Biomedical subjects

P Godement

Publications and source records attributed to P Godement.

At least 19 recordsLinked to original sources

PubMed2001

Ephrin-A6, a new ligand for EphA receptors in the developing visual system.

In the embryonic visual system, EphA receptors are expressed on both temporal and nasal retinal ganglion cell axons. Only the temporal axons, however, are sensitive to the low concentrations of ephrin-A ligands found in the anterior optic tectum. The poor responsiveness of nasal axons to ephrin-A ligands, which allows them to traverse the anterior tectum and reach their targets in the posterior tectum, has been attributed to constitutive activation of the EphA4 receptor expressed in these axons. EphA4 is highly expressed throughout the retina, but is preferentially phosphorylated on tyrosine (activated) in nasal retina. In a screen for EphA4 ligands expressed in chicken embryonic retina, we have identified a novel ephrin, ephrin-A6. Like ephrin-A5, ephrin-A6 has high affinity for EphA4 and activates this receptor in cultured retinal cells. In the embryonic day 8 (E8) chicken visual system, ephrin-A6 is predominantly expressed in the nasal retina and ephrin-A5 in the posterior tectum. Thus, ephrin-A6 has the properties of a ligand that activates the EphA4 receptor in nasal retinal cells. Ephrin-A6 binds with high affinity to several other EphA receptors as well and causes growth cone collapse in retinal explants, demonstrating that it can elicit biological responses in retinal neurons. Ephrin-A6 expression is high at E6 and E8, when retinal axons grow to their tectal targets, and gradually declines at later developmental stages. The asymmetric distribution of ephrin-A6 in retinal cells, and the time course of its expression, suggest that this new ephrin plays a role in the establishment of visual system topography.

Amino Acid Sequence↗

PubMed1999

The mouse homeodomain protein OTX2 regulates NCAM promoter activity.

The homeodomain transcription factor OTX2 is involved in defining regional identities in developing rostral brain. It appears to participate in morphogenetic processes leading to the formation of boundaries and substrates for early axon growth, processes which are in the end largely based on site-specific expression of cell adhesion molecules. Here, we present evidence that a candidate target of OTX2 is the gene encoding the neural cell adhesion molecule, NCAM. When Otx2 is transfected into NIH3T3 cells, NCAM protein expression is upregulated. Moreover, while mock-transfected cells display only the 140 kDa-isoform of NCAM, Otx2 transfected cells express also the two other major isoforms (NCAM-120 and -180), in agreement with the presence of the corresponding transcripts in Northern blots. In addition, transient expression of Otx2 in COS7 cells is able to dramatically enhance the transcriptional activity of the NCAM promoter. Taken together, our results argue for a regulation of NCAM expression by OTX2.

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PubMed1999

Positional specificities of retinal growth cones in the mouse superior colliculus.

In the developing retinotectal system, repulsive topographic tectal cues have been demonstrated to contribute to the final mapping. Here, we describe a novel response of nasal axons to growth-promoting cues expressed by anterior tectal cells. In in vitro experiments, contact of fibres from the nasal (but not temporal) pole of the mouse retina with anterior (but not posterior) tectal membranes leads to their adopting very elongated and filopodial morphologies, and to increase their growth rates. As previously demonstrated, fibres from the temporal pole of the retina are collapsed by posterior tectal membranes in vitro. In addition, a study of retinal growth cone morphologies in vivo, at early stages of target invasion, shows that growth cones of nasal fibres have streamlined morphologies, usually indicative of active elongation growth modes, in the anterior part of the embryonic mouse tectum, and more elaborate morphologies posteriorly. Vice versa, temporal fibres have mainly elaborate growth cones anteriorly, and collapsed growth cones posteriorly. These experiments demonstrate that nasal retinal fibres respond preferentially to permissive or growth-promoting cues in the embryonic mouse tectal environment, both in vitro and in vivo. This phenomenon might contribute to ingrowth of retinal fibres in their target area, and to promote the homing of nasal fibres towards the posterior aspect of the tectum, which is their normal target region.

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PubMed1998

A potential role for the OTX2 homeoprotein in creating early 'highways' for axon extension in the rostral brain.

Brain pattern formation starts with a subdivision of the neuroepithelium through site-specific expression of regulatory genes and, subsequently, the boundaries between presumptive neuromeres may provide a scaffold for early formation of axon tracts. In the mouse forebrain, the transcription factor OTX2 is strongly expressed at several such boundaries. Combining dye tracing and staining for OTX2 protein, we show that a number of early fibre tracts develop within stripes of OTX2 expression. To analyse a putative influence of OTX2 on the expression of molecules involved in neurite growth, we generated several clones of NIH3T3 cells stably expressing OTX2 protein at varying levels. As shown by immunoblotting, Otx2 transfection affects the expression of a variety of cell and substratum adhesion molecules, rendering the cells a favourable substratum in neurite outgrowth assays. Among the molecules upregulated with increasing levels of OTX2 are NCAM, tenascin-C and DSD-1-PG, which also in situ colocalize with zones of OTX2 expression at boundaries. These data suggest that Otx2 might be involved in defining local substrata for axon extension in the forebrain.

3T3 Cells↗

PubMed1996

The ganglionic eminence may be an intermediate target for corticofugal and thalamocortical axons.

In the nervous system of many species, growing axons associate transiently with cellular groupings along their path. Whether this mechanism applies to the development of corticothalamic and thalamocortical projections is unknown. Using carbocyanine dyes, we studied the early growth of both corticofugal and thalamocortical fibers in hamster embryos. At embryonic day 11.5 (E11.5), corticofugal fibers invade the lateral ganglionic eminence (LGE), and thalamocortical fibers invade the medial ganglionic eminence (MGE). At this age, both sets of fibers are not yet in contact with each other. At the same time, neurons in each subdivision of the GE grow toward the cortex and thalamus. During the next 24 hr, corticofugal and thalamocortical fibers remain within the confines of the GE, where they course at different radial levels and bear large and complex growth cones. In the LGE, corticofugal fibers are often found in close association with cells that are likely to be neuronal. Starting on E13.5, both early projections from the GE decrease, and corticothalamic and thalamocortical fibers invade their definitive target regions. To test whether the GE specifically orients the growth and trajectories of cortical fibers even in the absence of the reciprocal thalamic projection, we cocultured explants of cortex and GE from either hamster or mouse embryos. These experiments showed that the GE, but not other tested brain regions, is able specifically to orient the growth of cortical axons. We therefore suggest that the GE may be an intermediate target in the pathfinding of axons between the cortex and the thalamus.

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PubMed1995

Crossed and uncrossed retinal axons respond differently to cells of the optic chiasm midline in vitro.

In mouse, retinal axon divergence takes place within a cellular specialization localized at the midline of the optic chiasm. To test whether the cells in this locus present cues for differential retinal axon growth, retinal explants were cocultured with cells dissociated from the chiasmatic midline, both taken from day 14-15 embryos, during the principal period of retinal axon divergence. Compared with crossed axons from other retinal regions, axons from ventrotemporal retina, the sole source of uncrossed axons, were shorter, more fasciculated, and fewer in number when growing on chiasm cells. Furthermore, uncrossed axons avoided clusters of chiasm neurons and glia having the composition and arrangement of the midline specialization, but crossed axons readily grew over them. In contrast to the clusters of chiasm cells, however, individual neurons and glia did not elicit differential retinal axon growth. These data demonstrate that cues for divergence derive from cells resident to the chiasm and suggest that cellular interactions among resident midline cells are required to produce these cues.

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PubMed1995

Optical microscopy. 3. Tracking fluorescently labeled neurons in developing brain.

For decades, time-lapse microscopy has been used to track dynamic events associated with biological phenomena. Time-lapse studies of the developing nervous system have been restricted to analysis of dissociated cell cultures or of a series of static images from living organisms. The advent of new fluorescent dyes and video imaging technology has produced novel views of the behavior of neurons in the context of the developing nervous tissue, such as migrations within and away from proliferative zones and navigation of axonal processes to synaptic targets. After fixation of the tissue preparation, time-lapse monitoring can be followed by other analytical techniques and forms of microscopy, e.g., immunocytochemistry or electron microscopy, producing information on the interactions of individual cells whose behavioral histories are known. The power of video time-lapse microscopy of living brain tissue lies in the firsthand documentation of developmental patterning, which in turn can serve as an experimental assay.

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PubMed1995

Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse.

Axonal navigation during development requires that cues present in the extracellular environment be capable of modifying the structure of the cone in a dynamic way. Protein kinase C (PKC) has long been suspected to be one of the multiple molecular relays present in the terminal structure of the developing axon and involved in the transduction of extracellular signals. The latter proposal is, however, based on the use of drugs or of protocols leading to pleiotropic and often nonspecific effects. In the present study, we have taken advantage of the discovery of a peptide capable of translocating across biological membranes and to accumulate in the cytoplasm and nucleus of cells in culture, to internalize a highly specific peptidic inhibitor of PKC. We demonstrate that linking the two peptides (vector and PKC inhibitor) allows the internalization of the latter in live cells, specifically inhibits PKC and provokes a rapid modification of growth cone morphology. This set of data thus establishes that a peptidic inhibitor of PKC activity, once internalized, provokes a change in growth cone morphology, reminiscent of the collapse phenotype. In addition, the present study describes a new efficient and harmless way to introduce pharmacologically active substances in neural cells in culture.

Amino Acid Sequence↗

PubMed1995

Retinal axon divergence in the optic chiasm: uncrossed axons diverge from crossed axons within a midline glial specialization.

A long-standing question is how fiber pathways in the mammalian CNS project to both sides of the brain. Static and real-time analyses of dye-labeled retinal axons (Godement et al., 1990, 1994) have demonstrated that at embryonic day 15-17 in the mouse, crossed and uncrossed axons from each eye diverge in a zone 100-200 microns proximal to the midline of the optic chiasm. In this study, we identify cellular specializations in this zone that might serve as cues for retinal axon divergence. Second, using growth cone morphology as an indicator of growth cone destination, we analyzed how crossed and uncrossed retinal growth cones related to these cellular components. Monoclonal antibody RC2, a marker for radial glia in embryonic mouse CNS, revealed a palisade of radial glia straddling the midline. At the midline, a thin raphe of cells that appear morphologically distinct from the radial glia express a free carbohydrate epitope, stage-specific embryonic antigen 1 (SSEA-1). Sections containing Dil-labeled axons and immunolabeled cells indicated that all axons enter the radial glial palisade. Uncrossed axons turn within the palisade, but never beyond the raphe of SSEA-1-positive cells. In addition, ultrastructural analysis indicated that all growth cones contact radial glia, with projections of the growth cone interdigitating with glial fibers. These results demonstrate that retinal axons diverge within a cellular specialization centered around the midline of the developing optic chiasm, consistent with the hypothesis that cues for divergence are located in this zone.

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PubMed1994

Specific guidance and modulation of growth cone motility during in vivo development.

The hemidecussation of retinal fibers that occurs in mammals offers the opportunity to study several aspects of growth cone guidance in a single model system. Recent studies suggest that growth cones of crossed and uncrossed retinal fibers respond in differential manners when they contact cells at the optic chiasm midline, and that such contact interactions are the main event involved in their divergence. Observations of the in situ behaviors of these growth cones disclose that their guidance in this decision region involves two different processes: an orientational response, mediated by the selective guidance of the filopodia of frowth cones away or towards the midline of the optic chiasm, and a dynamic response, in which growth cones go through cycles of advance and pauses while in the optic chiasm. We hypothesize that these two aspects of growth cone motility represent two different aspects of the biology of growth cones in response to extrinsic cues, which are both used in their guidance during development.

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PubMed1994

Retinal axon divergence in the optic chiasm: dynamics of growth cone behavior at the midline.

To study how retinal ganglion cell axons diverge in the optic chiasm, the behavior of dye-labeled fibers was monitored in real time with video microscopy in an isolated preparation of embryonic mouse brain, with a focus on embryonic day 15-16. These real-time studies have revealed the dynamics of the growth of individual retinal axons, especially the tempo of extension and growth cone behaviors during divergence in the chiasm, a model for "decision" regions in developing pathways. Within the chiasm, retinal growth cones extend by saltatory growth, consisting of bursts of rapid advance alternating with pauses in extension. During pauses, growth cone appendages remain motile, and develop asymmetries prior to a change in the axis of growth. In a zone straddling the midline, retinal fibers, irrespective of destination, display long pauses for up to several hours, making small advances and retractions with no net extension. While crossed fibers ultimately progress through the midline, uncrossed fibers from inferior temporal retina develop wide-ranging branched growth cones, and then turn back to the ipsilateral side. Turns are effected by the selective retraction or micropruning of asymmetric foci of motile activity, and by the transformation of a backward-directed filopodium into a new growth cone. The behavior of retinal axons at the midline supports the hypothesis that this locus contains cues important for retinal axon divergence. Moreover, the observations of growth cone kinetics in the chiasm elucidate which growth cone forms seen in static preparations mediate growth cone turning, and suggest a model of axon navigation in decision regions in the intact nervous system.

Animals↗

PubMed1993

Guidance of retinal fibers in the optic chiasm.

At the optic chiasm of vertebrates, there occurs a sharp divergence in the destination of retinal ganglion cell axons as they are on their way to more central targets. Recent studies in the mouse indicate that the growth cones of each set of fibers diverge close to the midline of the optic chiasm, at the borders of a palisade of immature radial glia. Static and dynamic views of retinal growth cones in this decision region reveal that extensive exploratory behavior and selective retraction of parts of the growing tips of uncrossed fibers, in response to cellular cues at the midline, is a major event in the guidance of these fibers. In vitro experiments further show that presumptive crossed and uncrossed fibers differ in their responses to contact with cells from the optic chiasm. As with other instances of selective guidance of fibers at midline structures, the divergence of crossed and uncrossed retinal fibers therefore involves a selective remodeling of their growing tips and transitory axon-cell contacts during growth at the optic chiasm, presumably due to biochemical heterogeneity among crossed and uncrossed ganglion cell fibers.

Animals↗

PubMed1990

Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers.

In the developing mammalian visual system, retinal fibers grow through the optic chiasm, where one population crosses to the opposite side of the brain and the other does not. Evidence from labeling growing retinal axons with the carbocyanine dye Dil in mouse embryos indicates that the two subpopulations diverge at a zone along the midline of the optic chiasm. At the border of this zone, crossed fibers grow directly across, whereas uncrossed fibers turn back, developing highly complex terminations with bifurcating and wide-ranging growth cones. When one eye is removed at early stages, uncrossed fibers from the remaining eye stall at the chiasm midline. These results suggest that crossed and uncrossed retinal fibers respond differently to cues along the midline of the chiasm and that the uncrossed fibers from one eye grow along crossed fibers from the other eye, both guidance mechanisms contributing to the establishment of the bilateral pattern of visual projections in mammalian brain.

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PubMed1989

Spatial arrangement of radial glia and ingrowing retinal axons in the chick optic tectum during development.

Neuroanatomical tracing of retinal axons and axonal terminals with the fluorescent dye, DiI, was combined with immunohistochemical characterization of radial glial cells in the developing chick retinotectal system. Emphasis was placed on the mode of the tectal innervation by individual retinal axons and on the distribution and fate of the tectal radial glial cells and their spatial relation to retinal axons. It was obvious from fluorescent images obtained from anterogradely filled axons that these axons deserted the superficial stratum opticum (SO) to penetrate the stratum griseum et fibrosum superficiale (SGFS) by making right-angled turns within the SO. Frequently, axons which had invaded the SGFS were bifurcated and had a superficial branch which remained within the SO. Terminal axonal arborization occurred at various depths within the SGFS. Characterization of the tectal glial cells and their radial fibers by means of the anti-filament antibody, R5, and post-mortem staining with the fluorescent dye, DiI, revealed the following. (a) At least from day E8 to P1, tectal glial fibers traversed all tectal layers from the periventricular location of their somata to the superficial interface between SO and pia mater. In this interface they enlarged and formed characteristic endfeet. (b) Glial endfeet covered the whole tectal surface. They showed at early ages anterior-posterior differences having a higher density in the posterior tectum. These differences disappeared at embryonic day E13. (c) After innervation, glial endfeet of the anterior tectal third were arranged in rows parallel to the retinal fibers within the SO. This arrangement was not observed in eyeless embryos. (d) Radial glial fibers could be stained with R5 from day E8 to late embryonic stages throughout their entire length. (e) At the first posthatching days, only the segments of the radial glial fibers restricted to the thickness of the SO were R5-positive, although the fibers still traversed throughout the depth of the tectum. The results are discussed in context to the genesis of the retinotectal projection.

Animals↗

PubMed1989

Cross-species recognition of tectal cues by retinal fibers in vitro.

The retinae of vertebrates project in a topographic manner to several visual centers of the brain. The formation of these projections could depend on the existence of position-specific properties of retinal and target cells. In this study, we have tested the in vitro growth of mouse retinal fibers on membranes derived from various regions of the embryonic superior colliculus, a main target of the retina in this species. Fibers had the choice of elongating on membranes taken from either the anterior or the posterior half of the superior colliculus. Fibers from temporal areas of the retina prefer to elongate on anterior collicular membranes, while fibers from nasal areas do not show a preference. These phenomena are observed with membranes from embryonic (E15-E18) or young postnatal mice. In interspecies cultures where mouse retinal fibers had to grow on chick tectal membranes, or vice versa, the same preference for anterior tectal or collicular membranes in growth of temporal retinal fibers is observed, suggesting some similarities in the cues used in both species.

Animals↗

PubMed1988

The primary visual cortex in the mouse: receptive field properties and functional organization.

Receptive field (RF) characteristics of cells in primary visual cortex of the mouse (C57B16 strain) were studied by single unit recording. We have studied the functional organization of area 17 along both the radial and tangential dimensions of the cortex. Eighty seven percent of the visual neurons could be classified according to their responses to oriented stimuli and to moving stimuli. Cells which preferred a flashed or moving bar of a particular orientation and responded less well to bars of other orientations or to spots, were classified as orientation selective (simple RF 23%, complex RF 18%). The majority of them were, moreover, unidirectional (24%). All orientations were roughly equally represented. Cells with oriented RFs were recorded mostly in the upper part of cortical layers II-III, where they appeared to be clustered according to their preferred orientation. Neurons that responded equally well to spots and bars of all orientations (46%) were classified as "non-oriented"; among these neurons there were several subcategories. Cells which responded equally well to spots and bars but preferred stimuli moving along one or both directions of a particular axis were classified as non oriented asymmetric cells (unidirectional 14%, bidirectional 4%). They were recorded mainly in supra- and infra-granular layers. Cells unaffected by stimulus shape and orientation which responded equally well to all directions of movement were classified as symmetric units. They had receptive field classified as ON (11%), OFF (1%), ON/OFF (11%), or were unresponsive to stationary stimuli (5%). These cells were mostly found in layer IV, in which they constituted the majority of recorded cells. There was no apparent correlation between the functional type and size of RFs. However, the greatest proportion of small RFs was found in layer IV. In the binocular segment of the mouse striate cortex, the influence of the contralateral eye predominated. Ninety five percent of cells in this segment were driven through the contralateral eye. However, 70% of cells were binocularly activated, showing that considerable binocular integration occurred in this cortical segment. Ocular dominance varied less along the radial than along the tangential dimension of the cortex.

Animals↗

PubMed1987

Fate of uncrossed retinal projections following early or late prenatal monocular enucleation in the mouse.

In mammals binocular vision is made possible by the existence in the temporal retina of ipsilaterally projecting ganglion cells (IGCs) (with axons that do not cross the brain midline and join optic fibers from the opposite eye). To learn whether early interactions between fibers of each eye play a role in generating a mixed ipsi + contralateral projection pattern, we studied with horseradish peroxidase the origin of uncrossed retinal projections in mice that developed after one eye was destroyed at very early embryonic ages. One eye was removed on embryonic day 16 (E16; when optic fibers have grown past the chiasm bilaterally, but very few have grown into the visual centers) or on E13 or E12 (when few or no optic fibers have passed the presumptive chiasm region). Normal adult mice have a mean of 946 IGCs (range: 784-1,073) within the temporal sector of the retina, and less than 25 in the rest of the retina. In adult mice enucleated at E16, an average of 1,354 (1,215-1,484) IGCs are present within a clearly demarcated temporal sector of the remaining retina and 265 (152-312) are present throughout the rest of the retina. In both the temporal and nasal retina the excess IGCs in these mice have, generally, very small somas. In some of these mice the most peripheral part of the temporal sector contains fewer IGCs. In E12 or E13 enucleates, IGCs are also generally located in a narrow (often narrower than normal) region along the temporo-inferior retinal border, but their number is less than in normal or E16-enucleated mice: E13 enucleates have a mean of 639 cells (range: 361-875) in the temporal sector and 109 (8-275) in the rest of the retina. Following enucleation of one mouse at E12, the respective values are 349 and 31 cells. The reduction in numbers of IGCs in these mice is especially pronounced for ganglion cells with small cell bodies. These findings suggest that the development of uncrossed projections in mice depends on selective guidance mechanisms of axons from temporal retina through the chiasm. These may consist of interactions of optic axons with guidance cues distributed in the presumptive chiasm (possibly at early stages) and also of fiber-fiber guidance mechanisms, in particular between fibers from each eye.

Animals↗

PubMed1987

A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue.

Carbocyanine dyes, fluorescent lipophilic substances used for optical recordings of membrane voltage and for studies of membrane fluidity, have recently been shown to provide intense and long-lasting staining of neurones in vivo and in vitro (Schwartz & Agranoff, 1981; Honig & Hume, 1985, 1986; Catsicas, Thanos & Clarke, 1986; Landmesser & Honig, 1986; Thanos & Bonhoeffer, 1987). We report here that two of these dyes, diI (1,1',dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) and diO (3,3'-dioctadecyloxacarbocyanine perchlorate), can also label neurones in embryonic mouse and chicken brain tissue that has been previously fixed in aldehyde fixatives. Neuronal processes and perikarya can be labelled along considerable distances in both anterograde and retrograde directions. The staining of processes and cells, including their finest extensions is smooth and clear, rivalling intracellular injections of HRP or Lucifer Yellow. The appearance and time course of progression of the staining along axons suggest that the staining in fixed tissue occurs due to a process of diffusion of dyes along the plasma membranes of cells. This technique has allowed us to study the first stages in the development of optic fibres in mouse embryos, especially at the optic chiasm. The early retinal projection (E13-E13 1/2) is mainly crossed, but some optic fibres grow to the ipsilateral side of the brain at the outset. Retrogradely labelled ganglion cells from the dorsocentral area of the retina participate in the formation of both the ipsilateral and the contralateral projection. Thus, at early stages, crossed and uncrossed projections arise from identical subregions of the retina and the partition of the retina with respect to the laterality of its projection arises later.

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