Biomedical subjects

D L Becker

Publications and source records attributed to D L Becker.

31 records · Page 2Linked to original sources

PubMed1996

Expression of intercellular junctions during preimplantation development of the human embryo.

A total of 74 human embryos were stained with gap junction protein specific anti-peptide antibodies an antibodies to the desmosomal protein desmoplakin to reveal the expression pattern of intercellular junctions during preimplantation development. Prior to implantation, the human embryo expresses predominantly connexin (Cx43)-containing gap junctions. Gap junctions were first detected in apposing cell membranes at the 4-cell stage and became increasingly organized as development proceeded. In normal blastocysts, trophectoderm (TE) cells were linked by dense arrays of gap junctions while inner cell mass (ICM) cells were linked by small, punctate gap junctions. Gap junctions containing Cx32 or Cx26 were observed occasionally in the TE of late blastocysts. Desmosomes appeared between outer cells prior to cavitation and were retained in the TE, but not in the ICM. Levels of gap junction protein expression were variable in morphologically normal embryos at the same stage, suggesting that a normal appearance may not be a reliable indicator of future viability. Morphologically normal embryos often possessed multinucleate, apoptotic and decompacting cells. They could show either extensive, disorganized over-expression or reduced expression of gap junction protein. The results fit the view that only embryos destined to survive display an organized pattern of intercellular junctions.

Blastocyst↗

PubMed1995

Role of gap junctions in the development of the preimplantation mouse embryo.

We have taken several approaches to study the role of gap junctional communication during preimplantation mouse development. Firstly, the normal expression pattern of gap junctions has been characterized using immunostaining in conjunction with laser scanning confocal microscopy. Changes in junctional distribution have been correlated with developmental events. We have gone on to study development and junctional organization in mice which naturally exhibit reduced cell to cell communication (DDK syndrome), and in normal mice in which gap junction permeability has been artificially manipulated. Furthermore, anti-peptide antibodies have been tested for their ability to block gap junction communication and for the effects of such a block on subsequent development. Collectively, the results demonstrate that gap junctional communication plays an important role in the maintenance of compaction and the differentiation of an organized epithelium within an embryo, features which are vital for preimplantation development to progress successfully.

Animals↗

PubMed1995

Gap junctions in the vertebrate retina.

The vertebrate retina is a highly laminated assemblage of specialized neuronal types, many of which are coupled by gap junctions. With one interesting exception, gap junctions are not directly responsible for the 'vertical' transmission of visual information from photoreceptors through bipolar and ganglion cells to the brain. Instead, they mediate 'lateral' connections, coupling neurons of a single type or subtype into an extended, regular array or mosaic in the plane of the retina. Such mosaics have been studied by several microscopic techniques, but new evidence for their coupled nature has recently been obtained by intracellular injection of biotinylated tracers, which can pass through gap junctional assemblies that do not pass Lucifer Yellow. This evidence adds momentum to an existing paradigm shift towards a population-based view of the retina, which can now be envisaged both as an array of semi-autonomous vertical processing modules, each extending right through the retina, and as a multi-layered stack of interacting planar mosaics, bearing some resemblance to a set of interleaved neural networks. Junctional conductance across mosaics of horizontal cells is known to be controlled dynamically with a circadian rhythm, and other dynamically-regulated conductance changes are also likely to make important contributions to signal processing. The retina is an excellent system in which to study such changes because many aspects of its structure and function are already well understood. In this review, we summarize the microscopic appearance, coupling properties and functions of gap junctions for each cell type of the neural retina, the regulatory properties that could be provided by selective expression of different connexin proteins, and the evidence for gap junctional coupling in retina development.

Animals↗

PubMed1995

Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions.

Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding how this variation contributes to the control of intercellular signalling, both in the adult and during embryonic development, is an important problem that would be aided by reagents that interfere with gap junctional communication through specific connexins. We have begun to address this issue by raising antibodies to peptides derived from connexin43 and connexin32. Connexin43 peptides were located in the amino terminus, cytoplasmic loop and carboxytail. Connexin32 peptides came from the cytoplasmic loop and the first extracellular loop. Immunoblotting and immunostaining properties of purified IgGs were characterized on mouse heart, liver and the 8- to 16-cell mouse embryo. Effects on transfer through gap junctions were assessed in the fully compacted 8-cell mouse embryo by co-injection with Lucifer Yellow or Cascade Blue. Embryos were maintained in culture to assess the developmental consequences of injection. Peptide competition was used to confirm the specificity of immunostaining and inhibition of dye transfer. All connexin specific antibodies recognized their parent connexin on immunoblots and showed no 43/32 cross-reactivity. The connexin32 extracellular loop antibody recognized both connexin 32 and 43 on immunoblots, as predicted by the amino acid sequence homology in this region, but did not immunostain intact gap junctions. Connexin specific antibodies that immuno-stained showed the predicted connexin specificity. Antibodies to either connexin43 amino acids (AA) 1-16 (amino terminus) or AA 101-112 (cytoplasmic loop) neither immunostained nor prevented functional communication through 8-cell embryo gap junctions. Antibodies to AA 123-136 and AA 131-142 in the cytoplasmic loop immunostained heart and 8-cell embryo gap junctions and blocked transfer through them with high efficiency. Fab' fragments were equally effective. Peptide competition showed that both antibodies contained epitopes within AA 131-136 of connexin43. Antibodies against AA 313-324 in the carboxytail immunostained heart and the 8-cell embryo and, as IgGs, prevented dye transfer. Fab' fragments were ineffective. All connexin43 antibodies that blocked gap junctional communication between cells of the 8-cell mouse embryo induced non-communicating cells subsequently to withdraw from compaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Early postnatal changes in the somatodendritic morphology of ankle flexor motoneurons in the rat.

The development of locomotor function in the rat spans the first 3 postnatal weeks. We have studied morphological features of the soma and dendrites of motoneurons innervating the physiological flexor muscles of the ankle, tibialis anterior and extensor digitorum longus, by intracellular injection in vitro between the first and ninth postnatal days. We obtained serial optical sections of 96 adequately filled motoneurons in whole-mounted hemisected spinal cords by confocal microscopy, projected them onto a single plane and analysed them morphometrically. On the day after birth, the somatodendritic surfaces of most such motoneurons were covered in growth-associated spiny, thorny or hair-like appendages. These had disappeared from the soma by the fourth postnatal day and from most proximal dendrites by day 7, but were still common distally on day 9. During this period there was little or no net growth of either the soma (which was still much smaller than in the adult) or the dendritic tree. A dorsal dendritic bias was present and 'sprays' of long, loosely bundled dorsal dendrites were often seen. The mean number of primary dendrites remained constant at about eight, and their combined diameter was already significantly correlated with mean soma diameter, as in the adult cat. Thus, the critical neonatal period during which these ankle flexor motoneurons are known to change their electrophysiological properties and to be particularly sensitive to interference with neuromuscular interaction is characterized by major changes in the neuronal surface, presumably linked to synaptogenesis.

Animals↗

PubMed1992

Independent mosaics of large inner- and outer-stratified ganglion cells in the goldfish retina.

Goldfish retinal ganglion cells were filled with horseradish peroxidase and studied in flatmounts. Two regular mosaics of large neurons with many of the properties of mammalian alpha ganglion cells were found, differing from each other in spacing, size, and dendritic stratification. The existence of biplexiform ganglion cells with additional dendrites in the outer plexiform layer was also confirmed. One of the two alpha-like mosaics consisted of giant ganglion cells with thick primary dendrites and large, sparsely branched dendritic trees in the outer sublamina of the inner plexiform layer (IPL). In fish 55-65 mm long, about 300 formed a tessellated array across each retina. Their somata (mean area 277 +/- 6 microns 2) were displaced to varying degrees into the IPL, neighbours in the mosaic often occupying different levels. Their dendrites ramified in one stratum near the inner nuclear layer, at a mean depth of 70.8 +/- 0.5% of the IPL. The other alpha-like mosaic comprised about 900 large ganglion cells, with slightly smaller somata (mean area 193 +/- 4 microns 2) in the ganglion cell layer. Most of their dendrites lay in a narrow stratum at 41.9 +/- 0.5% of the depth of the IPL. However, deviations (usually into more vitread strata) were common, which was not true for similar cells in the distantly related cichlid fish Oreochromis. Measurements of nearest neighbour distance (NND) for 4 outer and 4 inner mosaics showed that they were at least as regular as the alpha cell mosaics of mammals: the ratio of the mean NND to the standard deviation ranged from 4.03 for the least regular outer mosaic to 6.47 for the most regular inner mosaic. The wide phylogenetic distribution of these paired, regular mosaics points to a fundamental role in vision. The presence of some variability in dendritic stratification even within the exceptionally regular inner-stratified mosaic suggests that classifications based entirely on the detailed morphology of individual neurons may not always correlate well with their primary functional roles. Where possible, neuronal morphology and spatial distribution should be studied together.

Animals↗

PubMed1992

The relationship of gap junctions and compaction in the preimplantation mouse embryo.

In the mouse embryo, gap junctions first appear at the 8-cell stage as compaction is about to take place. Compaction of the embryo is important for the differentiation of the first two cell types; the inner cell mass and the trophectoderm. Our studies examine the contribution of gap junctional communication at this stage of development. We have characterised the normal sequence of appearance of gap junction protein and its distribution. The extent of communication as shown by the passage of dye between cells has been recorded in both normal embryos and embryos treated with drugs that influence gap junctional communication. Comparisons have been made with embryos that express a lethal gap junction defect and attempts were made to rescue such embryos by increasing their gap junction communication.

Animals↗

PubMed1991

Regular mosaics of large displaced and non-displaced ganglion cells in the retina of a cichlid fish.

Large retinal ganglion cells in the tilapid cichlid fish Oreochromis spilurus (standard length 15-54 mm) were filled with horseradish peroxidase and studied in flatmounts. Three types, with distinct patterns of dendritic stratification, formed spatially independent, nonrandom mosaics. One type (about 0.3% of all ganglion cells) resembled the outer (off) alpha cells of mammals. They were very large, with thick primary dendrites and large, sparsely branched planar trees in the outer part of the inner plexiform layer (IPL). About 300 were arrayed regularly across each retina, their exact number and spacing depending on its size. Their somata were often displaced into the IPL, even where neighbours in the mosaic were orthotopic. Another type (0.8%) resembled the inner (on) alpha cells of mammals. These had slightly smaller somata that were never displaced and smaller trees in the middle layers of the IPL. About 800 were arrayed uniformly and regularly across each retina. A rarer type (0.06-0.08%) had two planar trees: one forming a coarse mosaic in the outer part of the inner plexiform layer (co-planar with the trees of outer alpha-like cells) and another in the outer plexiform layer. These "biplexiform" cells were smaller and rounder than alpha-like cells and always displaced. The dendrites were finer and less tapered. Cells in which we could identify an outer plexiform tree failed to cover the retina completely, but were nonrandomly distributed. We draw three main conclusions: (1) some nonmammalian vertebrates have separate inner and outer mosaics of large ganglion cells like those of mammals, (2) the vertical displacement of ganglion cell somata can vary widely within a single mosaic and may thus be functionally irrelevant, and (3) biplexiform ganglion cells exist in fish but differ in morphology from the biplexiform types described in some other vertebrates.

Animals↗

PubMed1991

Enhancing the laser scanning confocal microscopic visualization of Lucifer yellow filled cells in whole-mounted tissue.

The laser scanning confocal microscope (LSCM) is an extremely useful tool that allows fluorescently labelled cells to be visualized in whole-mount preparations. This is particularly advantageous, for example, in studying the dendritic trees of neurons with respect to their environment. One of the most popular, and easiest, ways to visualize a cell is to inject it intracellularly with the fluorophore Lucifer Yellow (LY). However, the argon gas lasers of most LSCM's are not well matched to the excitation spectrum of aqueous LY. When this largely inappropriate excitation is combined with standard filters, designed for fluorescein fluorescence rather than Lucifer Yellow, the resulting image is poor. We report that clearing LY-injected neurons in methyl salicylate and mounting them in Entellan, a non-aqueous medium of high refractive index, enhances their visualization on a Bio-Rad LSCM with standard fluorescein (FITC) filters to an unexpected degree. This technique also leads to a substantial reduction in photobleaching.

Animals↗

PubMed1990

Changes in goldfish retinal ganglion cells during axonal regeneration.

Recent work suggests that mammalian retinal ganglion cells may become more like developing ganglion cells in form while regenerating through a peripheral nerve graft. We have injected Lucifer Yellow into regenerating ganglion cells of goldfish to look for similar changes. Within three weeks of injury, we saw dye-coupling to nearby cells, which is a common developmental feature in many species. Dendrites and axons, which in most mature ganglion cells are smooth, became varicose and hairy, like those examined in mammalian development. Secondary axons arose later, not only as side-branches of the primary axon but also from the soma, as in mammalian development and regeneration. Since, in fish, these responses are clearly an intrinsic part of functional regeneration, their equivalence in fish and mammals strengthens the view that a similar regenerative competence may exist in the retinal ganglion cells of all vertebrates.

Amidines↗

PubMed1988

Divergent axon collaterals in the regenerating goldfish optic tract: a fluorescence double-label study.

In the normal goldfish, optic axons are distributed between the two arms (brachia) of each optic tract, in such a way that each axon enters the tectum close to its retinotopic termination site. We have shown previously that regenerating axons at first express little or no preference for their normal brachium. Later, however, a partial refinement of the brachial pathway takes place, implying that some axons must have sent out divergent collateral branches and then eliminated the least appropriate. We have now studied the formation and subsequent loss of axon collaterals in regeneration using retrogradely transported fluorescent dyes. We labelled the axons in the medial brachium with Fast Blue and those in the lateral brachium with Diamidino Yellow in a way that avoided cross-contamination. In normal fish, yellow-labelled ganglion cells dominated the dorsal retina and blue-labelled ganglion cells the ventral, with only a narrow zone of overlap. Double-labelled cells were not found. In fish labelled early in regeneration, however, both dyes were spread over the entire retina in single- and double-labelled ganglion cells. As regeneration progressed, each dye again came to dominate its appropriate retinal region; but much less strongly, confirming previous results. At the same time, double-labelled cells became harder to find. From 60 days after nerve cut onwards they were rare, and largely confined to the boundary zone between dorsal and ventral retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Retinotopic refinement of the regenerating goldfish optic tract is not linked to activity-dependent refinement of the retinotectal map.

The cut optic nerve of a goldfish can regenerate, restoring an orderly projection from the retina to the optic tectum. At first, regenerating axons make transient connections, many of them in inappropriate tectal locations. Later, their arrangement is gradually refined into an accurate retinotectal map by a process that depends on afferent activity. On their way to the tectum, many regenerating axons make erroneous choices between the two arms (brachia) of the optic tract. However, since they commonly possess divergent collateral branches, a secondary refinement of the brachial pattern can occur by selective collateral elimination. How or why a particular collateral is lost is not known, but we have previously suggested that sibling branches might compete to form stable tectal synapses, implying that there might be a causal link between refinement of the brachial pattern and refinement of the retinotectal map. In this paper, we have tested directly for such a link, blocking map refinement with tetrodotoxin (TTX) or stroboscopic light, verifying the effectiveness of the block and measuring the extent of brachial refinement by standard methods in experimental and control fish. Both TTX and stroboscopic light reliably prevented map refinement, their results being indistinguishable. However, neither had even the slightest detrimental effect on brachial refinement, either 42 days or 70 days after nerve cut. Evidently, neither activity nor a sharp retinotectal projection is necessary for brachial refinement. Theory and experiment both dictate that the basic projection pattern be controlled by a mechanism (such as chemoaffinity) that is independent of activity, and it would seem that selective collateral loss must depend on the same mechanism.

Animals↗

PubMedDate not supplied

Large retinal ganglion cells that form independent, regular mosaics in the bufonoid frogs Bufo marinus and Litoria moorei.

Population-based methods were used to study labeled retinal ganglion cells from the cane toad Bufo marinus and the treefrog Litoria moorei, two visually competent bufonoid neobatrachians with contrasting habitats. In both, cells with large somata and thick dendrites formed distinct types with independent mosaics. The alpha(a), alpha(ab), and alpha(c) mosaics of Bufo in all major respects resembled those of ranids, studied previously, and could be provisionally matched to the same functional classes. As in other frogs, some alpha(a) cells were displaced and many alpha-cells of all types were asymmetric, but within each type all variants belonged to one mosaic. Nearest-neighbor analyses and spatial correlograms confirmed that all three mosaics were regular and independent. In Litoria, monostratified alpha(a) cells were not found. Instead, two bistratified types were present, distinguished individually by soma size and dendritic caliber and collectively by membership of independent mosaics: the larger (approximately 0.8% of all ganglion cells) was termed alpha1(ab) and the smaller (approximately 2.2%) alpha2ab. An alpha(c) cell type was also present, although too inconstantly labeled for mosaic analysis. Nearest-neighbor analyses and spatial correlograms confirmed that the two alpha(ab) mosaics were regular and independent. Densities, proportions, soma sizes, and mosaic statistics are tabulated for each species. The emergence of a consensus pattern of alpha-cell types in fishes and frogs, from which this treefrog partly diverges, offers new possibilities for studying correlations between function, phylogeny, ecology, and neuronal form.

Animals↗