Biomedical subjects

S E Fraser

Publications and source records attributed to S E Fraser.

At least 73 records · Page 4Linked to original sources

PubMed1993

Proportion of proliferative cells in the tadpole retina is increased after embryonic lesion.

Little is known about the cellular mechanisms that cause some cells to stop dividing while leaving neighboring cells free to continue dividing. Such events occur during development of the Xenopus retina; all cells of the embryonic eyebud are mitotic, but by stage 37 (St 37), only cells at the ciliary margin continue to proliferate as neighboring cells become post-mitotic. The mechanisms that control these different proliferative fates remain unknown. One possibility is that total cell number regulates the initial number of proliferative cells at the ciliary margin. To test this hypothesis, we reduced the cell number by surgically removing a portion of the embryonic eyebud, including part of the prospective proliferative zone. Cell counts confirmed that the numbers of both the mitotic, undifferentiated cells and the post-mitotic, differentiated cells were reduced following the partial ablation. A regression analysis suggested that the initial number of undifferentiated cells was a fixed proportion of the total number, but that this proportion was increased by the partial ablation. This increase occurred for all stages that the partial ablation was performed, from early optic vesicle to mid optic cup stages. The proportion of undifferentiated cells was normal in sham-operated retinas, indicating that the increase in partially ablated retinas was induced by tissue removal and not by wound healing. Analyses of clones derived from single precursors, labeled with a fluorescent lineage tracer, indicated that the rate of proliferation was the same in partially ablated and sham-operated retinas. Measurements of bromodeoxyuridine incorporation directly confirmed that at the ciliary margin cell division time was unchanged after partial ablation. Our observations are most consistent with the hypothesis that the proportion of undifferentiated cells was increased because cells that would have become post-mitotic remained proliferative after the partial ablation. Furthermore, cell-cell interactions most likely play a major role in controlling the initial number of proliferative cells in the tadpole retina.

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PubMed1993

Biological microscopy: the emergence of digital microscopy.

The past few years have seen a tremendous renaissance in biological optical microscopy, mainly as a result of the application of digital image processing and video imaging techniques. We review recent developments in microscopy that are permitting unprecedented views of biological structure and function.

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PubMed1993

Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration.

Although the Xenopus embryo has served as an important model system for both molecular and cellular studies of vertebrate development, comparatively little is known about its neural crest. Here, we take advantage of the ease of manipulation and relative transparency of Xenopus laevis embryos to follow neural crest cell migration and differentiation in living embryos. We use two techniques to study the lineage and migratory patterns of frog neural crest cells: (1) injections of DiI or lysinated rhodamine dextran (LRD) into small populations of neural crest cells to follow movement and (2) injections of LRD into single cells to follow cell lineage. By using non-invasive approaches that allow observations in living embryos and control of the time and position of labelling, we have been able to expand upon the results of previous grafting experiments. Migration and differentiation of the labelled cells were observed over time in individual living embryos, and later in sections to determine precise position and morphology. Derivatives populated by the neural crest are the fins, pigment stripes, spinal ganglia, adrenal medulla, pronephric duct, enteric nuclei and the posterior portion of the dorsal aorta. In the rostral to mid-trunk levels, most neural crest cells migrate along two paths: a dorsal pathway into the fin, followed by presumptive fin cells, and a ventral pathway along the neural tube and notochord, followed by presumptive pigment, sensory ganglion, sympathetic ganglion and adrenal medullary cells. In the caudal trunk, two additional paths were noted. One group of cells moves circumferentially within the fin, in an arc from dorsal to ventral; another progresses ventrally to the anus and subsequently populates the ventral fin. By labelling individual precursor cells, we find that neural tube and neural crest cells often share a common precursor. The majority of clones contain labelled progeny cells in the dorsal fin. The remainder have progeny in multiple derivatives including spinal ganglion cells, pigment cells, enteric cells, fin cells and/or neural tube cells in all combinations, suggesting that many premigratory Xenopus neural crest precursors are multipotent.

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PubMed1993

Segmental migration of the hindbrain neural crest does not arise from its segmental generation.

The proposed pathways of chick cranial neural crest migration and their relationship to the rhombomeres of the hindbrain have been somewhat controversial, with differing results emerging from grafting and DiI-labelling analyses. To resolve this discrepancy, we have examined cranial neural crest migratory pathways using the combination of neurofilament immunocytochemistry, which recognizes early hindbrain neural crest cells, and labelling with the vital dye, DiI. Neurofilament-positive cells with the appearance of premigratory and early-migrating neural crest cells were noted at all axial levels of the hindbrain. At slightly later stages, neural crest cell migration in this region appeared segmented, with no neural crest cells obvious in the mesenchyme lateral to rhombomere 3 (r3) and between the neural tube and the otic vesicle lateral to r5. Focal injections of DiI at the levels of r3 and r5 demonstrated that both of these rhombomeres generated neural crest cells. The segmental distribution of neural crest cells resulted from the DiI-labelled cells that originated in r3 and r5 deviating rostrally or caudally and failing to enter the adjacent preotic mesoderm or otic vesicle region. The observation that neural crest cells originating from r3 and r5 avoided specific neighboring domains raises the intriguing possibility that, as in the trunk, extrinsic factors play a major role in the axial patterning of the cranial neural crest and the neural crest-derived peripheral nervous system.

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PubMed1992

Patterning of retinotectal connections in the vertebrate visual system.

Recent work on the retinotectal projection clearly establishes the roles of neuronal activity and position-based cues in the patterning of nerve connections. In some species, the high degree of spatial order has been shown to emerge from a continued process of terminal growth and refinement. The future challenge is now to determine how multiple cues work together to guide the sculpting of the final pattern.

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PubMed1992

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1991

Pattern formation in the vertebrate nervous system.

In recent years, the classical approaches of experimental embryology have been used in combination with more modern techniques to investigate aspects of neurogenesis. This combination has advanced our knowledge of several areas of neuronal development, including the lineages of neuronal precursors, the segmentation of the nervous system, and the patterning of the neural tube.

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PubMed1991

Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos.

We have used the vital dye, DiI, to analyze the contribution of sacral neural crest cells to the enteric nervous system in chick and mouse embryos. In order to label premigratory sacral neural crest cells selectively, DiI was injected into the lumen of the neural tube at the level of the hindlimb. In chick embryos, DiI injections made prior to stage 19 resulted in labelled cells in the gut, which had emerged from the neural tube adjacent to somites 29-37. In mouse embryos, neural crest cells emigrated from the sacral neural tube between E9 and E9.5. In both chick and mouse embryos, DiI-labelled cells were observed in the rostral half of the somitic sclerotome, around the dorsal aorta, in the mesentery surrounding the gut, as well as within the epithelium of the gut. Mouse embryos, however, contained consistently fewer labelled cells than chick embryos. DiI-labelled cells first were observed in the rostral and dorsal portion of the gut. Paralleling the maturation of the embryo, there was a rostral-to-caudal sequence in which neural crest cells populated the gut at the sacral level. In addition, neural crest cells appeared within the gut in a dorsal-to-ventral sequence, suggesting that the cells entered the gut dorsally and moved progressively ventrally. The present results resolve a long-standing discrepancy in the literature by demonstrating that sacral neural crest cells in both the chick and mouse contribute to the enteric nervous system in the postumbilical gut.

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PubMed1991

Migrating neural crest cells in the trunk of the avian embryo are multipotent.

Trunk neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Previously, we demonstrated that many premigratory trunk neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. The results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after leaving the neural tube either during their migration or at their sites of localization. Here, we test the developmental potential of migrating trunk neural crest cells by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells as they migrate through the somite. By two days after injection, the LRD-labelled clones contained from 2 to 67 cells, which were distributed unilaterally in all embryos. Most clones were confined to a single segment, though a few contributed to sympathetic ganglia over two segments. A majority of the clones gave rise to cells in multiple neural crest derivatives. Individual migrating neural crest cells gave rise to both sensory and sympathetic neurons (neurofilament-positive), as well as cells with the morphological characteristics of Schwann cells, and other non-neuronal cells (both neurofilament-negative). Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. Our data demonstrate that migrating trunk neural crest cells can be multipotent, giving rise to cells in multiple neural crest derivatives, and contributing to both neuronal and non-neuronal elements within a given derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

Segmental lineage restrictions in the chick embryo spinal cord depend on the adjacent somites.

We have investigated whether the developing spinal cord is intrinsically segmented in its rostrocaudal (anteroposterior) axis by mapping the spread of clones derived from single labelled cells within the neural tube of the chick embryo. A single cell in the ventrolateral neural tube of the trunk was marked in situ with the fluorescent tracer lysinated rhodamine dextran (LRD) and its descendants located after two days of further incubation. We find that clones derived from cells labelled before overt segmentation of the adjacent mesoderm do not respect any boundaries within the neural tube. Those derived from cells marked after mesodermal segmentation, however, never cross an invisible boundary aligned with the middle of each somite, and tend to be elongated along the mediolateral axis of the neural tube. When the somite pattern is surgically disturbed, neighbouring clones derived from neuroectodermal cells labelled after somite formation behave like clones derived from younger cells: they no longer respect any boundaries, and are not elongated mediolaterally. These results indicate that periodic lineage restrictions do exist in the developing spinal cord of the chick embryo, but their maintenance requires the presence of the adjacent somite mesoderm.

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PubMed1991

Macromere cell fates during sea urchin development.

This paper examines the cell lineage relationships and cell fates in embryos of the sea urchin Strongylocentrotus purpuratus leading to the various cell types derived from the definitive vegetal plate territory or the veg2 tier of cells. These cell types are gut, pigment cells, basal cells and coelomic pouches. They are cell types that constitute embryonic structures through cellular migration or rearrangement unlike the relatively non-motile ectoderm cell types. For this analysis, we use previous knowledge of lineage to assign macromeres to one of four types: VOM, the oral macromere; VAM, the aboral macromere, right and left VLM, the lateral macromeres. Each of the four macromeres contributes progeny to all of the cell types that descend from the definitive vegetal plate. Thus in the gut each macromere contributes to the esophagus, stomach and intestine, and the stripe of labeled cells descendant from a macromere reflects the re-arrangement of cells that occurs during archenteron elongation. Pigment cell contributions exhibit no consistent pattern among the four macromeres, and are haphazardly distributed throughout the ectoderm. Gut and pigment cell contributions are thus radially symmetrical. In contrast, the VOM blastomere contributes to both of the coelomic pouches while the other three macromeres contribute to only one or the other pouch. The total of the macromere contribution amounts to 60% of the cells constituting the coelomic pouches.

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PubMed1991

Microinjection of fluorescent tracers to study neural cell lineages.

The examination of cell lineages is an important step towards understanding the developmental events that specify the various cell types in the organism. The mechanisms that control which cell types are formed, their locations, and their numbers remain unknown. Analyses of cell lineage in the frog neural retina have revealed that individual precursors are multipotent and are capable of producing almost any combination of cell types. In addition to giving rise to a wide range of phenotypes, the precursors can give rise to a wide range of clone sizes. Cell lineage studies in other systems indicate that some precursors are multipotent, like those in the retina, while others appear to produce a more restricted range of descendants, perhaps even a single phenotype. These differences in the developmental potential of precursor cells suggest that the nervous system uses several strategies for producing its many cell types. Investigation of these strategies, at the cellular and molecular level, requires more than a description of the normal cell lineages. We are now exploiting the frog neural retina to perform the experimental manipulations needed to elucidate these strategies.

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PubMed1991

Cell lineage analysis of the avian neural crest.

Neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. A major unanswered question concerning the neural crest is when and how the neural crest cells become determined to adopt a particular fate. We have explored the developmental potential of trunk neural crest cells in avian embryos by microinjecting a vital dye, lysinated rhodamine dextran (LRD), into individual cells within the dorsal neural tube. We find that premigratory and emigrating neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. These results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after emigration from the neural tube either during their migration or at their sites of localization. To determine whether neural crest cells become restricted during their migration, we have microinjected individual trunk neural crest cells with dye shortly after they leave the neural tube or as they migrate through the somite. We find that a majority of the clones derived from migrating neural crest cells appear to be multipotent; individual migrating neural crest cells gave rise to both sensory and sympathetic neurons, as well as cells with the morphological characteristics of Schwann cells, and other non-neuronal cells. Even those clones contributing to only one neural crest derivative often contained both neurofilament-positive and neurofilament-negative cells. These data demonstrate that migrating trunk neural crest cells, like their premigratory progenitors, can be multipotent.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1990

Competitive and positional cues in the patterning of nerve connections.

The visual system of lower vertebrates has served as an important testing ground for the mechanisms that generate topographic neuronal connections. During both the outgrowth and the regeneration of the optic nerve, a smoothly ordered map of the retina is formed on its major target, the optic tectum (the retinotectal projection). Experiments performed on this projection have offered support for a variety of mechanisms, including the matching of positional cues in the retina and tectum, the guidance of nerve fibers by interactions between fibers, competition for synaptic space, and the refinement of connections based on neuronal activity. Unfortunately, individual experiments that support any one of these mechanisms have been taken at times as evidence against the involvement of any other mechanism; for example, experiments demonstrating the importance of positional cues have been thought mistakenly to indicate that activity-based interactions are unimportant. Computer simulations, in which multiple, somewhat opposed, mechanisms are allowed to operate in concert demonstrate that such a hybrid model is able to generate a full range of experimental results. More importantly, the elimination of any one of the mechanisms renders the model unable to fit entire classes of findings. Thus, the patterning of the retinotectal projection is best viewed as a process in which the optic nerve terminals attempt to satisfy multiple constraints in selecting their target sites.

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PubMed1990

Synaptic dynamics at the neuromuscular junction: mechanisms and models.

During development, the neuromuscular junction passes through a stage of extensive polyinnervation followed by a period of wholesale synapse elimination. In this report we discuss mechanisms and interactions that could mediate many of the key aspects of these important developmental events. Our emphasis is on (1) establishing an overall conceptual framework within which the role of many distinct cellular interactions and molecular factors can be evaluated, and (2) generating computer simulations that systematically test the adequacy of different models in accounting for a wide range of biological data. Our analysis indicates that several relatively simple mechanisms are each capable of explaining a variety of experimental observations. On the other hand, no one mechanism can account for the full spectrum of experimental results. Thus, it is important to consider models that are based on interactions among multiple mechanisms. A potentially powerful combination is one based on (1) a scaffold within the basal lamina or in the postsynaptic membrane which is induced by nerve terminals and which serves to stabilize terminals by a positive feedback mechanism; (2) a sprouting factor whose release by muscle fibers is down-regulated by activity and perhaps other factors; and (3) an intrinsic tendency of motor neurons to withdraw some connections while allowing others to grow.

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PubMed1990

Segregation of oral from aboral ectoderm precursors is completed at fifth cleavage in the embryogenesis of Strongylocentrotus purpuratus.

A specific set of founder cells uniquely gives rise to the oral and aboral ectoderms in the regularly developing sea urchin Strongylocentrotus purpuratus. We showed earlier that the polar No and Na (animal oral and animal aboral) blastomeres are specified by third cleavage, while the respective oral and aboral lineage contributions of the left and right NL (animal lateral) blastomeres have not yet segregated from one another at third cleavage. Here we demonstrate by iontophoretic injection of lysyl rhodamine dextran lineage tracer that segregation of oral vs aboral cell fates in the lineages of the NL blastomeres has still not occurred by fourth cleavage, but at fifth cleavage there arise from the NL sublineages founder cells whose progeny contribute exclusively to the aboral ectoderm. The sister cells of these fifth cleavage blastomeres are founder cells that contribute exclusively to oral structures. The aboral ectoderm tracts to which NL derivatives give rise occupy lateral regions of the anterior aboral ectoderm, while the oral structures deriving from the NL blastomeres are the lateral sectors of the ciliated bands. The cells of the ciliated bands do not express aboral ectoderm markers and are considered to constitute the border of the oral region. With these new findings we complete our knowledge of the origins, identities, and fates of the 11 founder cells, the progeny of which exclusively give rise to the aboral ectoderm, and of the 5 founder cells, the progeny of which exclusively produce the oral ectoderm and its derivatives.

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PubMed1990

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila.

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.

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PubMed1990

Cell migration in the formation of the pronephric duct in Xenopus laevis.

To determine if cell migration is involved in the formation of the pronephric duct in Xenopus, we used morphometry, ablation, and videomicroscopy of vitally stained cells to study duct formation. In St 23-24 (Nieuwkoop and Faber, 1956) embryos, a ridge of cells forms caudal to the pronephric rudiment. The ridge lengthens at approximately the same rate as the embryonic trunk from St 23 to St 31. Ablation experiments demonstrated that the ridge constitutes the pronephric duct rudiment (PDR); when the ridge was ablated at St 23-24, little or no duct formation occurred, whereas a duct formed when the pronephric rudiment was ablated and the ridge left intact. Vital dye injections showed that the PDR forms from the intermediate mesoderm ventral to myotomes IV-VIII. From St 29/30 to St 33/34, the PDR actively elongates along the ventral edge of the myotomes as far as myotome XIV, where it joins the cloaca as the pronephric duct. Videomicroscopy of vitally stained cells showed that the PDR elongates throughout its length and does not incorporate additional cells from the mesoderm over which it elongates. The results strengthen the case for a common mode of pronephric duct formation among amphibian species.

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