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S E Fraser

Publications and source records attributed to S E Fraser.

At least 37 records · Page 2Linked to original sources
PubMed2000

In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches.

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role.

Animals↗
PubMed1999

Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Actins↗
PubMed1999

Looking deeper into vertebrate development.

Magnetic resonance imaging (MRI) is a non-invasive imaging method that provides three-dimensional (3-D) images of the internal structure of opaque objects, such as humans and mice. In optimal situations, spatial resolution can approach the micron level. Arbitrarily oriented single-slice images can be obtained in seconds, with full 3-D volume images taking tens of minutes to collect. The exquisite sensitivity of MRI to the local physical and chemical environment provides a wide range of mechanisms giving rise to intrinsic contrast in the MR experiment, thus providing images with dramatic differences between different tissue types (e.g. white vs grey matter, myelinated vs unmyelinated fibres, and brain parenchyma vs ventricles). The recent advent of physiologically sensitive MRI contrast agents opens up a wealth of new avenues of study, even including the in vivo imaging of gene expression.

Animals↗
PubMed1998

Neural crest cell dynamics revealed by time-lapse video microscopy of whole embryo chick explant cultures.

DiI-labeled cranial neural crest cells were followed in whole embryo chick explant cultures using time-lapse confocal microscopy. Neural crest cells emerged along the dorsal midline of all rhombomeres. There was a small amount of mixing of neural crest cells between adjoining rhombomeres as cells emerged from the dorsal midline; this mixing persisted during their migration out of the neural tube. Neural crest cell-free zones lateral to rhombomere 3 (r3) and r5 resulted from neural crest cells migrating in either rostral or caudal directions to join other neural crest cells exiting adjacent to r2, r4, or r6. Neural crest cells migrated in a wide variety of individual cell behaviors, ranging from rapid unidirectional motion to stationary and even backward movement (toward the neural tube). Neural crest cells also migrated collectively, extending filipodia to form chain-like cell arrangements. In the midbrain and r1 region, many chains stretched from the dorsal midline to just beyond the lateral extent of the neural tube. In the r7 region, cells linked together and stretched laterally from the neural tube to other neural crest cells migrating into the third branchial arch. The unpredictable cell trajectories, the mixing of neural crest cells between adjoining rhombomeres, and the diversity in cell migration behavior within any particular region imply that no single mechanism guides migration. The regional differences in cell migration characteristics suggests that influential factors may vary spatially along the rostrocaudal axis in the head.

Animals↗
PubMed1998

A model for MRI contrast enhancement using T1 agents.

Contrast in MRI relies on differences in the local environment of water and is often enhanced by using contrast agents. We present a simple model for evaluating the minimal contrast agent concentration required to produce "satisfactory" contrast enhancement in magnetic resonance images. Previous strategies have been based largely on empirical results for specific systems. The present tissue contrast model (TCM) can be applied to "conventional," targeted, or biochemically responsive agents. The model results are formulated so that only a small number of parameters are required to analyze a given scenario. The TCM is a particularly useful tool in the development of new classes of magnetic resonance contrast media. These agents will have the ability to target specific cells or tissue, and perhaps be able to report on their physiological status. As an example of the applicability of the TCM, we test it against in vivo magnetic resonance microscopy results in frog embryos that have focal cell populations labeled with contrast agent by using calibrated single-cell microinjection techniques.

Animals↗
PubMed1998

Specification of the hindbrain fate in the zebrafish.

We determine the timing of neural commitment by hindbrain tissue in the zebrafish using microsurgical transplantation. When transplanted at shield stage to the ventral side of the embryo, presumptive hindbrain cells are not committed, as they can adapt to their environment and give rise to epidermis. In contrast, when transplanted at 80% epiboly, hindbrain cells retain their neural fate and express neural-specific antigens. Moreover, they are able to maintain regional fate, as is evident by the expression of the hindbrain-specific marker, Krox20. In addition, we observe that committed hindbrain tissues are able to induce presumptive ventral epidermis to form neural crest derivatives, otic vesicles, and neural tissues. We propose that hindbrain progenitors have acquired regional identity as a group at 80% epiboly even before making vertical contact with axial mesoderm. These results suggest that planar induction may constitute a significant component in the zebrafish neural patterning pathway.

Animals↗
PubMed1998

MR microscopy of transgenic mice that spontaneously acquire experimental allergic encephalomyelitis.

Pathology of fixed spinal cords from transgenic mice with a myelin basic protein (MBP) specific T cell receptor was investigated. These mice spontaneously acquire the demyelinating disease experimental allergic encephalomyelitis (EAE). Several complementary imaging modalities, all on the same tissues, were used to visualize lesions; these included high-field (11.7-T) microscopic diffusion tensor imaging (DTI), T2*-weighted imaging, and optical microscopy on histological sections. Lesions were predominantly in white matter around meninges and vasculature and appeared hyperintense in anatomical images. DTIs showed reduced diffusion anisotropy in the same hyperintense regions, consistent with inflammation and edema. Histology in the same tissues exhibited the characteristic pathology of EAE. Two techniques for visualizing the effective diffusion tensor fields are presented, which display direction, organization, and integrity of neuronal fibers. It is shown that DTI offers intriguing possibilities for visualizing axonal organization and lesions within white matter.

Algorithms↗
PubMed1998

Segmentation of the vertebrate hindbrain: a time-lapse analysis.

The chick hindbrain starts from a simple and relatively uniform axis and becomes segmented into repeating units, called rhombomeres. The rhombomeres become sites of cell differentiation into specific neurons and the location from which neural crest cells emerge from the neural tube to form the peripheral nervous system, which has only been analyzed at distinct time points due to the lack of a method to watch the neural tube as it is shaped into segments. We have developed a whole-embryo explant culture system in order to study cell and tissue movements with time-lapse video microscopy. Quantitative analyses of the neural tube during its segmentation show that not all rhombomeres are shaped by the same mechanism. In the rostral hindbrain, or first three segments, rhombomeres are shaped by an expansion in the lateral width of the mid-rhombomere; either a smaller expansion or a constriction takes place at the rhombomere boundaries. In the caudal hindbrain, the rhombomere boundaries constrict more than the mid-rhombomere lateral widths increase or decrease, leading to the shaping of the segments. Throughout the segmentation process the rostrocaudal lengths of all rhombomeres remain nearly constant indicating that shape changes are influenced by lateral expansions and constrictions of the neural tube.

Animals↗
PubMed1997

Heterogeneous expression of multiple putative patterning genes by single cells from the chick hindbrain.

The metameric organization of the vertebrate hindbrain into rhombomeres appears to result from the patterned expression of several transcription factors and putative signaling molecules. We have applied a refined single-cell reverse transcription-polymerase chain reaction strategy to examine the molecular logic proposed to pattern the hindbrain at the single-cell level. This technique allows analysis of the concurrent expression of several genes within an individual cell at higher sensitivity than by in situ hybridization. Our results demonstrate that cells in rhombomere (r) 4 and r5 are heterogeneous in their expression of Hoxa-3, Hoxb-2, Sek-1, and Krox-20, suggesting that single cells are dynamically regulating their rhombomere-specific gene-expression profiles. Furthermore, the strong correlation between Sek-1 and Krox-20 expression at stage 12 was greatly diminished by stage 16, suggesting that the proposed interdependence of these two genes is present only at early stages of hindbrain development.

Animals↗
PubMed1997

Specification of the zebrafish nervous system by nonaxial signals.

The organizer of the amphibian gastrula provides the neurectoderm with both neuralizing and posteriorizing (transforming) signals. In zebrafish, transplantations show that a spatially distinct transformer signal emanates from tissues other than the organizer. Cells of the germring (nonaxial mesendoderm) posteriorized forebrain progenitors when grafted nearby, resulting in an ectopic hindbrain-like structure; in contrast, cells of the organizer (axial mesendoderm) caused no posterior transformation. Local application of basic fibroblast growth factor, a candidate transformer in Xenopus, caused malformation but not hindbrain transformation in the forebrain. Thus, the zebrafish gastrula may integrate spatially distinct signals from the organizer and the germring to pattern the neural axis.

Animals↗
PubMed1997

Differentiation of the vertebrate neural tube.

The vertebrate nervous system arises through a series of inductive interactions, beginning with the induction of the neural plate and the rostrocaudal patterning of the neural tube. The process continues with dorsoventral patterning of the neural tube, during which floor plate cells and motor neurons are induced ventrally by interactions of the neural tube with the notochord, and dorsal cell types are induced via neural plate/ectodermal interactions. Later interactions result in the formation of interneurons as well as neuronal migrations. Recent progress, guided in part by knowledge of evolutionary conservation of transcription factors and signaling pathways, is beginning to reveal the cellular and molecular bases of each of these steps in neuronal patterning.

Animals↗
PubMed1996

The cellular patterns of BDNF and trkB expression suggest multiple roles for BDNF during Xenopus visual system development.

The temporal patterns of BDNF and trkB expression in the developing Xenopus laevis tadpole, and the responsiveness of retinal ganglion cells to BDNF, both in culture and in vivo, suggest significant roles for this neurotrophin during visual system development (Cohen-Cory and Fraser, Neuron 12, 747-761, 1994; Nature 378, 192-196, 1995). To examine the potential roles of this neurotrophin within the developing retina and in its target tissue, the optic tectum, we studied the cellular sites of BDNF expression by in situ hybridization. In the developing optic tectum, discrete groups of cells juxtaposed to the tectal neuropil where retinal axons arborize expressed BDNF, supporting the target-derived role commonly proposed for this neurotrophin. In the retina, retinal ganglion cells, ciliary margin cells, and a subset of cells in the inner nuclear layer expressed the BDNF gene. The expression of BDNF coincided with specific trkB expression by both retinal ganglion cells and amacrine cells, as well as with the localization of functional BDNF binding sites within the developing retina, as shown by in situ hybridization and BDNF cross-linking studies. To test for a possible role of endogenous retinal BDNF during development, we studied the effects of neutralizing antibodies to BDNF on the survival of retinal ganglion cells in culture. Exogenously administered BDNF increased survival, whereas neutralizing antibodies to BDNF significantly reduced baseline retinal ganglion cell survival and differentiation. This suggests the presence of an endogenous retinal source of neurotrophic support and that this is most likely BDNF itself. The retinal cellular patterns of BDNF and trkB expression as well as the effects of neutralizing antibodies to this neurotrophin suggest that, in addition to a target-derived role, BDNF plays both autocrine and/or paracrine roles during visual system development.

Animals↗
PubMed1996

Intravital imaging of green fluorescent protein using two-photon laser-scanning microscopy.

Imaging a fluorophore in a living tissue presents several unique problems. The fluorescence from the labeled cell(s) may be weak, the labeled cells may be buried deep within tissue and the presence of a fluorophore may render the cells photo-sensitive. Two-photon laser-scanning microscopy (TPLSM) offers several advantages in meeting these challenges. We show that TPLSM provides greater sensitivity, better resolution and less photo-bleaching, as compared to confocal laser-scanning microscopy. The dramatically reduced photo-bleaching makes it possible to image cells continuously for long periods of time. Therefore, TPLSM allows a safer and higher-resolution means of imaging living cells labeled with a variety of fluorophores, including green fluorescent protein.

Animals↗
PubMed1996

Characterizing the zebrafish organizer: microsurgical analysis at the early-shield stage.

The appearance of the embryonic shield, a slight thickening at the leading edge of the blastoderm during the formation of the germ ring, is one of the first signs of dorsoventral polarity in the zebrafish embryo. It has been proposed that the shield plays a role in fish embryo patterning similar to that attributed to the amphibian dorsal lip. In a recent study, we fate mapped many of the cells in the region of the forming embryonic shield, and found that neural and mesodermal progenitors are intermingled (Shih, J. and Fraser, S.E. (1995) Development 121, 2755-2765), in contrast to the coherent region of mesodermal progenitors found at the amphibian dorsal lip. Here, we examine the fate and the inductive potential of the embryonic shield to determine if the intermingling reflects a different mode of embryonic patterning than that found in amphibians. Using the microsurgical techniques commonly used in amphibian and avian experimental embryology, we either grafted or deleted the region of the embryonic shield. Homotopic grafting experiments confirmed the fates of cells within the embryonic shield region, showing descendants in the hatching gland, head mesoderm, notochord, somitic mesoderm, endoderm and ventral aspect of the neuraxis. Heterotopic grafting experiments demonstrated that the embryonic shield can organize a second embryonic axis; however, contrary to our expectations based on amphibian research, the graft contributes extensively to the ectopic neuraxis. Microsurgical deletion of the embryonic shield region at the onset of germ ring formation has little effect on neural development: embryos with a well-formed and well-patterned neuraxis develop in the complete absence of notochord cells. While these results show that the embryonic shield is sufficient for ectopic axis formation, they also raise questions concerning the necessity of the shield region for neural induction and embryonic patterning after the formation of the germ ring.

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