Hox-7 expression patterns in adjacent sections. This paper concentrates on the interesting expression patterns of Hox-8 during initiation and development of the molar and incisor teeth. Hox-8 expression domains are present in the neural crest-derived mesenchyme beneath sites of future tooth formation, in a proximo-distal gradient. Tooth development is initiated in the oral epithelium which subsequently thickens in discrete sites and invaginates to form the dental lamina. Hox-8 expression in mouse oral epithelium is first evident at the sites of the dental placodes, suggesting a role in the specification of tooth position. Subsequently, in molar teeth, this patch of Hox-8 expressing epithelium becomes incorporated within the buccal aspect of the invaginating dental lamina to form part of the external enamel epithelium of the cap stage tooth germ. This locus of Hox-8 expression becomes continuous with new sites of Hox-8 expression in the enamel navel, septum, knot and internal enamel epithelium. The transitory enamel knot, septum and navel were postulated, long ago, to be involved in specifying tooth shape, causing the inflection of the first buccal cusp, but this theory has been largely ignored. Interestingly, in the conical incisor teeth, the enamel navel, septum and knot are absent, and Hox-8 has a symmetrical expression pattern. Our demonstration of the precise expression patterns of Hox-8 in the early dental placodes and their subsequent association with the enamel knot, septum and navel provide the first molecular clues to the basis of patterning in the dentition and the association of tooth position with tooth shape: an association all the more intriguing in view of the evolutionary robustness of the patterning mechanism, and the known role of homeobox genes in Drosophila pattern formation. At the bell stage of tooth development, Hox-8 expression switches tissue layers, being absent from the differentiating epithelial ameloblasts and turned on in the differentiating mesenchymal odontoblasts. Hox-7 is expressed in the mesenchyme of the dental papilla and follicle at all stages. This reciprocity of expression suggests an interactive role between Hox-7, Hox-8 and other genes in regulating epithelial mesenchymal interactions during dental differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)