Carboxyfluorescein as a marker at both light and electron microscope levels to follow cell lineage in the embryo.
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Animals↗
Biomedical subjects
C M Griffith
Publications and source records attributed to C M Griffith.
Expanded and packed bed albumin adsorption on fluoride modified zirconia.
The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion. Bioeng 60: 333-340, 1998.
Adsorption↗
Fluidization characteristics of and protein adsorption on fluoride-modified porous zirconium oxide particles.
Porous zirconia particles of specific gravity approximately 3.2 g/ml, mean particle sizes of approximately 50 microns, and terminal settling velocity of approximately 2.8 mm/s in water, were synthesized using an oil emulsion method from 1000 A colloids and were evaluated for their potential use in expanded bed protein adsorption. Expanded beds of particles were stable even for small volume, shallow beds (settled bed: 10 ml, height to diameter ratio < 1.0) and even for fluidization velocities common to much larger particles (210 cm/h for a three-fold bed expansion). When the surface of these particles was modified by fluoride adsorption, the total bed capacity for bovine serum albumin (BSA) adsorption was 42 +/- 2 mg BSA/ml of settled bed volume at linear velocities of 109-210 cm/h. Residence time distribution studies of several solutes under non-binding conditions were performed to assess the degree of liquid mixing and channeling in the expanded bed as a function of fluidization velocity. Liquid mixing and channeling were also studied as a function of distributor design. With these very dense particles, the degree of channeling and mixing did not worsen with the degree of expansion. Elution of adsorbed BSA while the bed was expanded (by a step increase in ionic strength) was rapid resulting in a narrow peak at high fluidization velocities without resorting to settling of the bed. The dynamic binding capacity of BSA at 5% breakthrough (protein effluent concentration equal to 5% of the inlet concentration) was the same for a two-fold expanded bed as for a settled bed (22 +/- 2 mg BSA/ml of settled bed volume), though it decreased for higher bed expansions. BSA binding was reproducible following repeated cleaning of the adsorbent with 0.25 M sodium hydroxide.
Adsorption↗
Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.
We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.
Alternative Splicing↗
Glycoconjugates in normal and abnormal secondary neurulation.
In chick embryos, the anterior greater portion of the neural tube develops by the folding, apposition, and fusion of the neuroectoderm. The smaller caudal portion that forms the secondary neural tube (lumbosacral and coccygeal regions) is derived from the tail bud, an aggregate of mesenchymal cells located at the caudal limit of the body. Tail bud mesenchyme, arranged in a solid cord, undergoes mesenchymal-epithelial transformation to form the secondary neural tube. Previous evidence suggests that this transformation is accompanied by modulation of cell surface glycoconjugates in the differentiating tissues. In this study, we show by lectin histochemistry and lectin blotting of proteins isolated by SDS-PAGE, that Datura stramonium agglutinin (DSA) binds preferentially to differentiating tail bud cells. This lectin is specific for beta 1-4-linked N-acetylglucosamine oligomers, such as the oligosaccharides of the poly-N-acetyllactosamine series that have been previously implicated in cell differentiation. Ultrastructural lectin cytochemistry indicates that at least some of the proteins binding DSA are localized extracellularly. The use of DSA as a teratogen resulted in embryos showing a variety of neural tube and notochord defects. We have also examined the binding of DSA to embryos that were treated with teratogenic doses of retinoic acid by sub-blastodermal injection, and find that the DSA-binding patterns are perturbed. Analysis of DSA-treated embryos using the TUNEL technique indicated that cell death was not a factor in DSA teratogenesis. This strongly suggests that the glycoconjugates of the cell surface have a role in the normal differentiation of tail bud mesenchyme into the neuroepithelium of the secondary neural tube. Perturbations of glycoconjugate activity results in defects of the secondary neural tube and associated tail bud derivatives.
Abnormalities, Drug-Induced↗
Tissue-specific expression of type XII collagen during mouse embryonic development.
Polyclonal antibodies were raised in rabbits against a fusion peptide representing a portion of the amino-terminal non-triple-helical domain of mouse type XII collagen. The antibodies reacted with bands of 220 and 350 kDa on Western blots of mouse tissue extracts. Immunohistochemical analyses of mouse embryos demonstrated that type XII collagen is expressed mainly in dense connective tissues of tendons, ligaments, dermis, cornea, blood vessel walls, meninges, and developing membranous bones. Comparison of skin extracts and medium of cultured mouse skin fibroblasts by Western blotting showed that while tissue contain short 220 kDa type XII collagen polypeptides as well as the long form, cultured cells produce mainly the long form with 350 kDa polypeptides.
Animals↗
The vertebrate tail bud: three germ layers from one tissue.
The tail bud of amniote embryos comprises a mass of apparently undifferentiated mesenchymal cells located at the caudal limit of the embryo, representing the remains of Hensen's node and the primitive streak. These cells have the potential to give rise to a variety of different tissues including the posterior or 'secondary' neural tube, the tail gut, and somites and their derivatives. This seemingly homogeneous accumulation of cells therefore has the capacity to differentiate into tissues which in more cranial regions of the embryo are derived from cells of different germ layers. In this review, the tissue contributions of the tail bud in various vertebrate classes are discussed, with particular attention to the mesenchymal-to-epithelial transformation that characterizes the process of secondary neurulation, and which distinguishes it from the epithelial folding that occurs during primary neurulation in more cranial regions. Recent studies suggest that the transformation is accompanied by extensive changes in the cell surface oligosaccharide complement of the differentiating cells, and that the sialyted form of N-CAM is expressed both temporally and spatially in a manner that suggests a role for it in the process. The pluripotential nature of the tail bud mesenchyme may be revealed experimentally by grafting the tissue ectopically, or by culturing it on different substrata. In the latter case, the mesenchyme can be demonstrated to give rise to myocytes, chondrocytes, neuroepithelium and neural crest derivatives such as melanocytes, depending on the nature of the culture substratum. It is concluded that the tail bud mesenchyme represents a developing system which is readily amenable to experimentation and should provide insights into the general mechanisms of cell differentiation and transformation.
Amphibians↗
Epithelial-mesenchymal transformation during palatal fusion: carboxyfluorescein traces cells at light and electron microscopic levels.
During the fusion of rodent embryo palatal shelves, the cells of the outer epithelial layer slough off, allowing the cells of the medial edge basal layer to form a midline seam that undergoes epithelial-mesenchymal transformation, as judged by electron microscopy and immunohistochemistry. In this study, we analyze the fate of the transformed cells using a lipid soluble dye to label the medial edge epithelium in situ. Prefusion E14 mouse palates were exposed in vitro or in vivo to a fluoresceinated lipid soluble marker, carboxydichlorofluorescein diacetate succinimidyl ester (CCFSE), which localizes in epithelia as a lipid insoluble compound that does not pass into the connective tissue compartment. The midline seam that formed after 24 hours contained labelled epithelial cells that were replaced by individually labelled mesenchymal cells where the seam transformed. By light microscopy, the labelled cells were seen to contain intensely fluorescent bodies that do not react for acid phosphatase. We were able for the first time to identify these structures by electron microscopy as CCFSE isolation bodies. The cells with isolation bodies are clearly healthy and able to participate in subsequent development of the palate. At 4 days after labelling, individual CCFSE containing cells present in the palate mesenchyme occupy both midline and lateral areas and can clearly be classified as fibroblasts by electron microscopy. CCFSE is a far more useful marker than another lipid soluble marker, DiI, for following cells, because the cells can be fixed and identified both at the light and electron microscope levels. Interestingly, if labelled palatal shelves are not allowed to fuse in vitro, the basal epithelial cells do not form mesenchyme after sloughing, indicating that formation of the epithelial midline seam is necessary to trigger its epithelial-mesenchymal transformation.
Animals↗
Changes in glycoconjugate expression during early chick embryo development: a lectin-binding study.
A selection of lectins was used to investigate developmentally regulated changes in the distribution of cell surface oligosaccharides during the gastrulation and neurulation stages of early chick embryo development. Lectins from three specificity classes were used: glucose/mannose specificity (concanavalin A [Con A], Lens culinaris agglutinin [LCA], Pisum sativum agglutinin [PSA]); N-acetylglucosamine specificity (Lycopersicon esculentum agglutinin [LEA], wheat germ agglutinin [WGA], succinylated WGA [sWGA]); N-acetylgalactosamine/galactose specificity (Dolichos biflorus agglutinin [DBA], soybean agglutinin [SBA], Sophora japonica agglutinin [SJA], Bandeiraea (Griffonia) simplicifolia lectin I [BSL I], peanut agglutinin [PNA], Artocarpus integrifolia lectin [Jacalin], Ricinus communis agglutinin-1 [RCA-1], Erythrina cristagalli lectin [ECL]). At gastrulation stages, patterns of lectin binding could be distinguished in the epiblast, mesoderm, and endoderm cell layers. The primitive streak failed to bind any of the lectins, but LEA and WGA bound to the epiblast in regions lateral to the streak, indicating the loss of some glucosamine residues medially in preparation for the ingression movements of gastrulation. Several lectins showed marked binding to the mesoderm cells after their passage through the primitive streak; these were LCA, PSA, WGA, sWGA, BSL, and most particularly PNA. Therefore, the epithelial-mesenchymal transformation from epiblast to mesoderm at the primitive streak is accompanied by cell surface oligosaccharide changes in the epiblast and mesoderm that involve all classes of lectins including the PNA-binding sequence Gal beta 1-3GalNAc. Ultrastructurally, PNA was shown to bind extracellularly to matrix fibrils. Jacalin, having the same sugar specificity as PNA, but binding to serine/threonine linked chains rather than asparagine linked chains showed no binding to the mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of retinoic acid on chick tail bud development.
The present study describes the teratogenic effects of retinoic acid (RA) on the development of the chick tail bud. Chick embryos were recovered 48 hours after treatment at HH stages 11 to 16 with various dosages of RA by subblastodermal injection. At the gross level, RA treatment resulted in varying degrees of caudal regression, scoliosis, limb malformations, and open posterior neuropores among the survivors. Histological examination of tail buds from treated embryos revealed defects which included total dysplasia of caudal structures, the presence of accessory neural tube and notochord tissue, and abnormal fusions of the notochord to the neural tube and tailgut. The incidence, severity, and location of the defects were dependent on the dose of the teratogen, and the stage of development at the time of treatment. The defects resembled those induced in previous studies by treatment with sialic acid binding lectins such as wheat germ agglutinin and limulus polyphemus lectin (Griffith and Wiley, '90b).
Abnormalities, Drug-Induced↗
N-CAM, polysialic acid and chick tail bud development.
We have previously shown that the binding of the lectin wheat germ agglutinin (WGA) to developing tail buds results in a range of caudal axial defects, which were most likely due to the affinity of the lectin for sialic acid residues. In the present study, we examined the distribution and role of a sialic acid-containing glycoprotein, N-CAM, in chick tail bud development. In the early tail bud, anti N-CAM, staining was found in the medullary cord. However, there was no uptake of an antibody specific to N-CAM containing moderate to long chains of polysialic acid (5A5 monoclonal antibody). At later stages, while N-CAM localized throughout the neural tube, staining with the 5A5 antibody was restricted to the floor plate. Sub-blastodermal injection of the anti N-CAM antibody beneath the tail bud region of HH stages 13-14 embryos produced caudal axial malformations. These malformations included the presence of accessory segments of neural tube and/or notochord, and fusion between the neural tube and underlying segment of notochord. Our results suggest that N-CAM is present during the development of the secondary neuraxis from the tail bud, although the highly sialylated form of this molecule could not be visualized until relatively late stages. N-CAM probably plays a role in the normal course of tail bud development, since perturbation of the molecule with an antibody resulted in malformations. Since these malformations were similar to those we have previously reported when we treated similarly staged chick embryos with WGA, there is a possibility that the sialic acid residues recognized and bound by the lectin are those associated with the N-CAM molecule.
Animals↗
Differentiation of the chick embryo floor plate.
In a number of species, the floor plate of the developing neural tube and spinal cord has been ascribed specialized functions associated with the patterning of neuronal differentiation. The differentiation of the floor plate itself is believed to be closely related to the presence of the underlying notochord. Grafting experiments have previously shown that in the chick embryo an implanted segment of notochord is capable of inducing the adjacent host neural plate or neural tube to produce an additional floor plate, although the inductive effect diminishes with increasing age of the host. We have examined the potential of notochord to promote the appearance of floor plate-like structures from neural tube tissue in culture. To facilitate this, it was necessary initially to examine the immunoreactivity of the early neural tube and floor plate in situ and in vitro with a panel of antibodies to identify a suitable marker for floor plate differentiation in vitro. In situ, the differentiation of the floor plate was characterized by a lack of immunoperoxidase staining with antibody to neurofilaments and the monoclonal antibody HNK-1 throughout the period examined. This distinguished the floor plate from other regions of the neural tube, and was in contrast to its conspicuous affinity for antibodies to N-CAM and highly sialylated N-CAM, which also stained several closely adjacent regions of the neural tube over the period examined. We also found that oligodendrocytes occurred both in the floor plate and in the flanking ventral neural tube, and that astrocytes were too poorly represented throughout the neural tube at the stages examined to be useful markers of floor plate differentiation. We therefore concluded that only the anti-neurofilament and the HNK-1 antibodies were potentially useful markers for floor plate differentiation. When these antibodies were tested on cells in culture, neural tube tissue showed the presence of neurofilament- and HNK-1-positive neurites, while floor plate cultures showed few of these. These distributions were consistent with those demonstrated in situ. However, cells staining positively for N-CAM, sialylated N-CAM and the glial cell markers were relatively sparse in floor plate cultures, suggesting that these epitopes were not retained or were masked in cultured cells. As a result of these experiments, we selected the absence of neurofilament-positive cells as a marker for floor plate differentiation in culture.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Effects of extracellular matrix components on the differentiation of chick embryo tail bud mesenchyme in culture.
The mesenchymal cells of the chick tail bud comprise the remains of Hensen's node and the primitive streak after gastrulation. This mass of cells, situated at the caudal limit of the chick embryo, is morphologically homogeneous but pluripotent, with the ability to differentiate into a variety of tissues that are both ectoderm- and mesoderm-derived elsewhere in the embryo. These tissues include neuroectoderm, neurons, myoblasts and chondrocytes. As the factors regulating the differentiation of tail bud mesenchyme into so many cell types are unclear, and because the extracellular matrix (ECM) is known to have a profound effect on cellular differentiation in many embryonic systems, we studied the differentiation of tail bud mesenchyme explanted onto a variety of different ECM components as substrata. We report that the histogenetic potential of isolated tail buds in culture compares favourably with that in situ. Using various antibody markers, we have demonstrated that tail bud mesenchyme cultured upon different ECM components as substrata is able to differentiate into neurons, neuroepithelium, melanocytes, muscle and cartilage. Laminin and laminin-containing substrata (Matrigel) were found to promote the differentiation of neural crest derivatives (neurons and melanocytes) and neuroepithelial cells; type I collagen promoted both myogenesis and chondrogenesis; while type IV collagen promoted myogenesis only. We have therefore demonstrated that differentiation of tail bud mesenchyme in vitro is substratum-dependent.
Animals↗
Distribution of cell surface glycoconjugates during secondary neurulation in the chick embryo.
Lectin histochemistry was used to examine the expression of cell surface glycoconjugates during secondary neurulation in chick embryos. Fourteen lectins were applied to serial sections of the caudal region of embryos at the various stages of tail bud development. The lectins Bandeiraea simplicifolia, Dolichos biflorus agglutinin, Phaseolus vulgaris leukoagglutinin, soybean agglutinin, Sophora japonica agglutinin, Ulex europaeus agglutinin and succinylated wheat germ agglutinin (sWGA) showed very light or no binding to the developing medullary cord of the tail bud. With the other lectins, staining occurred throughout the early tail bud and solid medullary cord. During cavitation, however, differential expression of cell surface glycoconjugates by different cell populations was observed. The lectins concanavalin A, Lens culinaris agglutinin, Pisum sativum agglutinin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin and WGA showed basic similarities in the distribution of lectin binding. Of these, the binding pattern of WGA was the most striking. As the medullary cord cells were separating into central mesenchymal and peripheral epithelial populations, WGA bound preferentially to the epithelial cells and the notochord. The lectin PNA, however, became preferentially bound to the mesenchymal cells. Heavy staining by WGA (specific for N-acetylglucosamine and sialic acid) where sWGA staining (specific for N-acetylglucosamine only) was faint suggested that WGA binding was due to the presence of sialic acid containing glycoconjugates.
Animals↗
Effects of retinoic acid on the distribution of glycoconjugates during mouse tail bud development.
Retinoic acid (RA), a potent teratogen of caudal axial development in rodents, has been shown to alter glycoconjugates in a variety of embryonic tissues and teratocarcinomas. In this study, we examined its effects on the expression of cell surface and extracellular matrix glycoconjugates during tail bud development in mouse embryos by using lectin histochemistry. The lectins WGA, sWGA, and PNA showed striking differences in binding between RA-exposed and control embryos. Computer-assisted densitometry revealed a significant increase in binding of all three lectins to the extracellular material of the luminal and abluminal borders of the secondary neural tube and surrounding the notochord in RA-exposed embryos. RA-treated embryos also showed an increased binding affinity for the lectins sWGA and PNA to the cells of the notochord, while WGA showed increased binding to the neuroepithelial cells of the secondary neural tube. The results suggest that RA affects the expression of lectin binding sites during the early development of RA-induced caudal axial defects.
Animals↗
Sialoconjugates and development of the tail bud.
Using lectin histochemistry, we have previously shown that there are alterations in the distribution of glycoconjugates in the tail bud of chick embryos that parallel the developmental sequence of the caudal axis. If glycoconjugates or the cells bearing them play a role in caudal axial development, then, restriction of their availability by binding with lectins would be expected to produce abnormalities of caudal development. In the present study, we treated embryos at various stages of tail bud development by microinjection with a variety of lectins. Administration of WGA by sub-blastodermal injection resulted in high incidences of secondary neural tube and notochordal abnormalities in lectin-treated embryos. The incidence of malformations was dependent upon both the dose of WGA received and the stage of development at the time of treatment. Using an anti-WGA antibody, we have also shown binding of the lectin in regions where defects were found. The lectin WGA binds to the sialic acid residues of glycoconjugates and to N-acetylglucosamine. Treatment of embryos with Limulus polyphemus lectin (LPL), which also binds to sialic acid, produced results similar to those of WGA. Treatments using lectins with other sugar-binding specificities, including succinylated WGA (with N-acetylglucosamine specificity only) produced defects that differed from those produced by WGA and LPL, and only with the administration of much higher doses. The results suggest that glycoconjugates in general and sialoconjugates in particular, or the cells carrying them, may have a role in caudal axial development.
Animals↗
Direct effects of retinoic acid on the development of the tail bud in chick embryos.
Retinoic acid (RA) has been reported to induce vascular lesions and haematoma formation in the vicinity of the tail bud during the critical period for inducing abnormalities of tail bud development in hamsters (Wiley, '83; Tibbles and Wiley, '88), mice (Tibbles and Wiley, '88) and chicken embryos (Jelinek and Kistler, '81). Experiments were conducted to determine whether or not these vascular lesions were the primary cause of the malformations which they accompanied. Chick embryos were exposed for varying lengths of time to several dosages of RA. Primitive streaks or tail buds from treated embryos were then excised prior to vascularization and transplanted to the coelomic walls of untreated host embryos. The grafts were harvested at 3 or 6 days after grafting and processed for histological examination. Observations of serial sections of controls showed that the primitive streak and early (stage 13-14) tail bud were able to form neural tubes and a variety of other structures including ganglia, nerve fibres, and kidney tubules. Treatment of donor embryos with RA prior to grafting, however, affected the frequency and characteristics of the neural tubes and other tissues developing in the grafts. The effects of RA on development were correlated with both the dosage and length of exposure to the teratogen prior to grafting. Since the grafts were made before the appearance of blood vessels in the tail buds, we have concluded that the effects of RA on the development of tail bud tissues, and especially the secondary neural tube, are direct and are not mediated solely through the disruptive effects of vascular lesions seen in intact embryos.
Animals↗
The distribution of cell surface glycoconjugates during mouse secondary neurulation.
During secondary neurulation in the mouse, the neural tube develops from the tail bud by caudal extension of the primary neurocoele. The mesenchymal cells of the tail bud become radially arranged around the neurocoele and undergo a mesenchymal to epithelial transformation to form a neuroepithelium. In order to study the expression of glycoconjugates during the morphogenesis of the secondary neural tube, 14 lectins were applied to serial sections of tail buds at various stages of development. In general, binding was fairly homogeneous during the early stages of tail bud development. However, as development progressed, several lectins became localized to specific structures. The changes were observed to parallel the ongoing development of the secondary neuraxis. sWGA, which is N-acetylglucosamine (GlcNAc) specific, bound mainly to the luminal surface of the secondary neurocoele and to a lesser extent, the notochord. WGA, which has both GlcNAc and sialic acid specificities, showed most intense binding at the luminal and abluminal surfaces of the secondary neurocoele. Binding by the lectin PNA was restricted to the extracellular matrix around the developing secondary neural tube. A comparison of the lectin binding patterns in mouse with those previously reported in chick, demonstrates a less elaborate pattern of lectin binding in murine embryos. This may suggest a less complex expression of glycoconjugates in rodents, in keeping with their comparatively simpler mechanism of secondary neurulation.
Animals↗