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S E Fraser

Publications and source records attributed to S E Fraser.

At least 55 records · Page 3Linked to original sources
PubMed1996

Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.

We are developing a multielectrode silicon "neuroprobe" for maintaining a long-term, specific, two-way electrical interface with nervous tissue. Our approach involves trapping a neuron (from an embryonic rat hippocampus) in a small well with a stimulation/recording electrode at its base. The well is covered with a grillwork through which the neuron's processes are allowed to grow, making synaptic contact with the host tissue, in our case a cultured slice from a rat hippocampus. Each neuroprobe can accommodate 15 neurons, one per well. As a first step in studying neurite outgrowth from the neuroprobe, it was necessary to develop new staining techniques so that neurites from the probe neurons can be distinguished from those belonging to the host, without interference from non-specific background staining. We virtually eliminated background staining through a number of innovations involving dye solubility, cell washing, and debris removal. We also reduced photobleaching and phototoxicity, and enhanced imaging depth by using a 2-photon laser-scanning microscope. We focused on using the popular membrane dye, DiI, however a number of other membrane dyes were shown to provide clear images of neural processes using pulsed illumination at 900 nm. These techniques will be useful to others wishing to follow over time the growth of neurons in culture of after transplantation in vivo, in a non-destructive way.

Animals↗
PubMed1995

Effects of brain-derived neurotrophic factor on optic axon branching and remodelling in vivo.

Neurotrophins are thought to be important for the survival and differentiation of vertebrate neurons. Roles have been suggested for target-derived neurotrophins, based both on their expression in target tissues at the time of neuron innervation, and on their effects on axonal sprouting. However, direct in vivo evidence of their involvement in axon arborization has remained elusive. We have used in vivo microscopy to follow individual optic axons over time, and have examined the role of the neurotrophin brain-derived neurotrophic factor (BDNF) in their development. Here we show that injection of BDNF into the optic tectum of live Xenopus laevis tadpoles increased the branching and complexity of optic axon terminal arbors. In contrast, injection of specific neutralizing antibodies to BDNF reduced axon arborization and complexity. The onset of these effects was rapid (within 2 hours) and persisted throughout the 24-hour observation period. Other neurotrophins had little or no significant effects. These results demonstrate the involvement of neurotrophins in the dynamic elaboration of axon terminals, and suggest a direct role for target-derived BDNF during synaptic patterning in the developing central nervous system.

Animals↗
PubMed1995

Fate maps of the zebrafish embryo.

In the past few years, we have seen a surge of interest in the zebrafish as a model system for the study of embryonic induction and patterning. This review summarizes our current knowledge of the organization of zebrafish fate maps during early development. Recent advances have addressed the relationship between early cleavage planes and the future dorsal axis, the pattern of cell mixing during blastula and gastrula stages, and the morphogenesis of the trunk neural keel. In addition, refined fate maps have become available for the embryonic shield, the central nervous system, and the heart. In combination with recent advances in molecular and genetic manipulations, these fate maps set the stage for new, more incisive, experimental approaches.

Animals↗
PubMed1995

Receptor-targeted co-transport of DNA and magnetic resonance contrast agents.

BACKGROUND: Ligand molecules conjugated to polylysine can be electrostatically bound to DNA and can bind receptors or antigens on the surface of cells, delivering the DNA into specific cells and tissues. Several researchers have used this approach to generate non-viral vehicles for the efficient delivery of DNA to specific cells. We have attempted to adopt this general approach to the cell-specific delivery of magnetic contrast agents for use in magnetic resonance imaging (MRI). RESULTS: We have synthesized a new class of agents capable of both transfecting genes into cells and enhancing the contrast of the targeted cells for MRI. DNA is used both to encode a marker gene and as a molecular scaffold, which electrostatically binds polylysine conjugated to transferrin, an iron uptake protein, and polylysine modified with gadolinium chelated to diethylenetriaminepetaacetic acid. When cells displaying the transferrin receptor are treated with these particles, high levels of gene expression are observed, higher than with control particles composed only of transferrin, polylysine and DNA. The treated cells show specific MRI contrast enhancement, which did not require expression of the marker gene. CONCLUSIONS: The development of this class of particles permits the use of novel protocols by which genes for genetic therapy and agents for MRI contrast are co-transported. These protocols may allow non-invasive MRI monitoring of DNA delivery for gene therapy in real time.

Cells, Cultured↗
PubMed1995

Segmental migration of trunk neural crest: time-lapse analysis reveals a role for PNA-binding molecules.

Trunk neural crest cells migrate through the somites in a striking segmental fashion, entering the rostral but not caudal sclerotome, via cues intrinsic to the somites. Attempts to define the molecular bases of these cues have been hampered by the lack of an accessible assay system. To examine trunk neural crest migration over time and to perturb candidate guiding molecules, we have developed a novel explant preparation. Here, we demonstrate that trunk regions of the chicken embryo, placed in explant culture, continue to develop apparently normally for 2 days. Neural crest cells, recognized by prelabeling with DiI or by poststaining with the HNK-1 antibody, migrate in the somites of the explants in their typical segmental pattern. Furthermore, this paradigm allows us to follow trunk neural crest migration in situ for the first time using low-light-level videomicroscopy. The trajectories of individual neural crest cells were often complex, with cells migrating in an episodic mode encompassing forward, backward and lateral movements. Frequently, neural crest cells migrated in close-knit groups of 2-4 cells, moving at mean rates of migration of 10-14 microns/hour. Treatment of the explants with the lectin peanut agglutinin (PNA) both slowed the rate and altered the pattern of neural crest migration. Neural crest cells entered both the rostral and caudal halves of the sclerotome with mean rates of migration ranging from 6 to 13 microns/hour. These results suggest that peanut agglutinin-binding molecules are required for the segmental patterning of trunk neural crest migration. Because this approach permits neural crest migration to be both observed and perturbed, it offers the promise of more direct assays of the factors that influence neural crest development.

Animals↗
PubMed1995

Dynamics of thin filopodia during sea urchin gastrulation.

At gastrulation in the sea urchin embryo, a dramatic rearrangement of cells establishes the three germ layers of the organism. Experiments have revealed a number of cell interactions at this stage that transfer patterning information from cell to cell. Of particular significance, primary mesenchyme cells, which are responsible for production of the embryonic skeleton, have been shown to obtain extensive positional information from the embryonic ectoderm. In the present study, high resolution Nomarski imaging reveals the presence of very thin filopodia (02-0.4 micron in diameter) extending from primary mesenchyme cells as well as from ectodermal and secondary mesenchyme cells. These thin filopodia sometimes extend to more than 80 microns in length and show average growth and retraction rates of nearly 10 microns/minute. The filopodia are highly dynamic, rapidly changing from extension to resorption; frequently, the resorption changes to resumption of assembly. The behavior, location and timing of active thin filopodial movements does not correlate with cell locomotion; instead, there is a strong correlation suggesting their involvement in cell-cell interactions associated with signaling and patterning at gastrulation. Nickel-treatment, which is known to create a patterning defect in skeletogenesis due to alterations in the ectoderm, alters the normal position-dependent differences in the thin filopodia. The effect is present in recombinant embryos in which the ectoderm alone was treated with nickel, and is absent in recombinant embryos in which only the primary mesenchyme cells were treated, suggesting that the filopodial length is substratum dependent rather than being primary mesenchyme cell autonomous. The thin filopodia provide a means by which cells can contact others several cell diameters away, suggesting that some of the signaling previously thought to be mediated by diffusible signals may instead by the result of direct receptor-ligand interactions between cell membranes.

Animals↗
PubMed1995

Order and coherence in the fate map of the zebrafish nervous system.

The zebrafish is an excellent vertebrate model for the study of the cellular interactions underlying the patterning and the morphogenesis of the nervous system. Here, we report regional fate maps of the zebrafish anterior nervous system at two key stages of neural development: the beginning (6 hours) and the end (10 hours) of gastrulation. Early in gastrulation, we find that the presumptive neurectoderm displays a predictable organization that reflects the future anteroposterior and dorsoventral order of the central nervous system. The precursors of the major brain subdivisions (forebrain, midbrain, hindbrain, neural retina) occupy discernible, though overlapping, domains within the dorsal blastoderm at 6 hours. As gastrulation proceeds, these domains are rearranged such that the basic order of the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors is refined and becomes aligned with the primary axes of the embryo. Time-lapse video microscopy shows that the rearrangement of blastoderm cells during gastrulation is highly ordered. Cells near the dorsal midline at 6 hours, primarily forebrain progenitors, display anterior-directed migration. Cells more laterally positioned, corresponding to midbrain and hindbrain progenitors, converge at the midline prior to anteriorward migration. These results demonstrate a predictable order in the presumptive neurectoderm, suggesting that patterning interactions may be well underway by early gastrulation. The fate maps provide the basis for further analyses of the specification, induction and patterning of the anterior nervous system, as well as for the interpretation of mutant phenotypes and gene-expression patterns.

Animals↗
PubMed1995

Distribution of tissue progenitors within the shield region of the zebrafish gastrula.

The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish.

Animals↗
PubMed1994

A dual embryonic origin for vertebrate mechanoreceptors.

Neuromasts, the mechanoreceptors of the lateral line system of fishes and aquatic amphibians, have previously been thought to develop exclusively from embryonic epidermal placodes. Use of fate mapping techniques shows that neuromasts of the head and body of zebrafish, Siamese fighting fish, and Xenopus are also derived from neural crest. Neural crest migrates away from the neural tube in developing vertebrates to form much of the peripheral nervous system, pigment cells, and skeletal elements of the head. The data presented here demonstrate that neuromasts are derived from both neural crest and epidermal placodes.

Animals↗
PubMed1994

Magnetic resonance microscopy of embryonic cell lineages and movements.

Key events in vertebrate embryogenesis are difficult to observe in many species. High-resolution magnetic resonance imaging was used to follow cell movements and lineages in developing frog embryos. A single cell was injected at the 16-cell stage with a contrast agent, based on the gadolinium chelate gadolinium-diethylenetriamine pentaacetic acid-dextran. The labeled progeny cells could be followed uniquely in three-dimensional magnetic resonance images, acquired from the embryo over several days. The results show that external ectodermal and internal mesodermal tissues extend at different rates during amphibian gastrulation and neurulation.

Animals↗
PubMed1994

Mouse lemur microscopic MRI brain atlas.

We present a three-dimensional (3D) digital atlas of a mouse lemur head at 60-micron cubic voxel isotropic resolution. It was constructed from a 3D proton magnetic resonance image (MRI) acquired at 500 MHz. It shows views of 3D volume-rendering and movies of contiguous 2D plane cuts in three orthogonal directions. This MR data set was acquired using a spin-echo pulse sequence under conditions of strong T2 and significant diffusion weighting. Experimental parameters were optimized to provide strong intrinsic contrast between gray and white matter. Familiar anatomical structures are clearly identifiable. Fine fiber tracts, laminations of cortices, details of the inner ears, and layering in the lateral geniculate nuclei are all visible. Anatomical identifications of structures in representative slices selected from the atlas are presented. We also describe some difficulties and trade-offs encountered in microscopic resolution MRI. We note that this type of atlas does not suffer from the spatial distortions and slice registration problems introduced by standard histological techniques. Future in vivo longitudinal studies will provide atlases describing in detail the development of the primate brain.

Animals↗
PubMed1994

Imaging neuronal development with magnetic resonance imaging (NMR) microscopy.

An ideal technique for following the development of the vertebrate nervous system would allow cells to be followed at the resolution of light microscopy at depths of several millimeters into the tissue. This would permit critical events to be followed at cellular or sub-cellular resolution even deep within the developing organism. To date, no technique has emerged with all of the needed properties. Light microscopy can follow a cell and its descendants after they have been labeled by either the infection of embryonic cells with a recombinant retrovirus or the microinjection of individual precursor cells with enzymes or fluorescent dyes. However, light microscopy cannot image events deeper than a few hundred micrometers within an embryo due to light scattering and aberrations in the objective lenses and other optics. Magnetic resonance imaging (MRI) does not suffer from these limitations, routinely being used to image in 3 dimensions through specimens as large as adult humans. However, it is relatively slow and, as implemented to date, it cannot routinely achieve cellular resolution. Here, we present our attempts to meet the technical challenges posed by in vivo MRI microscopy. As an example of both the progress and the future challenges, we present images of cells within the developing frog embryo over a several day time course.

Animals↗
PubMed1994

BDNF in the development of the visual system of Xenopus.

To explore the role of BDNF during Xenopus visual system development, the expression of BDNF and TrkB, as well as the effect of BDNF during retinal ganglion cell (RGC) development in culture, was examined. BDNF mRNA was found to be expressed in both the developing eye and tectum, peaking at stage 45/46. The expression of BDNF coincided with RGC expression of full-length trkB transcripts, suggesting a functional role for BDNF. In culture, BDNF significantly increased the number of RGCs. The ability of BDNF to rescue differentiated RGCs that had projected to the tectum, the time course of the effect, and the lack of mitogenic response indicate that this neurotrophin promotes survival. The expression of BDNF and TrkB and the responsiveness of RGCs to BDNF coincide with retinal axon terminal arborization and patterning. Our results indicate that BDNF is a relevant neurotrophin for Xenopus RGC development and suggest that it plays a role during visual system patterning.

Amino Acid Sequence↗
PubMed1994

Rapid remodeling of retinal arbors in the tectum with and without blockade of synaptic transmission.

Dynamic rearrangements of axon terminal arbors may be critical for establishing appropriate connections in the developing nervous system. Here, the changes in complex retinal axon arbors in the tecta of live Xenopus larvae were followed during the formation of the topographic retinotectal projection. Three-dimensional reconstructions of terminal arbors made with a confocal microscope at hourly intervals revealed rapid remodeling of arbor extensions. Shorter branches were extended and retracted very rapidly, suggesting that they probe the environment for the optimal sites to form stable branches. About 27% of longer branches were present throughout the entire observation period and may be sites of stabilized synaptic contacts. Treatment of the animals to block postsynaptic activity resulted in increased rates of arbor rearrangements, which may coincide with decreased synapse stability. These studies reveal the dynamic behavior of nerve arbors and provide estimates for the lifetimes of retinotectal branches.

Animals↗
PubMed1994

Violation of cell lineage restriction compartments in the chick hindbrain.

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development.

Animals↗
PubMed1994

Developmental potential of trunk neural crest cells in the mouse.

The availability of naturally occurring and engineered mutations in mice which affect the neural crest makes the mouse embryo an important experimental system for studying neural crest cell differentiation. Here, we determine the normal developmental potential of neural crest cells by performing in situ cell lineage analysis in the mouse by microinjecting lysinated rhodamine dextran (LRD) into individual dorsal neural tube cells in the trunk. Labeled progeny derived from single cells were found in the neural tube, dorsal root ganglia, sympathoadrenal derivatives, presumptive Schwann cells and/or pigment cells. Most embryos contained labeled cells both in the neural tube and at least one neural crest derivative, and numerous clones contributed to multiple neural crest derivatives. The time of injection influenced the derivatives populated by the labeled cells. Injections at early stages of migration yielded labeled progeny in both dorsal and ventral neural crest derivatives, whereas those performed at later stages had labeled cells only in more dorsal neural crest derivatives, such as dorsal root ganglion and presumptive pigment cells. The results suggest that in the mouse embryo: (1) there is a common precursor for neural crest and neural tube cells; (2) some neural crest cells are multipotent; and (3) the timing of emigration influences the range of possible neural crest derivatives.

Animals↗
PubMed1994

Structure and expression of the nicotinic acetylcholine receptor beta subunit of Xenopus laevis.

A cDNA encoding the beta subunit of the Xenopus muscle nicotinic acetylcholine receptor (AChR) was cloned from an embryonic Xenopus cDNA library. The predicted mature polypeptide has 469 amino acids and four membrane spanning regions corresponding to the M1-M4 regions identified in other AChR subunit clones. The polypeptide bears greater homology to beta subunits of Torpedo and mouse than to alpha, gamma or delta subunits of Xenopus. The earliest beta subunit transcripts were detected by RNase protection assays at the neural plate stage of development (stage 14) and the level of transcripts, as a fraction of total RNA, continued to increase through the age of hatching (stages 34-36). Co-injection of Xenopus alpha, beta, gamma and delta cRNAs into Xenopus oocytes led to expression of functional AChRs. Micromolar concentrations of ACh activated depolarizing AChR currents which reversed at -5 mV and were blocked by alpha bungarotoxin. Injection of alpha, gamma and delta subunits alone did not yield detectable ACh responses. With the cloning of the Xenopus beta subunit, structure/function relations of AChRs can now be studied using receptors composed entirely of Xenopus subunits.

Amino Acid Sequence↗