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Biomedical subjects

C E Holt

Publications and source records attributed to C E Holt.

At least 37 records · Page 2Linked to original sources
PubMed1997

Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC.

Netrin-1 promotes outgrowth of axons in vitro through the receptor Deleted in Colorectal Cancer (DCC) and elicits turning of axons within embryonic explants when presented as a point source. It is not known whether netrin-1 alone can elicit turning nor whether DCC mediates the turning response. We show that Xenopus retinal ganglion cell growth cones orient rapidly toward a pipette ejecting netrin-1, an effect blocked by antibodies to DCC. In vitro, netrin-1 induces a complex growth cone morphology reminiscent of that at the optic nerve head, a site of netrin-1 expression in vivo. These results demonstrate that netrin-1 can function alone to induce turning, implicate DCC in this response, and support the idea that netrin-1 contributes to steering axons out of the retina.

Amino Acid Sequence↗
PubMed1997

cAMP-dependent growth cone guidance by netrin-1.

Netrin-1 is known to function as a chemoattractant for several classes of developing axons and as a chemorepellent for other classes of axons, apparently dependent on the receptor type expressed by responsive cells. In culture, growth cones of embryonic Xenopus spinal neurons exhibited chemoattractive turning toward the source of netrin-1 but showed chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A. Both attractive and repulsive responses were abolished by depleting extracellular calcium and by adding a blocking antibody against the netrin-1 receptor Deleted in Colorectal Cancer. Thus, nerve growth cones may respond to the same guidance cue with opposite turning behavior, dependent on other coincident signals that set the level of cytosolic cAMP.

Animals↗
PubMed1997

Essential role of heparan sulfates in axon navigation and targeting in the developing visual system.

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system.

Animals↗
PubMed1996

Expression and herbimycin A-sensitive localization of pp125FAK in retinal growth cones.

Proper elongation of Xenopus retinal ganglion cell (RGC) axons in the optic tract during development requires intact functioning of beta 1 integrin and tyrosine kinase (TK) activity. The cytoplasmic TK pp125FAK can directly associate with and become activated by beta 1 integrin in cultured fibroblasts. Here we demonstrate the presence of pp125FAK in the developing retina and in cultured retinal neurities, growth cones and filopodia. We show that pp125FAK immunoprecipitated from neural tissue is phosphorylated, and we compare the pattern of FAK and phosphotyrosine immunolabeling. Finally, we show that the localization of pp125FAK in filopodia depends upon TK activity sensitive to the TK inhibitor herbimycin A. These results indicate a role for pp125FAK in signaling the growth of retinal axons.

Animals↗
PubMed1996

Retrovirol gene transfer in Xenopus cell lines and embryos.

A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three different Xenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10(8) cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the beta-galactosidase gene into the neural tube lumen of 24-h embryos yielded beta-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer in Xenopus embryos and cell lines.

Animals↗
PubMed1996

Inhibition of FGF receptor activity in retinal ganglion cell axons causes errors in target recognition.

Native fibroblast growth factor receptor (FGFR) function was inhibited in developing Xenopus retinal ganglion cells (RGCs) by in vivo transfection of a dominant negative FGFR. Axons expressing the dominant negative protein advanced at 60% of the normal speed, but nevertheless navigated appropriately in the embryonic optic pathway. When they neared the optic tectum, however, many axons made erroneous turns, causing them to bypass rather than enter their target. By contrast, RGC axons expressing nonfunctional FGFR mutants entered the tectum correctly. These findings demonstrate a role for FGFR signaling in the extension and targeting of RGC axons and suggest that receptor tyrosine kinase/growth factor interactions play a critical function in establishing initial connectivity in the vertebrate visual system.

Animals↗
PubMed1996

Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo.

The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

Animals↗
PubMed1995

Postnatal changes in the uncrossed retinal projection of pigmented and albino Syrian hamsters and the effects of monocular enucleation.

Anterograde and retrograde tracing techniques have been used to study the uncrossed retinal projection in neonatal pigmented and albino Syrian hamsters. The total number of retinal ganglion cells projecting ipsilaterally peaks at postnatal days 2-4 (P2-P4) and declines to adult values by P12. The change in cell numbers has a similar time course in albino and pigmented animals. Although the population of uncrossed cells in the temporal retina of albino hamsters is always less than that in pigmented hamsters, no difference between the colour phases was found for the population of uncrossed cells in nasal retina. Differential cell death also contributes to the adult albino decussation pattern in hamsters: The relative loss of cells from temporal retina in albinos (72%) is greater than that in pigmented animals (56%). The additional loss in albinos does not appear to depend on binocular interactions: The same proportion (30%) of uncrossed cells is "rescued" from death by neonatal monocular enucleation in both colour phases. Flat-mount preparations showing the distribution of uncrossed fibres reveal that a distinct focus of terminals emerges in rostral superior colliculus, which is topographically appropriate for a binocular mapping, at the peak of uncrossed ganglion cell numbers (P4). Comparison of uncrossed terminal distributions and ganglion cell death reveals considerable refinement of the terminals prior to the main phase of cell death. Monocular enucleations performed some time after birth have a greater effect on uncrossed terminal distributions than on cell death. These observations suggest that independent mechanisms may be involved in the regulation of terminal distributions and of cell numbers in the developing uncrossed retinal pathways.

Animals↗
PubMed1995

Chimeric integrins expressed in retinal ganglion cells impair process outgrowth in vivo.

Integrin function in retinal ganglion cell (RGC) development was examined in vivo by transfecting genes encoding various dominant forms of the chicken beta 1 integrin subunit into intact eye primordia of Xenopus embryos. RGCs expressing the chimeric chicken/Xenopus integrin receptors exhibited a marked reduction in process outgrowth with only 27% extending an axon and 41% bearing dendrites compared to control levels of 85-88%. None of the integrin constructs impaired the ability of RGC axons to pathfind appropriately or of retinal precursors to migrate to different laminar positions. Chimeric integrin expression also impaired process outgrowth in cells of the inner nuclear layer, although to a lesser extent than RGCs. Transfected diencephalic neurons, by contrast, showed normal levels of process outgrowth. These findings show that beta 1 integrins play an important role in regulating the outgrowth of axons and dendrites from RGCs in the retina but that chimeric integrins do not impair growth cone steering in general.

Animals↗
PubMed1995

FGF signaling and target recognition in the developing Xenopus visual system.

We report that the growth cones of Xenopus retinal ganglion cells express fibroblast growth factor receptors (FGFRs) and that bFGF stimulates neurite extension from cultured retinal neurons. Furthermore, bFGF is abundant in the developing optic tract but is reduced in the optic tectum. To test whether FGF signaling plays a role in axonal guidance in vivo, bFGF was exogenously applied to the developing optic pathway in "exposed brain" preparations. FGF-treated retinal axons navigate normally through the optic tract, but the majority veer aberrantly at the tectal border and bypass the target. Our results implicate FGF signaling in target recognition and suggest that diminished levels of bFGF in the tectum cause arriving axons to slow their growth.

Animals↗
PubMed1995

Absence of topography in precociously innervated tecta.

The retinotectal map in Xenopus forms very early: retinal axons are topographically ordered along the dorsoventral axis of the tectum by stage 39, as they first arrive. To test whether topographic cues are present even earlier, we forced retinal axons to innervate the tectum prematurely by transplanting stage 28 eye primordia into stage 20 hosts, then assayed dorsoventral topography using focal injections of lipophilic dye into dorsal and ventral retina at donor stages 39-40. Unoperated and isochronic control projections showed normal dorsoventral ordering both in the optic tract and in the tectum. In contrast, projections from heterochronically transplanted eyes were ordered in the tract, but spread out upon entering the tectum and did not show significant dorsoventral ordering. Individual axons entering the tectum precociously often made abnormally abrupt and topographically incorrect turns. Thus, the topographical cues normally expressed in the tectum at stage 39 appear to be absent a few hours earlier. However, this lack of cues is only temporary, since heterochronic transplants allowed to survive to donor stages 45-46 showed normal topography. The absence of tectal topography at a stage when retinal axons can navigate to the young tectum strongly suggests that the molecules that provide tectal topographical signals are distinct from those used for pathfinding in the diencephalon and target recognition at the tectum.

Animals↗
PubMed1994

Cadherin-mediated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm.

Muscle progenitors in Xenopus interact in a community of 100 or more cells to activate their myogenic genes and the muscle differentiation pathway. We examine whether the cell adhesion molecule cadherin is involved in this process. Injections of dominant negative N-cadherin RNA into the region of 2- to 4-cell embryos that will give rise to muscle suppress MyoD expression in muscle progenitor cells. By contrast, Xbra expression is unaffected and levels of Xwnt-8 message rise with increasing doses of dominant negative cadherin RNA. MyoD inhibition in embryos injected with the dominant negative cadherin mRNA is rescued by coinjection of full-length cadherin RNA, showing that the inhibition of MyoD occurs through the cadherin pathway. These results show that cadherin-mediated cell interactions play a critical role in the signaling events required for muscle progenitor cells to differentiate, as judged by their stable activation of MyoD.

Animals↗
PubMed1993

Position, guidance, and mapping in the developing visual system.

Positional identity in the visual system affects the topographic projection of the retina onto its central targets. In this review we discuss gradients and positional information in the retina, when and how they arise, and their functional significance in development. When the axons of retinal ganglion cells leave the eye, they navigate through territory in the central nervous system that is rich in positional information. We review studies that explore the navigational cues that the growth cones of retinal axons use to orient towards their target and organize themselves as they make this journey. Finally, these axons arrive at their central targets and make a precise topographic map of visual space that is crucial for adaptive visual behavior. In the last section of this review, we examine the topographic cues in the tectum, what they are, when, and how they arise, and how retinal axons respond to them. We also touch on the role of neural activity in the refinement of this topography.

Animals↗
PubMed1993

Navigational errors made by growth cones without filopodia in the embryonic Xenopus brain.

We have developed an exposed brain preparation for observing growth cone pathfinding behavior while performing in vivo pharmacological manipulations, and we used it to test whether Xenopus retinal growth cones need filopodia to navigate. Time-lapse video observation showed that cytochalasin B acted quickly and reversibly when applied; cytochalasin B-treated growth cones lacked filopodia, but had active lamellipodia and continued to advance slowly. Whereas normal retinotectal axons visualized with horseradish peroxidase turn caudally in the mid-diencephalon to reach the tectum, cytochalasin B-treated axons grew past the normal turning point and, instead, continued straight within the diencephalon. In dose-response experiments, pathfinding became abnormal in the same concentration range in which filopodia disappeared. These results suggest that filopodia are necessary for retinal growth cones to respond to guidance signals in the diencephalon.

Animals↗
PubMed1990

Lipofection of cDNAs in the embryonic vertebrate central nervous system.

Neurons from the embryonic brain of Xenopus were transfected in vivo with a vector expressing luciferase cDNA using a simple lipofection procedure. Luciferase activity was monitored quantitatively, and the protein was immunolocalized in whole-mount embryonic brains. Luciferase-expressing neurons were often intensely labeled, displaying a Golgi-like filling of their dendrites, axons, and growth cones. Luciferase expression could be targeted to the retina by simply removing the skin epidermis covering the area and exposing the whole embryo to the DNA-lipofectin mixture. Luciferase activity in transfected embryos rose to peak values during the first 48 hr posttransfection and was still detectable 28 days later. Cotransfection experiments in which embryonic nervous tissue was exposed simultaneously to two different genes, luciferase and chloramphenicol acetyl-transferase, showed that transfected cells coexpressed the two genes at an extremely high frequency (85%-100%). This offers the possibility of targeting functionally significant genes along with benign reporter genes in the developing CNS.

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