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Biomedical subjects

J B Bard

Publications and source records attributed to J B Bard.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nephrogenesis and the development of renal disease.

Over the last few years there has been considerable progress in analysing the cellular basis of nephrogenesis and a start has been made towards elucidating the underlying molecular controls of this process. In this review we begin by describing how the kidney forms and then consider recent work on the mechanisms underlying these events. We review evidence implicating a neural basis for kidney induction and go on to show how the induced metanephric mesenchyme aggregates and forms condensations which themselves polarize to form epithelia and filtering nephrons. We then discuss how changes in the extracellular matrix are implicated in these processes and how the expression of nuclear transcription factors may regulate the final phenotype of the kidney. Finally, we show how the study of nephrogenesis is beginning to shed light on the aetiology of a range of disorders that include renal malformations, renal tumours, and inherited glomerular and polycystic kidney diseases.

Animals↗
PubMed1991

LIF, the ES-cell inhibition factor, reversibly blocks nephrogenesis in cultured mouse kidney rudiments.

Mouse kidney induction proceeds in vitro much as it does in vivo: the ureteric bud bifurcates to give collecting ducts while the mesenchyme condenses into aggregates which epithelialise and then elongate into tubules with glomerular and other nephron structures. We report here that the factor known as LIF (leukaemia inhibitory factor), which regulates the differentiation and growth of embryonic-stem (ES) and other cells in culture, has little effect in vitro on growth or on ureteric-bud morphogenesis other than to stimulate the bifurcation process. It does however exert a striking effect on the mesenchyme. At about four times the concentration required to inhibit ES-cell differentiation, LIF strongly but reversibly blocks the effects of metanephric mesenchyme induction: although mesenchyme condenses around growing duct tips, the number of mature nephrons that form over 6 days is reduced by 75% or more. The few nephrons that do develop in the presence of LIF probably come from mesenchyme already induced at the time of culture and are indistinguishable from those that form in controls as assayed by morphology, by X-gal staining of endogenous galactosidase and by antibodies to brush-border and CD15 antigens. There is a further unexpected feature of rudiments cultured in LIF which is absent in controls: they contain an unexpectedly high number of stable epithelialised aggregates that express laminin around their periphery and which do not develop further. These results argue that the process of nephrogenesis involves at least two distinct stages which can be blocked by LIF: the effect of the initial induction and the future development of epithelialised aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Traction and the formation of mesenchymal condensations in vivo.

Although the segregation of mesenchyme into distinct aggregates is the first step in the development of a range of tissues that includes bones, somites, feathers and nephrons, we still know very little about the mechanisms by which this happens. There are two obvious types of explanation: first, that there are global pre-patterns within the mesenchyme whose molecular expression leads to tissue fragmentation and, second, that the condensations arise spontaneously through the local morphogenetic abilities of the cells. The only known mechanism for the latter possibility is cell traction and this paper suggests that current studies are compatible with traction playing a primary role in the formation of nephrogenic condensations in the developing kidney and the separation of somites, but not for the generation of feather rudiments where there is evidence of a prepattern of adhesivity.

Animals↗
PubMed1989

Does chondroitin sulfate have a role to play in the morphogenesis of the chick primary corneal stroma?

This paper makes three points about how the chick corneal epithelium lays down the primary stroma, an orthogonally arranged array of well-spaced, 20-nm-diameter collagen fibrils. (1) Isolated corneal epithelia will, when cultured, lay down de novo stromas whose fibril-diameter distribution, fibril spacing, and proteoglycan profile are similar to those laid down in vivo. They differ from embryonic stromas in two ways: first, much of the chondroitin sulfate is released to the medium and, second, there is a relatively small amount of orthogonal organization. Epithelia seem only to lay down such stromas if they are separated from their original stromas with dispase, which leaves an intact basal lamina, and spread out, basal lamina downward, on a Nuclepore filter (poresize, 0.1 micron). (2) Chondroitin sulfate (CS), the predominant proteoglycan (greater than 85%), seems to play no significant role in collagen fibrillogenesis in vitro. Stromas laid down in its absence were indistinguishable from controls as assayed by fibril diameter, organization, and spacing and the amount of collagen synthesized. For these experiments, epithelia were cultured in the presence of hyaluronidase, which degrades CS, and p-nitrophenyl beta-D-xyloside, which inhibits the formation of links between the core protein and glycosaminoglycan side chains in the PG; the absence of intact CS was confirmed by gel filtration. We suggest that, in vivo, CS may facilitate the interfibrillar movement that takes place as the cornea grows. We have also found that keratinase, which degrades the very small amount of keratan sulfate present in the primary stroma, has no effect on stromal deposition. (3) There are substantial amounts of unidentified matrix components in primary stromas laid down both in vivo and in vitro. This conclusion was drawn from SEM observations on both types of stroma after they had been freeze-dried, a process which does not condense hydrated macromolecules. Even after being treated with hyaluronidase to remove the CS, substantial amounts of interfibrillar matrix were still present. Until these components are identified and their interactions with collagen are understood, the mechanisms responsible for stromal morphogenesis are unlikely to be understood.

Animals↗
PubMed1988

The extracellular matrix of the developing cornea: diversity, deposition and function.

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Mov 13 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Mov13 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro approximately 3 microns-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibril-diameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.

Animals↗
PubMed1987

The morphogenesis of the chick primary corneal stroma. I. New observations on collagen organization in vivo help explain stromal deposition and growth.

The primary stroma of the avian cornea contains collagen fibrils in orthogonal array. While investigating the processes underlying its morphogenesis, we have found that stromal organization is not as expected in three important respects. First, the fibrils are not uniform: those near the epithelium (newly laid down) have a maximum diameter of about 20 nm (mean: 17.7 nm), while those near the endothelium (laid down for approx. 40 h) have diameters up to 40 nm (mean: 22.8 nm). Fibrils thus grow rapidly to 20 nm and then continue to enlarge slowly, presumably by diffusion of collagen molecules from the epithelium. Second, the collagen, although orthogonally organized, does not contain layers of parallel fibrils. Instead, SEM observation shows that only a few fibrils lie in a parallel array before this short-range order is broken by orthogonal fibrils in the same plane. Furthermore, fibrils in corneas that had been freeze dried but not critical-point dried for SEM were widely spaced and the intervening gaps were filled by an extensive matrix that was probably composed of the proteoglycans known to be in the stroma. Third, we have shown experimentally that the stromal undulations seen in sections are not present in vivo but are shrinkage artifacts: the less corneas were shrunk for SEM preparation, the less pronounced were the stromal undulations. We also noted that, even after the distortions required for the stroma to undulate, the constituent fibrils remained orthogonally organized. These results give insight into the mechanisms underlying stromal morphogenesis and growth. The observations on the growth of collagen fibrils and on collagen organization show that stromal deposition is a more stochastic process than previously thought and, hence, provides support for the view that a complex self-assembly mechanism underlies both fibrillogenesis and the generation of orthogonal organization. The experiments on, and the analysis of, stromal folding show that fibrils slide over one another as undulations form, with the extensive matrix of hydrated proteoglycans being the likely lubricant. This fluidity of the stromal components probably explains how growth can occur without the structure being distorted.

Animals↗
PubMed1987

Corneal morphogenesis in the Mov13 mutant mouse is characterized by normal cellular organization but disordered and thin collagen.

This paper compares corneal development in the normal and in the Mov13 mutant mouse homozygote which does not synthesize type I collagen. During the period 12-14 days of development, there is no obvious difference between cellular organization in the normal and the mutant corneas or, indeed, elsewhere in the eye. In particular, there is normal colonization of the mutant cornea by the mesenchymal cells which will form the endothelium and the fibroblasts. In the early stages of stromal deposition (less than 14 days), when relatively little collagen is normally laid down, mutant and wild-type corneas differ only in that mutant collagen fibrils are less uniform than normal ones. Later development in the Mov13 mutant cannot usually be studied because almost all mutant embryos are dead by 14 days, but we now have two homozygous embryos from a single, 16-day litter. Their stromas obviously differed from those of their normal littermates: there was markedly less collagen in the mutant cornea and the collagen that was deposited lacked orthogonal organization. Fibril morphology also differed: the diameters of fibrils in the normal corneas peaked sharply at about 20 nm, whereas the diameters of mutant fibrils were spread over the range 5-15 nm, with only a small percentage overlapping the normal distribution. These results suggest that type I collagen is of negligible importance in controlling the cellular organization of the cornea, but has a dominant role in the formation of normal 20 nm fibrils and of normal stromal organization. They also show that, as collagen production is markedly lower in the mutant than in the wild-type cornea, the production of other collagens cannot compensate in any way for the lack of type I collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies.

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.

Animals↗
PubMed1985

THe acellular stroma of the chick cornea inhibits melanogenesis of the neural-crest-derived cells that colonize it.

Neural crest (NC) cells from the periorbital mesenchyme (POM) invade the acellular stroma of the chick cornea at stage 27 of development (approximately 6 days). The invading cells become collagen-producing fibroblasts while the NC cells remaining in the POM differentiate into a wide range of cell types, the most easily recognizable of which is the pigment-producing melanocyte. In this paper, we report observations on the differentiation in vitro of cells within and migrating from explants of corneal stroma and compare their behaviour with that of cells within and migrating from explants of the POM. In approximately 70% of cases, POM explants produced black, eumelanin pigmentation within 2-3 days in culture and gave rise to a mixed outgrowth of fibroblasts and melanoblasts that produced brown pigment. In no case, however, did a corneal explant produce black pigment (so demonstrating that any POM contamination was negligible). However, in 28% of cultures from stage-27 and -28 corneas, some of the cells in the outgrowth contained brown pigment indistinguishable from that produced by the POM control, although the majority of the cells in each case were fibroblasts. Two lines of investigation demonstrated that this pigment was melanin: first, transmission electron microscopy showed that the pigment organelles were incompletely melanized, granular melanosomes; second, tests designed to demonstrate the presence of lipofuscin, an alternative pigment, proved negative. Migrating cells from older corneas, in contrast, showed no evidence of even the first stages of melanogenesis. These results show, first, that some of the NC cells that invade the cornea are at least bipotent and hence representative of the POM population rather than being a unique subgroup and, second, that the acellular stroma of the cornea determines the state of differentiation of the NC cells that colonize it. The results thus provide an unequivocal demonstration that extracellular matrix can induce postmigratory NC cells to differentiate into fibroblasts.

Animals↗
PubMed1984

Butterfly wing patterns: how good a determining mechanism is the simple diffusion of a single morphogen?

The formation of the wing pigmentation patterns of three species of butterflies has been modelled using a mechanism based on a tripod of assumptions. First, that there may be morphogen sources in the foci of eyespots and morphogen sinks at some parts of the wing margin, all other cells being passive. Second, that the morphogen has a finite half life and diffuses simply and freely away from the sources throughout a wing of hexagonally packed cells. Third, that the overt pattern derives from cells interpreting the local morphogen concentration with respect to thresholds which determine scale colours. The final pattern thus follows lines of constant morphogen concentration and may, depending on the distribution of sources, comprise rings, curves or bands. With such a model, we have been able to compute stable patterns having the essential topology of the compound spots of Tenaris domitilla, the large rings of Diaethria marchalii and the pattern of eyespots, rings and asymmetric bands of Ragadia minoa. Quantitative analysis of the pattern-forming process shows that, with a biologically realistic diffusion constant (approximately 5.10(-7) cm2 sec-1) and a morphogen half life less than 6h, the patterns form within approximately 12h over a wing of approximately 1000 cells in length. The limitations of the model are that the exact morphology of the eyespots and bands do not match precisely those of the original wings, that there are edge distortions and that optimal patterns may be critically dependent on the exact positions of sources and sinks. An explanation for part of the discrepancy is that we have assumed an adult wing shape and foci coordinates in modelling a process that took place earlier in development. Nevertheless, the limitations of the model argue against a mechanism based on a single morphogen operating in vivo. However, as the model can generate many features of butterfly wing patterns, it may be considered as a degenerate case of that mechanism.

Animals↗
PubMed1983

Analysis of normal and abnormal amniotic fluid cells in vitro by cinemicrography.

Time-lapse cinemicrography has been used to study cell behaviour and movement in long- and short-term amniotic fluid cultures from various types of fetus. These included several with a neural tube defect (9 anencephalics, 5 spina bifidas, 2 encephaloceles), 1 with an abdominal wall lesion (omphalocele) and 9 controls with no open lesion. In short-term (less than 3 days) amniotic fluid cultures from normal fetuses, non-adherent squamous cells dominate, but there is a small population of cells which, in the longer term (approximately 4 weeks), will form colonies of epithelioid and fibroblastic cells. In addition, there is a further group, called 'AF' by Hoehn et al. (1974), which have the ability to form syncitial strands and are often multinucleated; these cells seem to be trophoblastic in origin. In contrast to the controls, short-term anencephalic cultures contained many adherent cells; these were mainly latex-particle-phagocytosing macrophages and neural cells. In long-term cultures, the neural cells dominate and differentiate into a range of recognizable forms whose type and behaviour seem to depend on cell density, time in culture and extent of cell-cell contacts. In the spina bifida and encephalocele cultures, there were initially far fewer adherent cells than in the anencephalic ones, but, after approximately 2 weeks in vitro, a range of neural cell types could be recognized in addition to those seen in the controls. In the context of prenatal diagnosis, the presence of neural cells certainly indicates that the fetus has a NTD, but the difficulty of standardizing culture conditions implies that any differential diagnosis on the basis or morphology and movement will be difficult. If, as seems likely, the AF cells of Hoehn et al. (1974) are indeed trophoblastic, amniotic fluids from the second trimester may provide a useful source of these cells.

Amniotic Fluid↗
PubMed1983

Partial retinal dysplasia and subsequent degeneration in a mutant strain of domestic fowl (rdd).

An inherited recessive form of retinopathy has been discovered in the domestic fowl (rdd) which is characterized by progressive deterioration of the retina, culminating in blindness by sexual maturity. Morphologically, the condition is recognizable by abnormalities in both the retinal pigment epithelium and the neural retina. Gaps in the pigment epithelium which are first detected macroscopically at nine days of incubation become larger and more numerous until the time of hatching, then disappear during the subsequent week. Undulations in the outer nuclear, outer plexiform, and inner nuclear layers are obvious by 11 days of incubation. There is a marked reduction of photoreceptors at 18 days of incubation as compared to normal controls. After hatching, the thickness of the retina decreases with age, primarily due to cell loss from the photoreceptor region and inner nuclear layer. Detachment of the atrophic retinas generally occurs in adults, and is followed in some adults by granulation and ossification of the vitreous. Problems concerning the site of the lesion are discussed.

Aging↗
PubMed1983

Morphology of hyaluronidase-sensitive cell coats as seen in the SEM after freeze-drying.

Many adherent cells in vitro are surrounded by a transparent exclusion zone or halo, several micrometers thick, which red blood cells, bacteria and carbon particles cannot penetrate. This halo is rapidly and specifically removed by hyaluronidase and its high degree of hydration is demonstrated by the fact that, although fixation does not eliminate the halo, solvent dehydration does. This latter observation means that the halo cannot be visualized by conventional electron microscopic techniques. We report here that the exclusion-zone material can, however, be seen in the scanning electron microscope if cells are fixed and frozen rapidly and then freeze-dried. Many cells in cultures from a murine fibrosarcoma or from human embryonic lung treated in this way appear to be covered by a matrix that obscures the microvilli that are visible on critical-point-dried or hyaluronidase-treated, freeze-dried cells. Only where the coat is, for some reason, missing can microvilli be seen on freeze-dried cells. The coat structure varies from amorphous to an assembly of fine fibres approximately 100 nm in diameter and its appearance is very similar to that of small drops of hyaluronic acid (10(-5) micrograms ml-1) treated in the same way. Halo material is fragile and detaches itself from the cell surface within an hour of fixation. These observations suggest that the halo phenomenon reflects only the production of extracellular matrix and its turnover. The fragility of the haloes implies that, if they do exist in vivo, they are unlikely to play any structural role. The results suggest that the technique will yield information on other highly hydrated, unstable structures.

Animals↗
PubMed1979

Hyaluronidase-sensitive halos around adherent cells. Their role in blocking lymphocyte-mediated cytolysis.

A variety of adherent sarcoma, carcinoma and normal cells are surrounded in vitro by thick, transparent zones (approximately equal to 9 micron thick) that spleen cells and a variety of other cells and particles cannot penetrate. Seven lymphoblastoid cell lines did not possess such halos. The presence of these halos around adherent fibrosarcoma cells appeared to protect them from lymphocyte-mediated cytolysis. Hyaluronidase treatment, which destroyed the halo and allowed lymphocytes to approach the tumor cell membrane, enhanced the cytotoxic action of immune but not of normal spleen cells. These observations, in addition to highlighting a little-known feature of the cell surface, may also be of general relevance to the in vitro and in vivo killing of tumor cells by immune effector cells.

Animals↗