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Biomedical subjects

D F Newgreen

Publications and source records attributed to D F Newgreen.

At least 37 records · Page 2Linked to original sources
PubMed1996

Migration of enteric neural crest cells in relation to growth of the gut in avian embryos.

Neural crest cell migration in the gut and the growth of the mid- and hindgut of avian embryos was investigated by a combination of whole-mount immunofluorescence of the HNK-1 neural crest marker epitope, chorioallantoic membrane grafting and morphometry. HNK-1-labelled cells advanced rostrocaudally in the gut of quail embryos (to the duodenum by stage HH 21, to the umbilicus by HH 25, to the ceca by HH 27, to the cloaca by HH 33). The timetable in chick embryos appeared to be slightly slower, but neural cells were obscured by background fluorescence in this species. More rostral regions of the gut commenced rapid growth earlier than more caudal regions (preumbilical small intestine after HH 26, postumbilical small intestine after HH 27 and colorectum after HH 28), and the small intestine and ceca grew most rapidly in length while the colorectum grew most rapidly in diameter. The rates of growth of the gut were low prior to the stage when HNK-1-labelled cells normally arrive in the small intestine, ceca and rostral colorectum, but increased dramatically after arrival. In the caudal colorectum rapid growth had commenced at the time of arrival of these cells. These data are consistent with the idea that a delay in arrival of vagal neural crest cells at any point in the intestine could jeopardize the ability of the cells to fully populate the remainder of the gut, due to the normal growth spurt causing the migration end-point to recede faster than the rate of neural crest cell migration. Thus, a mismatch in timing of neural crest cell migration and gut growth could play a role in the etiology of some forms of Hirschsprung's disease.

Animals↗
PubMed1995

Control of epitheliomesenchymal transformation. I. Events in the onset of neural crest cell migration are separable and inducible by protein kinase inhibitors.

Neural tubes isolated from quail embryos prior to epitheliomesenchymal transformation (EMT) and neural crest (NC) cell migration, when explanted onto fibronectin surfaces, replicated properties of normal NC morphogenesis such as (i) cell outgrowth, (ii) loss of A-CAM (N-cadherin) junctions and adoption of mesenchymal form, and (iii) development of HNK-1 immunoreactivity. The timetable of property (i) was essentially normal but the outgrowing cells were initially mainly epithelial, unlike NC outgrowth in vivo and in cultures of older neural tubes. Mesenchymal properties (ii) and (iii) were progressively and variably retarded relative to the in vivo timetable. Achievement of these properties by EMT was principally related to proximity to the neural tube and to preexisting mesenchymal cells, rather than being related temporally to the outgrowth timetable. This EMT, combined with a higher mitotic rate in the mesenchyme cells, resulted in the outgrowth passing from mainly epithelial at 16 hr to mainly mesenchymal at 48 hr in vitro. Immediate precocious EMT and outgrowth of A-CAM negative mesenchymal cells from pre-EMT neural tubes was stimulated by the protein kinase inhibitors staurosporine and bisindolymaleimide in a cycloheximide-independent manner. EMT could be induced not only on the dorsal (i.e., NC) side, but also on the ventral side of the neural tube, but the ventral cells were less sensitive than the dorsal cells, and with developmental age became still less sensitive while the dorsal cells became more sensitive. The results suggest that the complex events of EMT in the NC system are not obligatorily coregulated, can be triggered by epigenetic events involving differential protein phosphorylation, and may be controlled via intraneural signaling.

Animals↗
PubMed1995

Extracellular matrix and adhesive molecules in the early development of the gut and its innervation in normal and spotting lethal rat embryos.

The distribution of fibronectin (FN), laminin (LM), J1/tenascin, chondroitin sulphate proteoglycan (CSPG), neural cell adhesion molecule (NCAM), neurofilament (NF) and the HNK-1 epitope were studied immunohistochemically in the developing mid- and hindgut of E12.5-E16.5 rat embryos. Over this period the gut wall changed from a uniform mesenchyme to an annular organisation. FN and LM remained widely distributed, but J1/tenascin became concentrated in mesenchyme outside the nascent circular muscle layer, and CSPG declined in, and NCAM increased in, the circular layer. Protease and fixation treatments suggested that CSPG could mask other molecules such as LM. This re-organisation proceeded bidirectionally, as a rostrocaudal wave which was met in the colon by a caudorostral wave. The caecum, however, was conspiciously delayed in all maturation events and also showed mesenchymal and serosal epithelial labelling for the cell-adhesion-related HNK-1 epitope, which was absent elsewhere. This period also covered the appearance of enteric neurons, recognised by HNK-1 and NF antibodies. Cells labelled by these antibodies appeared in a unidirectional rostrocaudal wave, from the duodenum at E12.5 to the rectum at E16.5. This wave was not in exact synchrony with the wave of intestinal maturation, but lagged behind so that neuronal cells first appeared in increasingly mature micro-environment at progressively more caudal levels. These cells initially were positioned imprecisely about mid-way across the gut mesenchyme layer and were not clearly related spatiotemporally to any of the above molecules. Slightly later, however, this neural region was broadly defined by relatively low levels of both CSPG and J1/tenascin. The final position of the myenteric neurons was very precise, and was related to a thin J1/tenascin layer and to a step in NCAM labelling intensity. Litters of pups, of which 25% would be expected to be spotting lethal homozygous embryos which develop total colonic and caecal aganglionosis, showed no difference in any of the molecules studied, but in 4 embryos out of 12, the progress of the rostrocaudal wave of neuron appearance was distinctly slowed even in the duodenum and proximal small intestine, regions well outside the final aganglionic zone. The observations suggest that this Hirschsprung's-disease-like regionalised defect is a result of a generalised abnormality which does not involve gross changes in extracellular matrix and NCAM expression.

Animals↗
PubMed1993

Adhesion molecules in neural crest development.

Peripheral nerve cells, various endocrine and pigment cells and cranial connective tissue cells of vertebrates stem mainly from the embryonic neural crest. This originates with the central nervous system, but the crest cells detach from this tissue, via a decrease of cell-cell adhesion involving, particularly, a reduction of the adherens junction cell adhesive molecule A-CAM. This epithelio-mesenchymal transformation allows crest cells to migrate along pathways that are defined partly by the distribution of substrate adhesion molecules, the archetype being fibronectin, an extracellular matrix molecule recognized by integrin receptors on crest cells. Many other molecules, however, may act in the same way. In contrast, some molecules may define migration pathways by reducing adhesion; chondroitin sulfate proteoglycan is a candidate for this role. Pathway selection is most likely achieved by balanced combinations of molecules that promote and reduce adhesion. Cessation of migration, in the case of the nervous ganglia, correlated with re-expression of cell-cell adhesion molecules like A-CAM and others, consistent with an adhesive basis, although functional tests have not yet been performed. The development of the neural crest system provides a useful model that emphasizes the role of adhesion in morphogenesis.

Amino Acid Sequence↗
PubMed1991

Cytotactin expression in somites after dorsal neural tube and neural crest ablation in chicken embryos.

The spatiotemporal expression of the extracellular matrix protein cytotactin/tenascin during somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of neural crest (NC) development. In the present study, the spatial distribution and molecular forms of cytotactin in somites were examined using in situ hybridization, Western blotting, and immunohistochemistry during normal development and after injury. In situ hybridization showed that prior to NC cell invasion cytotactin mRNA was restricted to the caudal half of the newly formed epithelial somites. As each epithelial somite matured, giving rise to a sclerotome and dermamyotome, the mRNA was first restricted to the dermamyotome and later restricted to the rostral protion of the sclerotome, consistent with the previously reported protein distribution. Immunocytochemical analysis of the distribution of cytotactin and NC cells in embryos with ablations that removed NC cells, or with simple wounds that left NC cells in place, demonstrated that the presence of NC cells is neither necessary nor sufficient for the correct positioning of cytotactin. Immunoblotting analysis showed that cytotactin synthesized by sclerotomes in the absence of NC cells was of similar molecular mass to that produced in their presence. These findings are in accord with the notion that the abnormalities of cytotactin distribution are related to the wounding process. We conclude that, contrary to the suggestion of Stern et al. [Stern, C. D., Norris, W. E., Bronner-Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319], there is no causal link between the presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin.

Animals↗
PubMed1990

Spatiotemporal changes in HNK-1/L2 glycoconjugates on avian embryo somite and neural crest cells.

Neural crest cell migration was studied in trunks of quail and chick embryos using HNK-1 and L2 antibodies. At the intersegmental cleft, labeled crest cells were associated with the rostral wall of the somite rather than blood vessels. Migration into and through the rostral part of the sclerotomes was more rapid (40-70 microns/hr; quail) and the onset of localization was earlier (after 13-16 hr; quail) than previously supposed. Crest cells here were initially mono- to multipolar, scattered, and inconsistently oriented and formed numerous close (about 20 nm) homo- and heterotypic cell-cell contacts. In vitro models suggested that significant numbers of crest cells, however, could be unlabeled at this early phase. Somitic properties covarying with the hemisegmental pattern of crest cell immigration were investigated. Laminin distribution, although asymmetric in the somites, was not closely related to that of crest cells. Tenascin distribution matched that of crest cells, but only at the localization stage. Earlier, maximal tenascin expression occurred in the somite's caudal wall, a region avoided by crest cells. Chondroitin 6-sulfate proteoglycan expression was elevated in the caudal somite-half at the localization phase and also, at lumbar levels, at the immigration stage. This is consistent with tenascin and proteoglycan having a negative role in crest cell migration. The rostral somite-half was also labeled by HNK-1 and L2, but only in quails. This was associated with the cell surface, was transient, was stable to mild proteolysis, and was labile to cryoprocessing and lipophilic solvents. The spatial and temporal congruence with crest migration suggests that the HNK/L2 adhesion-related carbohydrate epitope on the somites indicates a molecule (possibly glycolipid) which acts via heterotypic cell-cell contacts to provide one cue in the patterned distribution of crest cells in the somites.

Animals↗
PubMed1990

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo.

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.

Ambystoma↗
PubMed1989

Physical influences on neural crest cell migration in avian embryos: contact guidance and spatial restriction.

Several ideas on how neural crest (NC) cell migration in bird embryos might be dependent on the physical qualities of the internal embryonic environment were studied. Contact guidance has been suggested to direct NC cells ventrally in the trunk, but this has been subject to doubt (see Newgreen and Erickson, 1986, Int. Rev. Cytol. 103, 118-119). On reexamination, in situ extracellular matrix (ECM) and cell processes on the medial face of the somites were found appropriately oriented for this function. In addition, tissue culture models of oriented ECM could induce orientation of NC cells which mimicked that observed in the embryo. It is concluded that in this situation, oriented structures contribute to directed migration of NC cells in vivo, but the mechanism of contact guidance (i.e., steric or adhesive guidance) could not be ascertained. Contact guidance, in the form of steric guidance, has also been suggested as limiting ventrad NC cell movement at the midbrain level due to an insurmountable ridge on the side of the midbrain. The presence of this ridge was confirmed but it is unlikely to be responsible for prevention of ventrad migration, because, although it subsides very rapidly, the cells still refuse to move ventrad, and because models of this ridge in vitro proved to be no obstacle to NC cells. NC cell migration is also described as being limited by gross space between other organs or tissues. In vitro, NC cells could penetrate Nucleopore filters with pore diameters of 0.86 micron or greater. Observation of cell-free spaces in embryos showed that these were almost all much larger than the minimum pore size established experimentally. It is therefore concluded that in general the dimensions of gross tissue spaces probably do not set important limits for NC cell migration, but that the dimensions of transiently distensible microspaces between ECM fibrils may be a critical physical parameter.

Animals↗
PubMed1986

Morphogenesis of sclerotome and neural crest in avian embryos. In vivo and in vitro studies on the role of notochordal extracellular material.

The distribution of sclerotome and neural crest cells of avian embryos was studied by light and electron microscopy. Sclerotome cells radiated from the somites towards the notochord, to occupy the perichordal space. Neural crest cells, at least initially, also entered cell-free spaces. At the cranial somitic levels they moved chiefly dorsal to the somites, favouring the rostral part of each somite. These cells did not approach the perichordal space. More caudally (i.e. trunk levels), neural crest cells initially moved ventrally between the somites and neural tube. Adjacent to the caudal half of each somite, these cells penetrated no further than the myosclerotomal border, but opposite the rostral somite half, they were found next to the sclerotome almost as far ventrally as the notochord. However, they did not appear to enter the perichordal space, in contrast to sclerotome cells. When tested in vitro, sclerotome cells migrated towards notochords co-cultured on fibronectin-rich extracellular material, and on collagen gels. In contrast, neural crest cells avoided co-cultured notochords. This avoidance was abolished by inclusion of testicular hyaluronidase and chondroitinase ABC in the culture medium, but not by hyaluronidase from Streptomyces hyalurolyticus. The results suggest that sclerotome and neural crest mesenchyme cells have a different distribution with respect to the notochord, and that differential responses to notochordal extracellular material, possibly chondroitin sulphate proteoglycan, may be responsible for this.

Animals↗
PubMed1985

Control of the onset of migration of neural crest cells in avian embryos. Role of Ca++-dependent cell adhesions.

To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of CA++ and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3-8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca++-and Mg++-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca++-free medium duplicated the effects of CMF, but neither Mg++-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane Ca++-channel antagonists D600 and Nifedipine, without any exernal Ca++-depletion. The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca++-depletion inactivates Ca++-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca++-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca++-channels suggest that Ca++ levels may be involved in regulating this system.

Animals↗
PubMed1983

Oriented axon outgrowth from avian embryonic retinae in culture.

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged varied with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explanation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.

Animals↗
PubMed1982

Adhesion to extracellular materials by neural crest cells at the stage of initial migration.

Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5-2 h incubation, the explants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.

Animals↗
PubMed1982

Differentiation of avian neural crest cells in vitro: absence of a developmental bias toward melanogenesis.

Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (greater than 400K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.

Animals↗
PubMed1982

Ultrastructural and tissue-culture studies on the role of fibronectin, collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo.

The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15-40nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin (greater than 3nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker (greater than 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen. Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates.

Animals↗
PubMed1980

Differentiation of sympathetic and enteric neurons of the fowl embryo in grafts to the chorio-allantoic membrane.

Sympathetic cells (adrenergic neurons, SIF cells and chromaffin cells) and enteric neurons differentiate from migratory cells derived from the neural crest. The development of these cell types was studied in chorio-allantoic membrane (CAM) grafts, using combinations of tissue from domestic fowl embryos. Neural anlagen (neural tube and crest) of the vagal, cervico-thoracic and lumbo-sacral axial levels were equally capable of sympathetic differentiation, but this required somitic tissue for its significant expression. However, the vagal somites possessed only slight sympathogenic activity, thereby accounting for the negligible contribution of the vagal neural crest to the sympathetic nervous system. The same three levels of the neural anlage could furnish enteric neurons when combined directly with the aneuronal colo-rectum. However, the scale of this line of differentiation varied with the level of origin of the neural anlage, in contrast to the apparent equivalence in the ability to diffentiate as sympathetic cells. The density of enteric neurons in combinations with the vagal neural anlage was estimated as 60 times greater than the neuron density in combinations with the cervico-thoracic neural anlage. The lumbo-sacral neural anlage gave results similar to those of the cervico-thoracic level. Moreover, neural crest-derived pigment cells, positioned ectopically in the wall of the colo-rectum, were rare in combinations with the vagal neural anlage, but common in grafts with the other levels. When tested physiologically, the colo-rectum grown with the vagal neural anlage showed non-adrenergic, non-cholinergic inhibitory nervous activity in addition to the expected cholinergic excitatory responses. The neurons derived directly from vagal neural anlagen were similar to those that had reached the colo-rectum via their normal migratory pathways, when studied in terms of histological appearance, density of distribution and physiological responses.

Allantois↗
PubMed1979

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.

Animals↗