Xenopus temporal retinal neurites collapse on contact with glial cells from
caudal tectum in vitro
ALEXANDER R. JOHNSTON* and DOUGLAS J. GOODAYt
MRC Neural Development and Regeneration Group, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EH9 3JT,Scotland
•Present address: Physiology Department, University of Edinburgh, Medical School, Teviot Place, Edinburght Author for correspondence
Summary
Nasal and temporal retinal neurites were confronted inculture with glial cells from the rostral and caudal partsof the optic tectum and with glial cells from thediencephalon. Twenty of each of the six classes ofencounter between individual growth cones and isolatedglial cells were analysed by time-lapse videorecording.The results show that growth cones from the temporalretina collapse when they contact glial cells from thecaudal tectum, but do not collapse when they contact gliafrom other areas. Growth cones of nasal retinal fibres do
not collapse on contact with any of the glial typesexamined. This suggests that the inhibitory phenomenadescribed by others are in part due to the cell surfacecharacteristics of glial cells, and that there are differ-ences between glia from the front and back of the optictectum.
During the development of the visual system ofXenopus laevis retinal fibres grow along the optic nerve,enter the brain and pass contralaterally up the optictract and terminate on the optic tectum. The terminalsare distributed in a topographically ordered fashionover the tectum, such that temporal retinal axons growto the most rostral parts of the tectum, nasal fibresgrow to caudal tectum, and ventral and dorsal fibresproject to medial and lateral tectum respectively. Theresult of this ordered growth is that a map of the retina,and thus of the visual field is projected onto the tectum.
Several mechanisms have been proposed to accountfor the development of neural maps, and all require theinteraction of growth cones with molecular cues presentin the target area (for a review of molecular mechan-isms see Dodd and Jessel, 1988). Recent experimentsusing chick and goldfish tissues in vitro have supportedthe idea that a graded series of cues on the tectum isrecognised by retinal growth cones (Bonhoeffer andHuf, 1982; Walter et al. 1987a; Vielmetter andStuermer, 1989). These workers have all used elegant invitro assays to examine the behaviour of retinal neuritesgrowing on cells or plasma membranes isolated fromdifferent parts of the optic tectum. The results showedthat temporal retinal axons recognise and respond to aproperty of the tectal cells that is gTaded rostrocaudally.
When presented with a choice of growing on eitherrostral or caudal tectal cells or membranes, temporalfibres always grew on the more rostral substratum;nasal fibres made no distinctions and grew equally wellon both types of substratum. The experiments of Walteret al. (19876) suggest that the graded property to whichtemporal fibres are responding is an inhibitory factorpresent on the surfaces of caudal tectal cells, andtherefore temporal fibres choose to grow on rostral cellsbecause they are inhibited from doing so on caudalcells.
In previous papers (Gooday, 1990; Jack et al. 1991),we have examined the growth of Xenopus neurites fromnasal and temporal retina on pure substrata of glial cellsisolated from the diencephalon and from the rostral andcaudal thirds of the optic tectum. On glial cells from thediencephalon, there were no differences in the growthof nasal and temporal fibres; both types of fibre grew aslong thin fascicles. On tectal glial cells, however,temporal fibres were restricted to growing in short fatfascicles. This property of tectal glial cells is moreconcentrated in glia from the caudal third of the tectum;nasal and temporal fibres show different growthpatterns on rostral tectal glia, although this difference isless marked. This restriction of the growth of temporalfibres by caudal tectal glial cells is in accordance withthe inhibitory theory proposed by Walter et al. (1987b).
The phenomenon of neurite inhibition has been
410 A. R. Johnston and D. J. Gooday
recognised for many years. The importance of growthcone inhibition in the formation of neuronal connec-tions during normal development and in regeneration,however, has only recently been realised (for a reviewsee Patterson, 1988). The in vitro experiments of Bray etal. (1980) and of Kapfhammer et al. (1986) showed thatembryonic chick retinal and sympathetic neurites didnot mix in culture; they suggested that active avoidanceof heterotypic neurites could serve as a guidancemechanism during development. Kapfhammer andRaper (1987) showed that this avoidance was caused bythe collapse of growth cones and retraction of neuriteson contact with heterotypic neurites. This worksuggests that growth cone collapse is an integral part ofneurite inhibition in vitro and may then serve as a usefulindicator in examining other inhibitory phenomena.
In this paper, we have analysed encounters betweenretinal growth cones and individual glial cells in vitro toexamine whether retinal neurites are inhibited by theglial cells that they may encounter as they grow to theirtargets in the optic tectum.
Materials and methods
Sparse cultures of glial cells were prepared from the tecta anddiencephalons of Xenopus laevis tadpoles at stage 54(Niewkoop and Faber 1967), the stage at which there is amaximal growth of retinal fibres (Jacobson 1976; Beach andJacobson 1979). Explants of retina from the most nasal andtemporal parts of the eye were cultured with the glial cells andencounters between growth cones and glial cells wererecorded by time-lapse videomicroscopy.
Preparation of substratumPlastic tissue culture Petri dishes (Nunc, 35 mm) were coatedwith laminin by applying a solution of laminin (20/^gmr1 inNiu Twitty saline, Gibco/BRL). The dishes were left for 2 h atroom temperature, and were rinsed in Niu Twitty salinebefore use.
Preparation of glial cellsThe tecta and diencephalons from four or five stage 54tadpoles were dissected aseptically and placed in 500 fA ofcalcium- and magnesium-free Niu Twitty saline. The tissueswere flushed three times through a 1 ml syringe fitted with a21G needle, three times through a 23G needle and finallytwice through a 26G needle. Each passage was performedslowly to avoid entrapment of air bubbles. The dissociatedtissues were centrifuged at 2000 revs min"1 for 2min and thepellet was resuspended in 1 ml of culture medium. Aliquots ofcell suspension (100 il) were placed in microculture chambersmade by sealing a 13 mm diameter silicone rubber ring(Sylgard 184 Silicone rubber elastomer, Dow Corning) to thebase of a laminin-coated Petri dish. The suspensions were leftfor 3—4 days at 20°C in a refrigerated incubator; the rings wereremoved and the dishes were filled with 2ml of medium.Before use, the neuronal cells were removed by flushing withmedium expelled from a Pasteur pipette. The cultures wereallowed to grow until the cell density was around6 cells mm"2.
Preparation of retinal explantsThe optic nerves of stage 54 tadpoles were cut bilaterally
under MS222 anaesthesia to stimulate later outgrowth offibres (Agranoff et al. 1976). The animals were allowed torecover for 10 days. They were then killed, the eyes removedand the retinas dissected free. Small parts of the retina, about250/an square, were cut from the most nasal, and temporalregions and were placed in the middle of the glial cell cultures.The explants were held in place by small squares ofpolyacrylamide gel weighted with fragments of glass slide.The gels and glass were removed 24 h later.
Tissue culture mediumTissue culture medium consisted of 60% L15 (Flow), 6g l - 1
D-glucose (BDH), O^gl"1 L-glutamine (Flow), ^ m g T 1
insulin (Sigma) and 10% foetal calf serum (Sera Lab).
Immunocytochemical identification of glial fibrillaryacidic proteinTissue for anti-GFAP staining was fixed in methanol at — 20°Cfor 30min and was then immersed in a cryoprotectant solutionof 30 % sucrose in PBS and left overnight. The tissue was thensnap-frozen in a mixture of acetone and dry ice, mounted onchucks with OCT compound (BDH), and 20/an cryostatsections were cut and mounted on subbed slides. The sectionswere post-fixed in methanol at -20°C for 6min, rinsed threetimes in PBS containing 0.1% Triton X-100 for 5min eachand then transferred to 10% horse serum in PBS for 30min,drained of excess serum and incubated overnight at 4°C inanti-GFAP (1:50, clone G-A-5, Boehringer and Sigma);control slides were incubated in 0.1% BSA in PBS. Afterincubation in primary antiserum slides were rinsed three times15 min each in PBS, incubated in biotinylated anti-mouse IgG1:100 (Vector) for 30min, rinsed again three times andincubated with ABC complex (Vector) for 30 min, then rinsedthree times in PBS. The HRP was visualised using DAB as thechromogen.
To confirm the glial nature of the cultured cells, some of thecultures were stained with anti-GFAP after photographing agrowth cone collapse. The cells in question were identified forfuture location by scratching the plastic dish around the cellsusing a fine needle. The cultures were then fixed in methanolat —20°C for 6 min and were then treated according to theabove protocol for tissue sections, with the exception thatAvidin-Texas Red 1:100 (Vector) was used to visualise thecells.
Recording of resultsAfter addition of the retinal explants, the cultures were leftfor 24 h before being examined and recorded. Videorecord-ings were commenced when retinal growth cones were aboutto contact individual glial cells. To ensure that growth coneshad not already contacted glial cells, situations were chosen inwhich there were no glial cells near the axon. In addition onlythose glial cells that were at least 200-300 fan from the explant
Table 1. Number of growth cones to collapse andretract (r) out of a total (t) for each class of encounter
Origin of glia
DiencephalonRostral tectumCaudal tectum
Origin of
Nasal
t r
19 219 021 2
retinal explant
Temporal
t r
21 223 320 20
Inhibitory effects of Xenopus tectal glia 411
Figs 1 and 2. Sequences of photographs of temporal retinal growth cones contacting caudal tectal glial cells. The numbersat the top right of each photograph refer to the time in minutes since the sequence started. Fig. 1A-D. Temporal growthcone (A) advancing, (B) contacting, (C) collapsing and (D) retracting from an elongated caudal tectal glial cell. Scale barrepresents 50/im. Fig. 2A-D. Temporal growth cone contacting and retracting from a highly flattened glial cell. Scale barrepresents 50/fln.
412 A. R. Johnston and D. J. Gooday
Fig. 3. A-D. Two temporal retinal growth cones retractingfrom two caudal tectal glial cells. (A) Fibre 1 has alreadycontacted and retracted from cell a and a fine stretchedprocess can be seen linking the two (arrowed), fibre 2 hasjust contacted cell b. (B) Twenty-four minutes later fibre 1has again contacted cell a, and fibre 2 has retracted fromcell b. (C) The growth cone of fibre 1 has collapsed 8minafter contacting cell a, and has begun to withdraw from thecell. Fibre 2 has developed a new growth cone. (D) Fibre 1has completely retracted from cell a, and fibre 2 has begunto advance towards cell b again. Scale bar represents 50 j/m.
were chosen to ensure that neurites were not encounteringretinal glia. Recordings were made using a Leitz Fluovertmicroscope, a Panasonic WV-1500 TV camera and PanasonicNV-8055 time-lapse video recorder with NV-F85 time dategenerator. Photographs were taken at selected intervals usingIlford FP4 35 mm film and a Wild MPS 45 camera.
Results
Time-lapse videorecordings were made of growthcone-glial cell encounters of each of six types: nasal andtemporal growth cones were confronted with glia fromthe diencephalon, the rostral third of the optic tectumand the caudal third of the tectum. The number ofrecordings made and the number of growth cones toshow collapsing behaviour and retraction are listed inTable 1.
Contact of temporal retinal neurites with caudal tectalglial cellsTwenty encounters of this sort were recorded and, in allcases, the growth cone of the advancing neuritecollapsed on contacting the glial cell. Growth conescollapsed and retracted from both flattened andelongated glial cells. Encounters of this type are shownin Figs 1 and 2. Typically temporal fibres advanced at amean rate of 78/xmh"1. The growth cones of both nasaland temporal retinal neurites consisted of largeirregular lamellipodia with a few filopodia, there tendedto be more filopodia associated with the rear of thegrowth cones rather than at the leading edge. Thelamellipodia exhibited extensive ruffling activity at theleading edge. On contact with caudal glial cells thisruffling activity did not immediately cease, and thegrowth cones continued to advance until extensivecontact was made with the cell. Two or three minutesafter initial contact, growth cone morphology began tochange and forward advance ceased. The lamellipodiawere withdrawn and the growth cone shrank in size andafter 4-5 min began to withdraw from the cell. After amean time of 8 min in contact with the cells, the growthcones retracted completely from the glial cells and, insome cases, the neurites were seen to collapse andassume an irregular wavy profile as they withdrew fromthe cells. On withdrawing from the cells, fine stretchedprocesses were often left attached to the glial cells as theneurites retracted up to 100 ^m.
In some cases, we recorded retracted axons that
Fig. 4. A-D. Temporal retinal growth cone contacting adiencephalic glial cell. (A) The growth cone advancestowards the cell, (B) makes extensive contact with the celland (C) continues to grow over the cell (the micrographswere taken using an inverted microscope with the cell-substratum interface as the focal plane). (D) The growthcone progresses in contact with an extended process of theglial cell. Scale bar represents 50/un.Fig. 5. A-D. Nasal growth cone encountering a rostraltectal glial cell. (A) The growth cone advances towards thestretched process of a glial cell. (B) Contact is made withthe cell and (C) the growth cone grows over the cellprocess. Scale bar represents 50/an.
Inhibitory effects of Xenopus tectal glia 413
414 A. R. Johnston and D. J. Gooday
Figs 6 and 7. Temporal growth cones collapsing and retracting from caudal tectal glial cells. After retraction, the cultureswere fixed and stained with anti-GFAP. Scale bars represent 50 /an.
organised new growth cones and advanced again tocontact, collapse and retract from the same cell. Fig. 3shows such an example; fibre 1 has already retractedfrom cell a, and a fine stretched process can be seenlinking the two. In Fig. 3B, fibre 1 has developed a newgrowth cone and it has advanced again to contact the
cell. In Fig. 3C and D, it collapses and retracts from thecell again. Meanwhile fibre 2 contacts and withdrawsfrom cell b.
All other encountersIn encounters between temporal retinal growth cones
Inhibitory effects of Xenopus tectal glia 415
Fig. 8. (A) Anti-GFAP staining of frozen section of tadpole tectum. This antibody (clone G-A-5) only stains radial glialcells which span the width of the brain. Scale bar represents 100/zm. (B,C) Higher power views of the areas outlined in Ato show the glial endfeet at the pial margin. Scale bar represents 100/im.
and glial cells from rostral tectum and diencephalon,and in encounters of nasal growth cones with gh'a fromall three tissues, the majority of growth cones did notshow any collapsing or retracting behaviour (Table 1).Examples of a temporal growth cone contacting adiencephalic glial cell, and a nasal growth coneencountering a rostral tectal cell are shown in Figs 4 and5. In most cases, the rate of forward advance of thegrowth cones slowed on contact with glial cells, butruffling activity of the lamellipodia continued. This gavethe impression that the growth cones were 'exploring'the surfaces of the glial cells. The growth conescontinued to advance over the surface of the cells andon crossing the cells continued growing on the plasticsurface.
Identity of the glial cellsThe cells in our cultures have been identified as glia inprevious publications by staining with anti-GFAP(Gooday, 1990; Jack et al. 1991). In this paper, we havefixed and immunocytochemically characterised ident-ified cells after they had induced the collapse oftemporal retinal growth cones Figs 6 and 7; this was
performed on six separate occasions and all the cellsstained with anti-GFAP. In frozen sections of brain, theanti-GFAP stained only radial glial cells. Thesespanned the width of the tectum from the ventricle tothe pial margin, where they terminated in enlargedendfeet (Fig. 8).
Discussion
The results of this study show that in vitro the growthcones of temporal retinal neurites collapse and retractfrom caudal tectal glial cells, but not from rostral cells,or from cells from the diencephalon. Nasal fibres rarelyretract from any of the glia and continue to grow overthe surface of the cells. The results suggest thattemporal retinal fibres are inhibited from growing to aninappropriate target, the caudal optic tectum, due tothe repulsive nature of the glia; nasal fibres do not findcaudal glia repulsive. As yet we cannot say whether thisrepulsive effect is graded across the tectum. Due to thesmall size of the Xenopus brain it is feasible to dissect itinto only three parts, the middle part was discarded toensure no overlap of the cell types isolated. The stripe
416 A. R. Johnston and D. J. Gooday
assays of Walter et al. (1987a) on chick and ofVielrnetter and Stuermer (1989) on goldfish suggest thatthis effect does operate in a rostrocaudal gradient.
It is unclear at present how this collapsing activityrelates to the in vivo guidance of retinal axons on thetectum, the culture methods in this paper detectcollapsing activity only in the caudal third of the tectum,a part that is not encountered by temporal fibres innormal growth. Our earlier work (Gooday, 1990; Jacket al. 1991), which involved culturing retina onconfluent monolayers of glia, showed that temporalfibres were highly growth-restricted on caudal tectal gliaand, to a lesser extent on rostral tectal glia. However,the present study shows that rostral tectal glial cells donot cause the collapse of any temporal growth cones.The previous studies would have led us to expect thatrostral cells would have caused a proportion oftemporal growth cones to collapse. The lack of collapseinduced by rostral glia in this present work could be anartefact of culture; the expression of an inhibitorymolecule by the glia, or the threshold of its recognitionby growth cones may be lower in vitro than in vivo. Thediscrepancy between the experiments could also be dueto a quantitative effect due to the numbers of glial cells:the previous experiments were performed using con-fluent monolayers in contrast to the individual glial cellsused in this study. The sections of brain stained withanti-GFAP (Fig. 8) show that there are many hundredsof radial glia in the tectum, so it is probable that growthcones in vivo are in contact with more than one glial cellat any time. In this respect the collapse assay is perhapsa more artificial environment than monolayer culture.Therefore, assuming that a gradient of inhibition existsover the tectum, a higher level of inhibitory activity percell may be required to elicit a response in the collapseassay than in the monolayer assay or in vivo.
Recent work from several laboratories suggests thatthe collapsing activity is caused by a cell surfacecomponent. Raper and Kapfhammer (1990) have foundcollapsing activity in enriched, detergent-soluble ex-tracts of chick brain plasma membranes of putativerelative molecular mass 50X103. Davies et al. (1990)have isolated a Peanut agglutinin-binding glycoproteinfraction from chick somites, which causes the collapseof dorsal root ganglion growth cones in vitro. They haveproposed that this molecule restricts the growth ofsensory and motor nerves to the anterior half of eachsclerotome, and so brings about the segmental patternof spinal nerves characteristic of vertebrates. Cox et al.(1990) prepared suspensions of plasma membranesfrom anterior and posterior chick tectum. Posteriormembranes, and to a lesser extent anterior membranes,caused the growth cone collapse and retraction oftemporal retinal fibres, whereas nasal fibres remainedunaffected by either preparation.
AH these extracts have so far been made from wholetissues, and the precise cellular locations of anyinhibitory molecules are so far unknown. Our resultsusing purified populations of glial cells firmly indicatethat such molecules are present on the surfaces of glialcells from the caudal part of the optic tectum at least.
Our results also suggest that glial cells may providepositional information for nerve fibres in target areas inaddition to their role as general mediators of axonalguidance. Further evidence for the provision ofpositional information comes from the recent work ofSuzue et al. (1990) who raised monoclonal antibodiesagainst rostrocaudally graded molecules in mammaliansympathetic ganglia. One antibody ROCA1 bound to aglial cell-surface component in rostral ganglia and thebinding intensity decreased in caudal ganglia.
The role of glia in facilitating axon growth and inguiding neuronal trajectories during development iswell documented (reviewed by Hatten, 1990), andrecently the significance of inhibitory glial neuronalinteractions has been realised. The failure of mam-malian CNS neurons to regenerate may be due in partto two cell-surface components of 35 and 250 xlO3,found in CNS myelin and oligodendrocyte membranes,which repel neurites (Schwab and Caroni, 1988).Oligodendrocyte-type cells in the optic nerve ofgoldfish, however, support the growth of retinalneurones, and promote axonal regeneration in vitro.Goldfish retinal axons recognise and are inhibited bymammalian oligodendrocytes; however, goldfish oligo-dendrocytic cells also support the growth of chickretinal neurones in vitro (Bastmeyer et al. 1991). Thissuggests that a fundamental difference exists betweenthe oligodendrocytes of birds and mammals and theirequivalents in fish and amphibia, in the ability of thesecells to support axon growth. The glial cells in ourcultures are either elongated cells or fibroblast-like andare unlike cultured mammalian oligodendrocytes mor-phologically. They stain with an antibody to pig GFAP,and this same antibody only stains radial glial cells infrozen sections of Xenopus tectum. The developmentallineages of amphibian glial cells, and for that matter,goldfish glial cells, are not known and little antigeniccharacterization has been done, unlike the situation inthe rat optic nerve. Therefore we are reluctant to assigna glial category to these cells. The cells in our culturesmay be equivalent to radial glial cells and it is probablethat growing retinal fibres contact radial glia in vivo, asthe glial processes extend to the pial margin of thetectum, where they terminate in enlarged endfeet.
The role of glia in organising fibre pathways in thevisual systems of several classes of vertebrates hasalready been suggested by others. Several lines ofevidence suggest that at key positions in the opticpathway, where fibre reordering or rerouting takesplace, there is a change in glial cell phenotype. In theoptic system of cichlid fish, there is a change inintermediate filament expression in glial cells at theboundary between the nerve and the tract (Maggs andScholes, 1986). A change in glial type between nerveand tract has also been found in the ferret (Guillery andWalsh, 1987).
Silver (1984) suggested that some glial structurescould act as barriers to axon growth. He demonstratedthe presence of two distinct glial structures in the opticchiasm of embryonic mice. Fibres from ventrotemporaland ventronasal retina occupy opposite sides of the
Inhibitory effects of Xenopus tectal glia 417
tract and come into contact with different glialstructures. Fibres from ventrotemporal retina encoun-tered a maze-like system of glial processes and channelsthat seemed to direct growth ipsilaterally, whereasfibres from more nasal retina encountered a dense glial'knot' which appeared to impede growth and deflectaxons contralaterally. Other axon-deflecting glial struc-tures have been identified: the roof plate of thedeveloping spinal cord and brain may serve to maintainthe side-specificity of developing axonal tracts, whichdo not normally cross the midline. The roof plate glialcells secrete keratan sulphate glycosaminoglycan (Snowet al. 1990a), which has been shown to be inhibitory forneurite growth in vitro (Snow et al. 19906). Interferencewith the roof plate of the mesencephalon in hamstersleads to the misrouting of axons across the midline(Poston et al. 1988; So, 1979; Wu et al. 1989).
With the current level of interest in the role of glialcells and inhibitory phenomena in the development ofthe nervous system, it seems probable that research intoother aspects of neural development will result in othersuch phenomena being described.
We are grateful to Professor R.M. Gaze for help and adviceand to James Jack, Maida Davidson and Christine Virtue fortechnical assistance. The project was funded by the MedicalResearch Council of Great Britain, and by a Summer Bursaryfrom the Nuffield Foundation awarded to A.R.J.
References
AGRANOFF, B. W., FIELD, P. AND GAZE, R. M. (1976). Neurite
outgrowth from explained Xenopus retina: an effect of prioroptic nerve section. Brain Res. 113, 225-234.
BASTMEYER, M., BECKMANN, M., SCHWAB, M. E. AND STEURMER,
C. A. O. (1991). Growth of regenerating goldfish axons isinhibited by rat oligodendrocytes and CNS myelin but not bygoldfish optic nerve tract oligodendrocyte like cells and fish CNSmyelin. / . Neuroscience 11, 626-640.
BEACH, P. M. AND JACOBSON, M. (1979). Pattern of cellproliferation in the retina of the clawed frog duringdevelopment. J. comp. Neurol. 183, 603-611.
BONHOEFFER, F. AND HUF, J. (1982). In vitro experiments on axonguidance demonstrating an anterior-posterior gradient on thetectum. EMBO J. 1, 427-431.
BRAY, D., WOOD, P. AND BUNGE, R. P. (1980). Selectivefasciculation of nerve fibres in culture. Expl Cell Res. 130,241-250.
Cox, E. C , MULLER, B. AND BONHOEFFER, F. (1990). Axonalguidance in the chick visual system: posterior membranes inducecollapse of growth cones from the temporal retina. Neuron 4,31-37.
DAVIES, J. A., COOK, G. M. W., STERN, C. D. AND KEYNES, R. J.(1990). Isolation from chick somites of a glycoprotein fractionthat causes collapse of dorsal root ganglion growth cones.Neuron 4, 11-20.
DODD, J. AND JESSEL, T. (1988). Axon guidance and thepatterning of neuronal projections in vertebrates. Science 242,692-699.
GOODAY, D. J. (1990). Retinal axons in Xenopus laevis recognisedifferences between tectal and diencephalic glial cells in vitro.Cell Tissue Res. 259, 595-598.
GUILLERY, R. W. AND WALSH, C. (1987). Changing glial
organisation relates to changing fibre organisation in thedeveloping optic nerve of ferrets. J. comp. Neurol. 265,203-217.
HATTEN, M. E. (1990). Riding the glial monorail: a commonmechanism for glial-guided neuronal migration in differentregions of the developing mammalian brain. Trends inNeurosciences 13, 179-184.
JACK, J. L., GOODAY, D. J., WILSON, M. A. AND GAZE, R. M.
(1991). Retinal axons in Xenopus show different behaviourpatterns on various glial substrates in vitro. Anat. Embryol. 183,193-203.
JACOBSON, M. (1976). Histogenesis of retina in the clawed frogwith implications for the pattern of development of retinotectalconnections. Brain Res. 103, 541-545.
KAPFHAMMER, J. P., GRUNEWALD, B. E. AND RAPES, J. A. (1986).
The selective inhibition of growth cone extension by specificneurites in culture. /. Neuroscience 6, 2527-2534.
KAPFHAMMER, J. P. AND RAPER, J. A. (1987). Collapse of growthcone structure on contact with specific neurites in culture. /Neuroscience 7, 201-212.
MAGGS, A. AND SCHOLES, J. (1986). Glial domains and nerve fibrepatterns in the fish retinotectal pathway. / . Neuroscience 6,424-438.
NIEWKOOP, P. D. AND FABER, J. (1967). A Normal Table ofXenopus laevis (Daudin). North Holland, Amsterdam.
PATTERSON, P. (1988). On the importance of being inhibited, orsaying no to growth cones. Neuron 1, 263-267.
POSTON, M. R., JHAVERI, S., SCHNEIDER, G. AND SILVER, J. (1988).
Damage of a midline boundary and formation of a tissue bridgeallows the misguidance of optic axons across the midline inhamsters. Soc. Neurosci. Abstr. 14, 595.
RAPER, J. A. AND KAPFHAMMER, J. P. (1990). The ennchement ofa neuronal growth cone collapsing activity from embryonic chickbrain. Neuron 4, 21-29.
SCHWAB, M. E. AND CARONI, P. (1988). Oligodendrocytes andCNS myelin are non-permissive for neurite growth andfibroblast spreading in vitro. J. Neuroscience 8, 2381-2393.
SNOW, D. M., LEMMON, V., CARJUNO, D. A., CAPLAN, A. L. AND
SILVER, J. (1990£>). Sulfated proteoglycans in astroglial barriersinhibit neurite growth in vitro. Expl Neurol. 109, 111-130.
SNOW, D. M., STEINDLER, D. A. AND SILVER, J. (1990a).
Molecular and cellular characterisation of the glial roof plate ofthe spinal cord and optic tectum: a possible role for aproteoglycan in the development of an axon barrier. Devi Biol.138, 359-376.
So, K-F. (1979). Development of abnormal recrossing retinotectalprojections after superior colhculus lesions in newborn Syrianhamsters. J comp. Neurol. 186, 241-258.
SUZUE, T., KAPRIELIAN, Z. AND PATTERSON, P. H. (1990). A
monoclonal antibody that defines rostrocaudal gradients in themammalian nervous system. Neuron 5, 421-431.
VIELMETTER, J. AND STUERMER, C. A. O. (1989). Goldfish retinalaxons respond to position-specific properties of tectal cellmembranes in vitro. Neuron 2, 1331-1339.
WALTER, J., HENKE-FAHLE, S. AND BONHOEFFER, F. (1987b).
Avoidance of posterior tectal membranes by temporal retinalaxons. Development 101, 909-913.
WALTER, J., KERN-VEITS, B., HUF, J., STOLZE, B. AND
BONHOEFFER, F. (1987a). Recognition of position-specificproperties of tectal cell membranes by retinal axons in vitro.Development 101, 685-696.
Wu, D-Y., JHAVERI, S. AND SCHNEIDER, G. (1989). Recrossing ofretinal axons after early tectal lesions in hamsters occurs onlywhere vimentin- and GFAP-positive midline cells are damaged.Soc. Neurosci. Abstr. 15, 873.
