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Mol. Cell. Neurosci. 25 (2004) 56–68

EphA/ephrin-A interactions during optic nerve regeneration:

restoration of topography and regulation of ephrin-A2 expression

J. Rodger,a,b,* P.N. Vitale,a L.B.G. Tee,a C.E. King,a C.A. Bartlett,a A. Fall,a C. Brennan,c

J.E. O’Shea,a S.A. Dunlop,a,b and L.D. Beazleya,b

aSchool of Animal Biology, The University of Western Australia, Crawley 6009, Western Australia, AustraliabWestern Australian Institute of Medical Research, Western Australia, AustraliacQueen Mary College, University of London, London, UK

Received 2 June 2003; revised 15 September 2003; accepted 18 September 2003

During visual system development, interactions between Eph tyrosine

kinase receptors and their ligands, the ephrins, guide retinal ganglion

cell (RGC) axons to their topographic targets in the optic tectum. Here

we show that Eph/ephrin interactions are also involved in restoring

topography during RGC axon regeneration in goldfish. Following optic

nerve crush, EphA/ephrin-A interactions were blocked by intracranial

injections of recombinant Eph receptor (EphA3-AP) or phospho-

inositol phospholipase-C. Topographic errors with multiple inputs to

some tectal loci were detected electrophysiologically and increased

projections to caudal tectum demonstrated by RT-97 immunohisto-

chemistry. In EphA3-AP-injected fish, ephrin-A2-expressing cells in the

retino-recipient tectal layers were reduced in number compared to

controls and their distribution was no longer graded. The findings,

supported by in vitro studies, implicate EphA/ephrin-A interactions in

restoring precise topography and in regulating ephrin-A2 expression

during regeneration.

D 2003 Elsevier Inc. All rights reserved.

Introduction

Eph tyrosine kinase receptors and their ligands, the ephrins, are

cell-membrane bound proteins with tightly regulated expression

that mediate cell–cell interactions both during development and in

the adult (Drescher, 1997; Mc Laughlin et al., 2003). Eph/ephrins

are classified into two families: EphAs bind to glycosylphospha-

tidylinositol (GPI)-linked ephrin-As and EphBs to transmembrane

ephrin-Bs (Flanagan and Vanderhaeghen, 1998). Spatially and

chronologically restricted interactions between Eph/ephrin-As

and/or Bs control many aspects of development including rhom-

bomere formation and blood vessel patterning (Brantley et al.,

2002; Conover et al., 2000; Cooke and Moens, 2002; Cooke et al.,

2001; Durbin et al., 1998; Wang et al., 1998). In addition, Ephs/

1044-7431/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2003.09.010

* Corresponding author. Department of Zoology, The University of

Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia,

Australia. Fax: +61-8-9380-1029.

E-mail address: jrodger@cyllene.uwa.edu.au (J. Rodger).

Available online on ScienceDirect (www.sciencedirect.com.)

ephrins play important roles in the nervous system by controlling

development of topographic organisation and aspects of synaptic

plasticity throughout life (Contractor et al., 2002; Gao et al., 1998;

Gerlai, 2002; Marın et al., 2001; Rogers et al., 1999; Vanderhae-

ghen et al., 2000).

In the developing visual system, complementary gradients of

Eph/ephrins define the projection of retinal ganglion cell (RGC)

axons within the major primary visual centre, the optic tectum

(superior colliculus in mammals), fulfilling the predictions of

Sperry’s chemoaffinity hypothesis (Karlstrom et al., 1996; Mc

Laughlin et al., 2003; Sperry, 1963; Trowe et al., 1996). Eph/

ephrins guide RGC axons and control branch formation (Connor et

al., 1998; Yates et al., 2001) by interactions that are primarily

repulsive (EphA/ephrin-As) or attractive (EphB/ephrin-Bs; Holm-

berg and Frisen, 2002). Gradients and counter-gradients of EphAs

and ephrin-As are expressed along the naso-temporal retinal and

rostro-caudal tectal axes (Brennan et al., 1997; Cheng et al., 1995;

Hornberger et al., 1999); gradients of EphBs and ephrin-Bs define

the orthogonal dorso-ventral retinal to medio-lateral tectal axes

(Hindges et al., 2002; Mann et al., 2002). In addition, Eph/ephrins

guide outgrowth and fasciculation of RGC axons within the retina,

optic nerve and tract (Braisted et al., 1997; Caras, 1997; Marcus et

al., 1996; Nakagawa et al., 2000; Sefton et al., 1997).

A key role of Eph/ephrin interactions in the development of

topography has been revealed using mutant mice lacking or over-

expressing one or more of the Eph/ephrin proteins (Brown et al.,

2000; Feldheim et al., 2000; Hornberger et al., 1999; Park et al.,

1997). RGC axon guidance is also abnormal in wild-type animals

following in vivo viral mis-expression of ephrin-A2 in the retina or

tectum (Hornberger et al., 1999; Nakamoto et al., 1996), or when

Eph/ephrin interactions are prevented in vivo (Mann et al., 2002)

or in vitro (Ciossek et al., 1998; Hornberger et al., 1999; Nakamoto

et al., 1996; Winslow et al., 1995). Less is known about the role of

Eph/ephrin interactions in the restoration of topography following

injury in the normal adult. During optic nerve regeneration in

goldfish, specific EphAs and ephrin-As are up-regulated coincident

with restoration of retino-tectal topography (King et al., in press;

Rodger et al., 2000). The result suggests that, as in development,

EphA/ephrin-A interactions are required for the restoration of

topography.

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–68 57

To test the hypothesis, we prevented EphA/ephrin-A interac-

tions within the goldfish tectum during optic nerve regeneration

and examined subsequent topography. We treated the tectum with

recombinant EphA3 protein linked to alkaline phosphatase

(EphA3-AP) to mask ephrin-As from endogenous EphA receptors.

In other animals, we applied phospho-inositol phospholipase-C

(PIPLC) to remove all GPI-linked proteins from cell membranes

(Low, 1989). The treatments affect all ephrin-As expressed on

RGC axons and tectal cells, including the two main contributors to

retino-tectal topography, ephrin-A2 and ephrin-A5; the relative

contributions of these molecules to optic nerve regeneration remain

unknown. We confirmed that both techniques were successful

across the entire tectum by detecting either injected (EphA3-AP)

or endogenous (PIPLC injected) alkaline phosphatase activity

(Cheng et al., 1995; Drawbridge and Steinberg, 2000). Endoge-

nous alkaline phosphatase activity can be used to indicate the

effectiveness of the PIPLC treatment since AP is a GPI-linked

Fig. 1. (A,B) Whole-mounted goldfish brains with the forebrain removed stained fo

tecta in AP-injected (A) or PBS-injected (B) controls. Following EphA3-AP inject

Activity was maximum at 24–48 h and began to decrease at 72 h (A). Following PI

(experimental) tectum at 24 h but had returned by 72 h (B). (C) Photomicrographs

between the rostral and caudal poles. At 24 h post injection, ephrin-A2 immunopo

but absent in that of PIPLC-injected fish. (D) Histogram showing the time course

each of the histograms represent four equidistant sampling locations spanning the

cells compared to normal by 72 h after PIPLC injection. T: tectum, C: cerebellum

enzyme (Low, 1989). Errors in RGC axon projections were

revealed electrophysiologically; neurofilament immunohistochem-

istry using RT-97, a monoclonal antibody that preferentially binds

to regenerating RGC axons (Velasco et al., 2000), verified that

RGC axons had regenerated to retino-recipient tectal layers. In

vitro explant cultures using recombinant ephrin-A5-AP supported a

role for EphA/ephrin-A interactions in guiding regenerating RGC

axons.

We also tested the possibility of an additional function of EphA/

ephrin-A interactions. Transcription of many receptor– ligand pairs

is regulated by a feedback loop signalling via one or both members

of the pair. Examples are the growth factor receptor TrkB that, like

EphA, is a member of the tyrosine kinase receptor family, and the

NMDA glutamate receptor (Frank et al., 1996; Goebel and Poosch,

2001). We propose that similarly, EphA/ephrin-A interactions

regulate ephrin-A2 expression in the tectum. The hypothesis is

supported by the observation that ephrin-A2 up-regulation coin-

r alkaline phosphatase (AP) activity. No differences were observed between

ion, recombinant AP activity was detected in the left (experimental) tectum.

PLC injection, endogenous AP (a GPI-linked enzyme) was absent in the left

of ephrin-A2 immunohistochemistry in the sfgs of goldfish tectum, midway

sitive cells were present in normal numbers in the sfgs of PBS-injected fish

of ephrin-A2 expression in the sfgs after PIPLC injection. The four bars in

rostral to caudal tectal extent. Ephrin-A2 was re-expressed in roughly 1/4 of

. Scale bars: A,B: 1 mm; C: 50 Am.

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–6858

cides with the arrival of regenerating RGC axons (Rodger et al.,

2000). Using immunohistochemistry and in situ hybridisation, we

show that tectal ephrin-A2 expression is abnormal when endoge-

nous EphA/ephrin-A interactions are blocked during optic nerve

regeneration; we examined EphA3-AP-injected fish only since

PIPLC treatment directly reduces ephrin-A2 protein levels (Cheng

et al., 1995; Low, 1989). Some of the results have been presented

in abstract form (Vitale et al., 2003).

Results and discussion

Here, we investigated the role of EphA/ephrin-A signalling in

the generation of topographic order in regenerating RGCs projec-

ting to the tectum. Disruption of EphA/ephrin-A signalling by

injection of either EphA3-AP or PIPLC during optic nerve

regeneration resulted in aberrant retino-tectal topography with

multiple inputs to some tectal loci. Increased projections to caudal

tectum were detected anatomically. Normal topography was re-

stored in uninjected fish and in those injected with AP or

phosphate-buffered saline (PBS) as controls. The findings, sup-

ported by in vitro evidence, implicate EphA/ephrin-A interactions

in restoring precise topography during regeneration. In EphA3-

AP-injected fish, ephrin-A2-expressing cells in the retino-recipient

stratum fibrosum griseum et superficiale (sfgs) were reduced in

number compared to controls and their distribution was no longer

graded.

Efficacy of injections

A time course study measuring levels of endogenous or

recombinant alkaline phosphatase (AP) confirmed that, in normal

fish, the effects of EphA3-AP and PIPLC injections persisted for

48 h. For EphA3-AP, we were unable to confirm whether all

ephrin-As were blocked by the recombinant protein. However,

recombinant AP activity was highest between 24 and 48 h after

injection and decreased thereafter, suggesting that maximum

blocking activity was obtained during this time (Fig. 1A). Activity

was no longer detected by 72 h after injection (Fig. 1A). Following

PIPLC injection, endogenous AP activity was completely absent

from the targeted tectum for up to 48 h after the injection (Fig. 1B).

At 72 h, AP activity was weak and had returned to normal levels

by 120 h (Fig. 1B). In confirmation, ephrin-A2 immunopositive

Fig. 2. Two-dimensional representations of the projection from the retina (large

experimental fish and graphical representations of order across each projection ax

positions across the tectum and the retinal location of RGCs projecting to each

plotted on an AIMARK perimeter using visual field coordinates. Tectal poin

Experimental methods). The method results in the disorder of the projection bein

electrode placements. For each animal, tectal points are joined in the medio-lateral

the retinal representations expose errors in the naso-temporal (NT) to RC projectio

ML projection axis (blue: lower retinal representation). One map is shown for eac

EphA3-AP-injected and PIPLC-injected fish. EphA3-AP map 2 (points 7 and 13)

loci and the more inappropriately projecting point is joined to the row by a dotted

medial; L: lateral. Small crosses indicate no responses. Graphical representations

from normal, EphA3-AP- and PIPLC-injected fish. NT coordinates of retinal poin

and ML coordinates are plotted similarly in separate graphs. A trendline is shown

injected fish, analysis of the NT/RC axis reveals that RGC axons project abnor

normal trendline. Similarly, analysis of the DV/ML axis reveals that RGC axon

underneath the normal trendline. For PIPLC-injected fish, RGC axon projections a

in the DV/ML axis.

cells were not detected in the sfgs of PIPLC-injected fish at 48

h after injection, but up to 25% of the expected cell number could

be detected after 72 h (Fig. 1C). The time course for PIPLC

corresponds to that previously reported in developing axolotl

(Drawbridge and Steinberg, 2000; Zackson and Steinberg, 1989).

For experimental fish receiving multiple injections, AP activity

indicated the extent and localisation of the affected area at 24

h following the final injection. Whole brain preparations stained

with NBT/BCIP confirmed that the actions of EphA3-AP and

PIPLC were consistently localised to the left (experimental) tectum

(Figs. 1A,B); there was no difference in AP activity between

normal, uninjected and control-injected fish.

We also performed morphological and histological analysis of

injected tecta to ensure that observed phenotypes were not due to

injection damage. Injected tecta retained a normal size and shape

and pyknotic nuclei were not observed, suggesting that there was

no significant damage to the tissue. Moreover, total cell numbers in

the sfgs were normal in experimental and control groups with a

uniform distribution across the rostro-caudal axis (Rodger et al.,

2000; Fig. 6A).

Topography

The projection from the retina to the contralateral tectum was

mapped electrophysiologically to examine topographic order.

Responses in normal fish were strong and reliable at all tectal

loci (Fig. 2). In uninjected and control-injected fish, regenerate

responses were strong and reliable from rostral and medial tectum,

but weaker and less reliable caudally and laterally, presumably

reflecting the rostro-caudal sequence of reinnervation (Stuermer,

1986). In all three groups, topography was normal as previously

described (Fig. 2; Meyer, 1977): the naso-temporal (NT) and

dorso-ventral (DV) visual field axes mapped precisely across the

rostro-caudal (RC) and medio-lateral (ML) tectal axes with high

order factors (normal: 0.072 F 0.02; AP: 0.084 F 0.02; PBS:

0.075 F 0.01).

Tectal responses were present in 11/11 EphA3-AP-injected

fish recorded 24 or 48 h following the final injection; similar to

controls, responses were more readily elicited from rostral than

caudal tectum. One EphA3-AP-injected fish had normal topog-

raphy and was excluded from further analysis; we suspect

injections failed due to an accumulation of adipose tissue at

the injection site. For PIPLC-injected fish, 7 fish were mapped

open circles) to the tectum (small closed ovals) in normal, control and

is. To obtain the maps, an electrode was placed serially in regularly spaced

tectal position determined using a moving stimulus. Retinal points were

ts were plotted on a standard grid used for placing the electrode (see

g illustrated in the retina, since order is imposed on the tectal grid by the

(ML; green) and rostro-caudal (RC; blue) axes. The corresponding rows in

n axis (green: upper retinal representation), or in the dorso-ventral (DV) to

h of normal, AP-injected and PBS-injected fish. Three maps are shown for

and PIPLC map 2 (point 14) illustrate multiple responses to a single tectal

line. N: nasal; T: temporal; D: dorsal; V: ventral; R: rostral; C: caudal; M:

reveal order across each projection axis separately for all points recorded

ts are plotted (Y axis) against RC coordinates for tectal points (X axis). DV

in black for normal fish and in red for experimental fish. For EphA3-AP-

mally caudally since the experimental trendline is located underneath the

s project abnormally medially since the experimental trendline is located

re slightly rostrally shifted in the NT/RC axis, but strongly laterally shifted

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–68 59

24 or 48 h following the final injection and all lacked responses.

RT-97 immunohistochemistry detected regenerating RGC axons

in the caudal tectal pole of all 7 fish, ruling out the possibility

that regeneration was delayed or inhibited. In the likely event

that PIPLC digestion was removing GPI-linked proteins required

for synaptogenesis and synapse maturation, such as NCAM,

fasciclin2 and cpg15 (Cantallops et al., 2000; Walsh and

Doherty, 1991; Walsh et al., 2000; Wright and Copenhaver,

2001), we recorded 3 additional fish 72 h after the final

injection, since our time course suggested that some GPI-linked

proteins would be re-expressed at this time. Tectal responses were

present in all 3 fish (maps for these fish are shown in Fig. 2) and

similar to controls, were more readily elicited from rostral than

caudal tectum.

Fig. 3. Phase-contrast micrographs of retinal explants and associated

pigment epithelium (black) from temporal (A,C) and nasal (B,D) retina

cultured at 1 week following optic nerve crush. Dotted circles represent the

location of the source of ephrin-A5-AP (A,B) or AP alone (C,D). Temporal

axons are repelled from ephrin-A5-AP, while nasal axons are not affected.

AP alone had no effect on axon trajectory. Scale bar: 250 Am.

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–6860

In both experimental groups, topography was abnormal with

errors in the projection of both the NT and DV retinal axes

across the RC and ML tectal axes (Fig. 2). Order factors were

low (EphA3-AP: 0.042 F 0.005; PIPLC: 0.039 F 0.01) and

were significantly decreased compared to controls (P < 0.05)

but did not differ between groups (P > 0.05).

For EphA3-AP-injected fish, analysis of the NT retinal axis

revealed that most points projected to abnormally caudal tectal

locations, illustrated by their location beneath the normal animal

trendline. The most significant errors were of central and nasal

RGC axons making errors of up to 30% of the tectal extent

(Fig. 2). The abnormally caudal projections were presumably

due to the reduction in repulsive cues within caudal tectum. A

small number of retinal points were observed to project to

abnormally rostral locations (Fig. 2), errors that could be due to

the blocking or removal of ephrin-As from RGC axons (Horn-

berger et al., 1999; McLaughlin and O’Leary, 1999). These

terminals could also be filling space created by caudal shifting

(Brown et al., 2000). Analysis of the DV retinal axis revealed

that errors were less systematic; however, there was a weak

trend for RGC axons to project abnormally laterally (Fig. 2).

For the PIPLC-injected group, RGC axon projection errors

differed from those in the EphA3-AP-injected group. Trendlines

for PIPLC-injected fish were similar to normal for the NT retinal to

RC tectal projection, but were located above normal trendlines for

the DV retinal to ML tectal projection, suggesting that RGC axons

projected abnormally laterally. However, we consider that it is

inappropriate to analyse the projection errors in this experimental

group extensively, or compare them with the EphA3-AP-injected

group, since low levels of GPI-linked proteins were allowed to be

re-expressed before mapping, confounding interpretation of the

results.

Aberrant topography across the RC tectal axis was similar to

that seen previously in ephrin-A2/ephrin-A5 mutant mice (Feld-

heim et al., 2000). The ectopic projection of temporal axons into

caudal tectum and other examples of loss of order along the

rostral–caudal axis in both models can presumably be explained

by blocking (present study) or removal (Feldheim et al., 2000)

of the rostro-caudal ephrin-A gradient. The hypothesis is sup-

ported by our in vitro study. In a retinal explant model using

retinal tissue at 1 week following optic nerve crush, temporal

RGC axons avoided a source of ephrin-A5-AP, whereas nasal

RGC axons maintained a straight trajectory (Figs. 3A,B). In

control cultures, RGC axons from both nasal and temporal

quadrants maintained straight trajectories when ephrin-A5-AP

was replaced by AP alone (Figs. 3C,D). The differential behav-

iour of nasal and temporal RGC axons resembles the results of

developmental studies using stripe assays: in both situations,

temporal but not nasal RGC axons strongly avoid high concen-

trations of ephrin-As (Brennan et al., 1997; Caras, 1997;

Ciossek et al., 1998).

The observation of aberrant DV retinal to ML tectal projec-

tions in our experimental fish is similar to the phenotype of

ephrin-A2/ephrin-A5 mutant mice. In both models, the result

was unexpected since EphA/ephrin-A interactions have been

primarily implicated in mapping NT/RC topography. For the

mutant mice, it has been suggested that loss of the weak ML

ephrin-A gradient present during normal development in mouse

may underlie the result (Feldheim et al., 2000). However, in

experimental fish, we found that DV retinal to ML tectal

projection errors were mostly random with only a weak ten-

dency for RGC axons to project more laterally, a finding that

does not support a requirement for graded medialhigh to later-

allow expression as has been observed in mouse and chick

(mouse: Feldheim et al., 2000; chick: Marın et al., 2001). It

is unlikely that EphB/ephrin-B signalling was affected since

EphA3 does not bind to any ephrin-B (Flanagan and Vander-

haeghen, 1998). However, it is possible that EphA and EphB

receptors co-cluster in the membrane of RGC axon terminals

and/or tectal cells as has been described in hippocampus

(Buchert et al., 1999); in this case, disruption of EphA/ephrin-

A interactions may have consequences on EphB/ephrin-B sig-

nalling by affecting receptor crosstalk or recruitment of proteins

to the signalling complex (Buchert et al., 1999; Halford et al.,

2000; Stein et al., 1998).

In a minority of experimental fish, some tectal loci received

input from multiple visual field locations; of the two responses,

one was topographically appropriate and the other inappropriate

(Fig. 2, EphA3-AP map 2 and PIPLC map 2). The result is

reminiscent of the inappropriately located multiple terminal

arbors of adjacent RGC axons observed using anatomical

tracing in ephrin-A2/ephrin-A5 mutant mice (Feldheim et al.,

2000). Electrophysiology in mutant mice would reveal whether

the inappropriately located arbors are functional. However, RGC

axon tracing in experimental fish is unlikely to reveal errors in

RGC axon trajectory since high levels of disorder are present

during regeneration in goldfish, even in the absence of further

manipulation (Stuermer 1986; Stuermer and Easter, 1984;

Schmidt et al., 1988). For this reason, it is highly likely that

silent connections (either appropriately or inappropriately locat-

ed) are present in addition to the inappropriately located

functional ones demonstrated electrophysiologically (Meyer and

Kageyama, 1999).

Fig. 4. Histograms showing magnification factors (A) and receptive field dimensions (B) for normal, control and experimental groups. Values significantly

different from normal are marked by an asterisk.

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–68 61

Despite the aberrant topography in experimental fish, some

degree of rostro-caudal and medio-lateral order was restored (Fig.

2). Whilst the result might be attributable to incomplete actions of

EphA3-AP and/or PIPLC, a similar result was obtained in ephrin-

A2/ephrin-A5 mutant mice. The studies indicate that molecules

other than ephrin-As define the overall axes of developing and

regenerating retino-tectal projections (Feldheim et al., 2000). A

candidate in the developing system is the GPI-linked repulsive

guidance molecule (RGM, Monnier et al., 2002) expressed as an

ascending rostro-caudal gradient in embryonic chick tectum and

considered to be important in establishing topography. A similar

role for RGM during regeneration, however, is unlikely since

overall order persisted after cleavage of GPI-linked proteins in

PIPLC-injected fish. Although the result is far from clear-cut since,

by necessity, PIPLC treatment was incomplete for up to 3 days in

these fish, the implication is that non-GPI-linked molecules may

play a role in restoring the axes of topography and may be unique

to regeneration.

Magnification factors and receptive field dimensions

Magnification factors across both the RC and ML axes were

normal in control and experimental fish (Fig. 4A). However,

receptive fields were enlarged compared to normal (P < 0.05) in

all fish undergoing optic nerve regeneration, irrespective of

whether they were in the control or experimental groups (Fig.

Fig. 5. Analysis of RT-97 immunohistochemistry at the caudal tectal pole. (A

immunopositive axons. Values in control and experimental groups are compar

immunonegative for RT-97. Significant differences are marked with an asterisk.

EphA3-AP (C)-injected fish. Scale bar: 20 Am.

4B). As in normals, receptive fields remained symmetric in all

groups. The measures are thought to reflect activity-dependent

processes: magnification factors indicate the extent to which

neighbour–neighbour relations are preserved, whilst receptive

field dimensions reflect terminal arbor size (Schmidt, 1993;

Schmidt and Edwards, 1983). Our result accords with activity-

dependent refinement taking place at stages beyond those studied

here. Blocking EphA/ephrin-A interactions at later stages of

regeneration would determine whether these molecules are in-

volved in activity-dependent refinement as suggested by their

function in the adult hippocampus (Gao et al., 1998; Gerlai,

2002; Gerlai et al., 1999).

Projection depth

RT-97 immunohistochemistry revealed abnormalities in the

distribution of regenerating RGC axon terminations in experi-

mental fish (Figs. 5A–C). Regenerating RGC axons were

labelled throughout the stratum opticum (so) and sfgs of control

and experimental fish, including those lacking responses in the

EphA3-AP- and PIPLC-injected groups. In control fish, RGC

axons in the sfgs formed a band that tapered from rostral to

caudal. However, in experimental fish, the band was of uniform

width across the RC tectal axis: the thickness was similar to

controls in the rostral three-quarters but was increased in the

caudal-most quarter (Figs. 5A–C). Presumably, when EphA/

) Histogram showing the proportion of the tectum occupied by RT-97

ed to those for uninjected animals since normal RGC axons are mostly

(B,C) Photomicrographs of RT-97 immunohistochemistry in AP (B)- and

Fig. 6. Analysis of ephrin-A2 expression in EphA3-AP- and AP-injected

fish. (A) Histogram showing the total number of cresyl violet-stained cells

(grey) and ephrin-A2 immunopositive cells (black) in the sfgs at four

equidistant locations across the tectum from rostral to caudal for normal,

control and experimental groups. Hashes indicate where the distribution of

ephrin-A2 immunopositive cells is graded from rostral to caudal. Asterisk

indicates that the number of ephrin-A2 positive cells is decreased compared

to the AP-injected control. (B,C) Photomicrographs of ephrin-A2

immunohistochemistry in the caudal tectal pole. The number of ephrin-

A2 immunopositive cells is decreased in EphA3-AP (B)- compared to AP

(C)-injected fish. (D,E) Photomicrographs of ephrin-A2 in situ hybrid-

isation in the caudal tectal pole. Ephrin-A2 expression is decreased in

EphA3-AP (D)- compared to AP (E)-injected fish. (F) Dot blots to detect

ephrin-A2 immunoreactivity in the presence of bound EphA3-AP. (i)

Ephrin-A2-Fc detected with an anti-ephrin-A2 antibody (Santa Cruz

Biotechnology). (ii) Ephrin-A2-Fc incubated in EphA3-AP, fixed with

4% paraformaldehyde and detected with an anti-ephrin-A2 antibody. (iii)

Ephrin-A2-Fc incubated in EphA3-AP, fixed with 4% paraformaldehyde

and developed for AP activity. Note that the intensity of dots in (i) and (ii) is

similar, indicating that EphA3 binding does not prevent the ephrin-A2

antibody from recognising ephrin-A2. The anti-ephrin-A2 antibody did not

bind to ephrin-A5-Fc (commercially available from R&D Systems; iv) or

ephrin-A5-AP (zebrafish protein sequence; v). Scale bars: B,X: 50 Am;

D,E: 250 Am.

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–6862

ephrin-A interactions were prevented, RGC axons were no

longer repulsed from caudal tectum. In addition, RGC axon

fasciculation may have been reduced (Winslow et al., 1995) and/

or branching increased (Sakurai et al., 2002; Yates et al., 2001).

Nevertheless, in control and experimental fish, regenerated

projections did not venture into non-visual brain regions; the

result contrasts with the development of abnormal RGC axon

projections to the inferior colliculus in ephrin-A5 mutant mice

(Frisen et al., 1998).

Ephrin-A2 expression

Immunohistochemistry indicated that the pattern of ephrin-A2

expression was abnormal in the EphA3-AP-injected group (Figs.

6A–C). Expression was increased compared to normal but only

rostrally and, as a consequence, was uniform across the RC tectal

axis (Fig. 6A, Table 1). The results contrast with those for control

groups in which ephrin-A2 expression was increased rostrally and

to a greater extent caudally, sharpening the gradient as reported

previously (Rodger et al., 2000; Fig. 6A, Table 1). The results of

in situ hybridisation matched the immunohistochemistry (Figs.

6D,E, Tables 1 and 2), indicating that changed ephrin-A2

expression is due to transcriptional regulation rather than loss

of protein by, for example, metalloprotease cleavage (Hattori et

al., 2000). We are currently investigating whether other ephrin-

As, in particular ephrin-A5, are regulated by a similar mecha-

nism. Regardless of the patterns of ephrin-A expression, these

changes cannot be responsible for the reported abnormal topog-

raphy, since injected EphA3-AP masked the abnormal pattern of

tectal ephrin-A2.

Abnormal ephrin-A2 expression may be an indirect conse-

quence of aberrant topography, but a more parsimonious expla-

nation is that Eph/ephrin interactions themselves regulate ephrin-

A2 expression. The best characterised role of Eph/ephrin

signalling is to regulate cytoskeletal dynamics; however, there

is also evidence that the proteins can affect transcription. A best

example is the regulation of NMDA receptor-mediated gene

expression by EphB (Battaglia et al., 2003; Takasu et al,

2002). In addition, the Eph/ephrins activate signalling pathways

known to affect gene transcription such as the MAP kinase

pathway (Pratt and Kinch, 2002; Tong et al., 2003) and the Rho

GTPases (Lawrenson et al., 2002; Miralles et al., 2003). It is

therefore possible that the abnormal ephrin-A2 expression shown

here is a result of EphA3-AP interfering with EphA/ephrin-A

signalling. EphA3-AP binding to ephrin-As will prevent ligands

from binding to endogenous EphAs and thus inhibit forward

signalling. In addition, by its masking effect, EphA3-AP will

prevent endogenous EphAs from activating reverse signalling via

ephrin-As. However, EphA3-AP may itself activate reverse

signalling. Since EphAs and ephrin-As are expressed on both

RGC axons and tectal cells (Hornberger et al., 1999), the effect

of injected EphA3-AP on ephrin-A2 expression may occur by

inhibition of forward signalling via EphAs, and/or by inhibition

or stimulation of reverse signalling via ephrin-As (Davis et al.,

1994; Davy et al., 1999; Himanen and Nikolov, 2003; Stein et

al., 1998).

Many receptor– ligand interactions have been demonstrated to

feedback upon their own expression patterns. As an example,

BDNF binding to the TrkB tyrosine kinase receptor induces

down-regulation of the receptor mRNA (Frank et al., 1996).

Similarly, we suggest that EphA/ephrin-A interactions have the

potential to feedback upon tectal ephrin-A2 expression. The

implication is that an initial weak gradient of EphA/ephrin-As on

RGC axons and/or tectal cells provides a substrate from which

expression can be differentially increased across the rostro-caudal

axis. Such a mechanism could act in concert with graded tran-

Table 1

Ephrin-A2 immunohistochemistry

Normal Uninjected AP-injected EphA3-AP-injected

Rostral 4.43 F 1.40 (25.13 F 2.12) 12.76 F 1.98 (23.21 F 1.41) 12.67 F 1.04 (20.33 F 3.51) 13.58 F 2.91 (22.00 F 4.58)

7.50 F 1.38 (22.50 F 0.71) 16.27 F 2.69 (23.36 F 3.21) 14.29 F 1.06 (24.67 F 0.71) 13.12 F 3.54 (20.28 F 3.53)

9.57 F 1.27 (23.33 F 1.41) 18.43 F 2.74 (22.67 F 1.53) 16.83 F 0.76 (24.67 F 2.89) 14.12 F 3.34 (22.54 F 3.61)

Caudal 10.60 F 2.07 (21.96 F 0.94) 21.90 F 2.50 (19.82 F 3.61) 22.33 F 3.40 (23.33 F 6.66) 16.83 F 1.29 (24.19 F 4.58)

Numbers of ephrin-A2 immunopositive cells are shown at each tectal location from rostral to caudal. Numbers of cresyl violet-stained cells are shown in

parentheses. Values are FSD.

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–68 63

scription factors (Logan et al., 1996; Picker et al., 1999; Yuasa et

al., 1996; Ziman et al., 2000) to sculpt the shape and intensity of

gradients during the development and regeneration of any topo-

graphically organised system.

Experimental methods

Animals and anaesthesia

Goldfish, 7–9 cm in length, purchased locally were kept in

gravel bottomed tanks containing aerated tap water at 22jC.Terminal anaesthesia was by immersion in 0.4%MS222; for surgery

and electrophysiology, gills were perfused with 0.2% and 0.005%

MS222, respectively. Procedures conformed to the National Health

and Medical Research Council Guidelines for the Care and the Use

of Experimental Animals and with approval from the Animal Ethics

and Experimentation Committee of The University of Western

Australia.

Optic nerve crush

The right eye was deflected forward and connective tissue

removed to expose the optic nerve that was crushed with watch-

maker’s forceps 1 mm from the back of the eye; the procedure

severs all RGC axons but leaves the nerve sheath intact as a

conduit for regeneration (Meyer and Kageyama, 1999). On recov-

ery from anaesthesia, animals were returned to their tanks. Return

of optomotor responses (Northmore and Masino, 1984) by 4

weeks, together with RT97 immunohistochemistry (see Results

and discussion), confirmed that RGC axons had regenerated to the

tectum in all control and experimental fish.

Synthesis of recombinant proteins

Constructs were used encoding zebrafish (Danio rerio) EphA3-

alkaline phosphatase (EphA3-AP), ephrin-A5-alkaline phosphatase

(ephrin-A5-AP) or recombinant alkaline phosphatase (AP) alone as

Table 2

Ephrin-A2 in situ hybridization

Normal Uninjected AP-injected EphA3-AP-injected

Rostral + +++ +++ +++

+ ++++ ++++ +++

++ ++++ ++++ +++

Caudal +++ +++++ +++++ +++

Estimation of ephrin-A2 mRNA-expressing cells detected by in situ

hybridisation.

a control (Brennan et al., 1997). Recombinant proteins were syn-

thesised using standard procedures (Brennan et al., 1997). Briefly,

plasmids were transfected into COS 7 cells using Lipofectamine

2000 (Invitrogen Life Technologies). Culture medium was collected

after 3 days and filter-sterilised using a 0.45-Am filter. Concentra-

tions of protein were estimated on a dot blot using 5-bromo-4-

chloro-3-indolyl phosphate/nitro blue tetrazolium (NBT-BCIP tab-

lets, Sigma) to detect AP activity.

Tectal injections

A time course study examined fish at 24, 48, 72, 96 and 120

h after a single injection of EphA3-AP, PIPLC or AP or PBS as

their respective controls. The results indicated that the actions of

EphA3-AP and PIPLC were maximal between 24 and 48 h (see

Results and discussion). Therefore, for experimental fish, injec-

tions for EphA3-AP, PIPLC and their respective controls were

performed at 48-h intervals for 2–3 weeks starting 2 weeks after

optic nerve crush coinciding with the arrival in the tectum of

regenerating RGC axons.

Anaesthetised fish were placed in a support and a 30-gauge

needle was used to pierce the skull above the left (experimental)

tectum. Injections were made of 10 Al of 20 nM EphA3-AP or

PIPLC (0.1 units/ml; experimental fish), or their respective

controls (20 nM AP or 0.1 M PBS; control-injected fish).

The small aperture generated by the first injection was used

for repeat injections. Injected fish were maintained in Holtfre-

ter’s solution (60 mM NaCl, 6.7 mM KCl, 0.3 mM CaCl2, 2.3

mM NaHCO3; pH 7.2) to prevent infection and promote

healing. An additional control group received no injections

(uninjected controls).

Whole brain histochemistry

To detect recombinant and endogenous AP activity, fish were

sacrificed between 24 and 120 h after injection. Brains were

dissected in Hanks’ buffer (Sigma), fixed in 4% paraformalde-

hyde for 20 min and washed in Hanks’ buffer overnight.

Experimental and control brains were processed together to avoid

experimental variation. To detect the distribution of injected

EphA3-AP and AP, brains were heat-treated at 65jC for 1 h,

inactivating endogenous AP, but not the heat-resistant form fused

to EphA3 or injected as a control (Brennan et al., 1997). Brains

were cooled in Hanks’ buffer, washed in Detection buffer [100

mM Tris (pH = 9.5), 100 mM NaCl, 50 mM MgCl2, levamisole

24 mg/ml] for 10 min at room temperature and stained with NBT/

BCIP. Endogenous AP activity was used as a marker for the

extent of PIPLC digestion. Brains were washed in Detection

buffer for 10 min at room temperature and stained with NBT/

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–6864

BCIP. Colour development was monitored by eye and brains

photographed at 16� magnification.

Electrophysiology

Fish were prepared for in vivo recording (1–3 days after the

final injection) and maintained under light anaesthesia (Meyer,

1977). A small circle of skull (approximately 2 mm in diameter)

was removed with iridial scissors to expose the optic tectum.

Fish were placed in a small water-filled hemisphere at the centre

of a translucent hemisphere (40 cm in diameter) displaying

circumferential and radial coordinates. The position of the fish

was adjusted to centre the experimental eye on the apex of the

hemisphere as assessed by reflection of the optic disk. A

tungsten microelectrode (9–11 MV resistance), lowered into

the superficial 50–200 Am of the left (experimental) tectum,

was used to record extracellular multi-unit responses; recordings

involve both pre- and post-synaptic components (Kolls and

Meyer, 2002). The electrode was positioned serially at the

intersections of a grid projected onto dorsal tectum. The location

of maximal response and the extent of receptive fields (circum-

ferential and radial axes) were assessed using the edge of a small

black rectangle moved slowly across the visual field. Responses

were amplified, displayed on an oscilloscope and stored using

MacLab. Fish were excluded from further analysis if no

responses were elicited in the experimental tectum despite strong

responses via the left (non-experimental) eye to the right (non-

experimental) tectum. Final numbers of animals were: uninjected:

n = 5; AP: n = 5; PBS: n = 5; EphA3-AP: n = 11; PIPLC: n =

10. Upon completion of mapping, fish were terminally anaes-

thetised, perfused with 4% paraformaldehyde and the brain

immersed in sucrose.

Analysis of electrophysiological maps

Maps of the retinal projection onto the tectum are shown

according to standard conventions (Meyer, 1977). An orderly

distribution of tectal points is imposed by experimenter’s control

of electrode placement, therefore the distribution of points in the

retina displays the extent of topographic order or disorder. To

reveal disorder in the naso-temporal (NT) retinal to rostro-caudal

(RC) tectal projection, tectal points are joined in rows following

the medio-lateral (ML) axis. The corresponding retinal rows reveal

points that project abnormally rostrally (nasal to the row) or

caudally (temporal to the row). Similarly, to reveal disorder in

the dorso-ventral (DV) retinal to ML tectal projection, tectal points

are joined in rows following the NT axis. The corresponding retinal

rows reveal points that project abnormally laterally (dorsal to the

row) or medially (ventral to the row).

To more readily illustrate the abnormalities in separate axes,

tectal recording loci and visual field projections were overlain by a

Cartesian grid, aligning the midpoint of dorsal tectum with the

corresponding visual field location. Remaining points were allocat-

ed x and y coordinates expressed as % total tectal dimensions (to

control for small inter-fish variations). The correlation between

location of retinal and tectal points was analysed separately across

the NT retinal to RC tectal projection and DV retinal to ML tectal

projection axis by plotting tectal points (X axis) against retinal points

( Y axis). For each projection axis, the trendline for normal fish was

overlain on the trendline for experimental ones. The location of

experimental points relative to the normal trendline revealed in

which way projections were abnormal: for the NT axis, points

located above the line project abnormally rostrally and those below

the line abnormally caudally. For the DVaxis, points located above

the line project abnormally medially and those below the line

abnormally laterally.

Overall extent of topography

The difference between the x and y coordinates for each tectal

locus (xt, yt) and the visual response location (xv, yv) allowed

analysis of overall topographic order, as shown by the equation:

Order = 1/average (Abs(xt � xv) + Abs( yt� yv)), where Abs is the

absolute value. High values indicate normal, and low values

abnormal, topography. Multiple F tests were used to analyse the

values between projections.

Magnification factors

In normal goldfish, magnification factors are uniform through-

out the tectum, reflecting the approximately uniform distribution of

RGCs across the retina (Jacobson, 1991). In other words, distances

between tectal loci are proportional to distances between visual

responses. Distances between tectal loci were plotted against the

distance between their corresponding visual responses and the

coefficient of determination (R2) was used to estimate magnifica-

tion factors in experimental and control fish using regression

analysis (Statview); P-values were calculated using the Bonfer-

roni/Dunn post hoc test.

Receptive field dimensions

Sizes for the circumferential (normalised for radial position)

and radial axes were compared between experimental and control

fish using ANOVA (Statview) and P-values calculated using the

Bonferroni/Dunn post hoc test. Symmetry was assessed as the ratio

between the circumference and radius.

Retinal explants

Fish were terminally anaesthetised 1 week after optic nerve

crush and right (experimental) retinae with pigment epithelium

attached were dissected on ice. Nasal and temporal quadrants were

isolated and treated with 0.25% trypsin, 1 mM EDTA in Hanks’

buffer (calcium and magnesium-free) for 30 min. The trypsin

solution was removed and the tissue washed twice by resuspending

in culture medium (Neural basal medium with 20% FCS and 20

mg/ml penicillin and streptomycin). Ephrin-A5-AP or AP (20 nM

solutions) was mixed with collagen (1:1 volume) and a droplet of

the mixture placed in the centre of a glass cover slip coated with

collagen (Bornstein, 1973). A nasal and a temporal quadrant were

explanted equidistant (0.5 mm) from the Ephrin–collagen droplet.

Explants were incubated for 5–7 days at 22jC in the culture

medium and photographed using phase-contrast microscopy.

Immunohistochemistry

Following electrophysiological mapping, fish were perfused

transcardially with saline followed by 4% paraformaldehyde. Brains

were dissected (n = 5 per group), post-fixed in 4% paraformaldehyde

for 6 h and stored in 15% sucrose in PBS (pH 7.2) overnight before

cryosectioning. Tissue was embedded in tissue-tek medium and

brains sectioned horizontally at 16 Am to visualise the rostro-caudal

tectal axis. Slides were stored at � 80jC. Before use, sections wereair-dried for 1–2 h at room temperature. Tissue was rehydrated in

J. Rodger et al. / Mol. Cell. Neurosci. 25 (2004) 56–68 65

PBS containing 0.2% Triton X-100 for 10 min; endogenous perox-

idases were inhibited in PBS containing 0.3% H2O2 for 10 min.

Sections were rinsed in PBS and incubated in blocking serum (10%

horse serum, 0.01% Tween 20 in PBS) for 30 min at room

temperature. Sections were incubated in RT-97 (all fish; Chemicon,

diluted 1:1000 in PBS + 0.2% Triton X-100) or in ephrin-A2 (AP-

and EphA3-AP-injected fish only; Santa Cruz Biotechnology,

diluted 1:500 in PBS + 0.2% Triton X-100) and slides incubated

in a humid chamber overnight at 4jC. Antibody binding was

visualised using a biotin–streptavidin–HRP system (Dako) and a

diamino-benzidine (DAB)–metal complex (Pierce). Slides were

rinsed in PBS, dehydrated in increasing alcohol concentrations,

cleared in xylene and mounted in DEPEX. Adjacent sections were

stained with cresyl violet to estimate total cell numbers.

Antibody specificity

We used a dot-blot procedure to examine binding specificity of

the ephrin-A2 antibody to complement previous studies of anti-

body specificity using negative controls (Rodger et al., 2000),

Western blots and peptide competition (Rodger et al., 2001). We

confirmed that EphA3-AP binding to ephrin-A2 did not prevent

binding of the ephrin-A2 antibody (Fig. 6F). Ephrin-A2-Fc (1 Ag;R&D Systems) was attached to three nylon membranes in

duplicate. Membranes were blocked for 30 min in PBS containing

1% BSA. The first membrane was incubated in anti-ephrin-A2

(Santa Cruz Biotechnology) and signal detected by anti-rabbit

linked to horseradish peroxidase and DAB. The second and third

membranes were incubated in EphA3-AP and proteins were fixed

in 4% paraformaldehyde to prevent the antibody from competing

out bound receptor. The protocol was designed to mimic the

procedure followed in vivo whereby tissue was fixed by perfusion

with paraformaldehyde before antibody detection. The second

membrane was incubated in anti-ephrin-A2 (Santa Cruz Biotech-

nology) and signal detected by anti-rabbit linked to horseradish

peroxidase and DAB. The third membrane was developed with

BCIP-NBT to confirm binding of EphA3-AP. Intensity of signal

was identical in the first and second membranes, confirming that

EphA3-AP binding did not mask the epitope detected by anti-

ephrin-A2 (Fig. 6F). We also confirmed that the anti-ephrin-A2

antibody was specific for ephrin-A2 and did not cross-react with

commercially available recombinant ephrin-A5-Fc (human se-

quence; R&D Systems) or ephrin-A5-AP (zebrafish sequence;

Fig. 6F).

Analysis

RT-97 expression

Sections (n = 3 per animal) were examined at a final magni-

fication of 200�. Within the sfgs of the experimental tectum, the

depth of the RGC axon input was measured at four equidistant

locations from far rostral to far caudal (termed positions 1–4). To

control for variations in size between fish, at each location, the

depth of the RGC axon input was expressed as a percentage of

tectal thickness (from the pial surface to the stratum periventricu-

lare). Data were analyzed using ANOVA (Statview) and P-values

calculated using the Bonferroni/Dunn post hoc test.

Cresyl violet staining and ephrin-A2 expression

Sections were examined at a final magnification of 400�.

Sections (three to five per animal) were analysed using the optical

dissector method (Coggeshall and Lekan, 1996). Cresyl violet-

stained cell bodies within the sfgs were counted in four 100 Am �100 Am strips directly beneath the stratum opticum (so) at equidis-

tant locations from far rostral to far caudal (termed 1–4). Similar

analyses were carried out for ephrin-A2 immunopositive cells, since

their distribution in the sfgs presumably reflects that of protein

transported to dendritic arbors, responsible for guiding regenerating

RGC axons (Meek and Schellart, 1978; O’Benar, 1976; Schmidt et

al., 1988). Vascular elements were excluded from counts. Data were

analysed by ANOVA and P-values calculated using the Scheffe F-

test (Statview).

In situ hybridisation

Tissue was prepared as for immunohistochemistry. Sections

were hydrated in a graded series of alcohol and 2� SSC,

treated with proteinase K (40 Ag/ml) for 4 min, washed in

DEPC water and incubated in 0.1 M triethanolamine with acetic

anhydride. Slides were washed in 2� SSC and dehydrated in a

graded series of alcohol. RNA probes to rat ephrin-A2 sequence

were labelled with Digoxygenin-UTP (Roche). Sections were

hybridised in probe (1 ng/Al final concentration) overnight at

56jC. Slides were washed in 2� SSC at 37jC for 30 min, 2�SSC in 50% formamide at 60jC for 30 min and 2� SSC at

37jC (2 � 10 min). Slides were incubated in RNase A (20 Ag/ml) at 37jC for 30 min and washed in RNase buffer (0.5 M

NaCl, 10 mM Tris, 1 mM EDTA) at 60jC for 30 min. Slides

were blocked in 2� SSC; 0.05% Triton X-100; 2% blocking

solution (Roche), washed in Maleate buffer (2% Normal horse

serum, 100 mM Maleic acid, 150 mM NaCl, pH 7.5; 2 � 5

min) and incubated with anti-Dig-alkaline phosphatase overnight

at 4jC. Slides were washed in Maleate buffer (2 � 10 min) and

detection buffer (10 min), signal was developed with NBT-BCIP

(Sigma), washed in AP-substrate wash (100 mM Maleic acid,

150 mM NaCl, 0.3% Tween 20, pH 7.5) and sections cover-

slipped in gel mount.

Acknowledgments

Funded by a National Health and Medical Research Council of

Australia Program Grant (993219), the Medical Research Fund of

Western Australia and the Neurotrauma Research Program (Western

Australia). We thank Michael Archer, Abbie Fall, Sherralee

Lukehurst, Truc Quach, Andreia Schineanu and Vicky Stirling for

technical assistance. We are very grateful to David Willshaw,

Stephen Eglen and Kar Lee Yeap for assistance with mathematical

and statistical analyses.

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