/ . Embryol. exp. Morph. Vol. 31, 1, pp. 123-137, 1974 1 2 3Printed in Great Britain

The retinotectal projection from a double-ventralcompound eye in Xenopus laevis

By K. STRAZNICKY,1 R. M. GAZE2 AND M. J. KEATING2

From the National Institute for Medical Research, London, and theDepartment of Physiology, University of Edinburgh

SUMMARY

The retinotectal projection was mapped in 22 post-metamorphic Xenopus in which theeye under investigation had been made double-ventral by operation at stage 32. The contra-lateral retinotectal projection from a double-ventral eye is neither normal nor does it showthe type of abnormality predicted from previous work on double-nasal and double-temporaleyes. In the case of double-ventral eyes, the nasal part of the field projection tended to bereduplicated about the horizontal midline and those field positions corresponding to latero-medial rows of electrode positions on the tectum ran ventrodorsally in the field. As theelectrode rows on the tectum progressed more caudally, so the corresponding rows ofstimulus positions in the field tended to curl in a temporal direction. These observationshave been interpreted as indicating that the nasotemporal and dorsoventral polarities of theeye are not irreversibly determined at stage 32 and that the mechanisms generating thenasotemporal and dorsoventral axes of the eye may interact with each other.

INTRODUCTION

In a normal Xenopus there is a well-ordered fibre projection from the retinalganglion cells to the contralateral optic tectum. The ordering of synapticconnexions in this projection is believed to reflect processes of axial polarizationof the retina (Sperry, 1943; Stone, 1944; Szekely, 1954, Jacobson, 1968) andwhat has been called the 'specification' of retinal ganglion cells. Some insightsinto these processes may be gained by observing the effects of surgical inter-ference with the developing eye.

In Xenopus with one 'compound eye' (Gaze, Jacobson & Szekely, 1963;1965; Gaze, Keating, Szekely & Beazley, 1970) made up of two nasal (NN)or two temporal (TT) half-retinae, the projection pattern differs from that seenin the normal animal. The projection from each half of the 'compound retina'spreads out evenly to cover the entire dorsal surface of the tectum, instead ofbeing restricted to the rostral (TT eye) or caudal (NN eye) half of the tectum,as might be expected from the normal pattern. Although it is important to

1 Present address: Department of Anatomy, School of Medicine, The University of Zambia,P.O. Box RW110, Lusaka, Zambia.

2 Authors' address: National Institute for Medical Research, London NW7 1AA, U.K.

124 K. STRAZNICKY, R. M. GAZE AND M.J.KEATING

account for these findings an unequivocal interpretation has not yet been possible(Straznicky, Gaze & Keating, 1971).

It has been inferred from earlier experiments that the nasotemporal anddorsoventral retinal axes become polarized at separate times during develop-ment (Szekely, 1954; Jacobson, 1968). This led us to hope that some light mightbe shed on the problem of the specification of retinal ganglion cells by investi-gating the projections formed by other varieties of compound eye. Accordinglywe constructed compound eyes made up from two ventral halves or two dorsalhalves in Xenopus embryos. The present paper reports on the nature of theretinotectal projection from these eyes. An abstract of some of this work hasbeen published elsewhere (Gaze, Keating & Straznicky, 1971) and a shortdiscussion of parts of it was included in another work (Gaze, 1970).

METHODS

The animals used were Xenopus laevis, bred in the laboratory and stagedaccording to the normal tables of Nieuwkoop & Faber (1956). After operationthe animals were reared at 20 °C and were fed, while tadpoles, on strainedHeinz baby soup (beef and liver) and, after metamorphosis, on chopped heartor liver.

The operations to produce double-ventral (VV) eyes were performed inHoltfreter's solution with minimal addition of MS222 (Tricaine methane-sulphonate, Sandoz) to prevent movement of the animal. Two embryos atstage 32 were placed head-to-head in an operating dish. With tungsten needlesthe dorsal half of one eye aniage was excised and discarded and was replacedby the ventral half of the eye aniage of opposite laterality taken from the otheranimal. In this way a VV eye was produced in which both the normal and thetransplanted ventral half-retina had normal nasotemporal polarity. In acomparable fashion double-dorsal (DD) eyes were produced.

Operated animals were raised through metamorphosis and, when they hadreached a body length of 3-5 cm, were used for electrophysiological analysisof the retinotectal projection. The techniques used for this procedure havebeen described in previous papers (Gaze et al. 1963, 1970). At the end of therecording experiment the head of the animal was fixed in Susa fixative and15/im sections, stained by Holmes' silver method, were prepared for histo-logical analysis.

RESULTS

Both DD eyes and VV eyes developed in a fashion that appeared, by externalcriteria, normal. In all DD eyes, however, the optic nerve failed to form; afailure that may be attributed to the absence of a ventral fissure in these eyes.DD eyes therefore did not form a retinotectal projection.

Histological examination of VV eyes shortly after operation showed that

Retinotectal projection in Xenopus 125

3B

Fig. 1. Double optic nerve leaving the back of the retina in a Xenopus larva whichhad been given a double ventral eye as described in the methods. Bar, 100/tm.Fig. 2. Eye showing two ' ventral' fissures. This eye gave the map illustrated in Fig. 7.Fig. 3 (A) Normal optic nerve head, adult Xenopus. Bar, 100/tm. (B) Residualreduplication of the optic nerve head shown in an animal with a compound doubleventral eye. Bar, 100 /̂ m.

126 K. STRAZNICKY, R. M. GAZE AND M.J.KEATING

19 IS 17 16 15 14 13

Fig. 4. Normal contralateral retinotectal projection in adult Xenopus, right visualfield to left tectum. The other diagram represents the tectum seen from above. Themidline is to the left, rostral (R) in front and caudal (C) behind. The numbers on thediagram represent electrode positions. The lower diagram is a chart of the rightvisual field extending from the centre of the field out to 100°. N, Nasal; T, temporal;S, superior; I, inferior. The numbers on the field chart indicate the optimal responsepositions for the corresponding electrode positions on the tectum. Figs. 5-9 use thesame conventions.

Retinotectal projection in Xenopus 127

Fig. 5. Projection from the right eye (double ventral) to the left tectumin Xenopus VV 16.

there was, in some cases, a double optic nerve leading from the eye (Fig. 1),the two parts of which united to form one nerve shortly after leaving the eye.At the time of recording W eyes commonly (but not always) had two ' ventral'fissures (Fig. 2). A second 'ventral' fissure in the dorsal half of the eye was notseen, however, in all the animals which showed a reduplicated projection of thenature described below. Again, at the time of recording, VV eyes often showedresidual histological evidence of the earlier operation, in the form of a doubleoptic nerve head (Fig. 3).

128 K. STRAZNICKY, R. M. GAZE AND M.J.KEATING

31 30 29 28 27

26 25 24 23 22 2

20 19 IS 17 16 15

14 13 12 II 10 9

R

Fig. 6. Projection through the right eye (double ventral) to the left tectumin Xenopus VV 18.

Electrophysiological mapping of the visual projection was restricted to theexposed dorsal surface of the optic tectum. In normal animals this part of thetectum receives input from the inferior two-thirds of the retina and thus receivesthe projection of the superior two-thirds of the visual field (Fig. 4). The fibresfrom the most dorsal or superior retinal regions (most ventral or inferior field)project round the lateral edge of the tectum in a position that is not readilyaccessible for electrode placing.

The projection from the W eye to its contralateral (left) optic tectum wasmapped in 22 animals. In 18 of these there was clear electrophysiological

Retinotectalprojection in Xenopus 129

30 29 2S 27 26 2523 22 21 20 19 1816 15 14 13 12 11 10

Fig. 7. Projection through the right eye (double ventral) to the left tectumin Xenopus VV 12.

evidence that the transplanted ventral retina (or retina occupying this site, dorsalin these animals) had formed connexions with the dorsal surface of the tectum.In the remaining four animals the projection from the VV eye only arose fromthe superior visual field, i.e. from the original ventral retina.

In the first group (18 animals) the pattern obtained is illustrated in Fig. 5,with minor modifications of this pattern being seen in some animals of thegroup. With the exception of its most nasal part the entire visual field projectsto the dorsal surface of the tectum. In a normal Xenopus (Fig. 4) a lateromedialrow of tectal recording positions gives a row of corresponding field positionsthat runs ventrodorsally; and the projection from a double-nasal or double-

9 EMB 31

130 K. STRAZNICKY, R. M. GAZE AND M.J.KEATING

- N

Fig. 8. Projection through the right eye (double ventral) to the left tectumin Xenopus VV 10.

temporal eye (Gaze et al. 1963, 1965) shows reduplication of these rows of fieldpositions, the tectal projection from the two halves of the retina being mirror-symmetrical about the vertical midline. Thus for a VV eye it could have beenexpected that for each lateromedial row of tectal positions there would betwo rows of field positions, running ventrodorsally in the superior field anddorsoventrally in the inferior field, the two rows being mirror images of eachother about the horizontal meridian. Fig. 5 shows that this relatively simplestate of affairs does not obtain in the actual projection from a VV eye.

Retinotectal projection in Xenopusc

131

S 20

Fig. 9. Projection through the right eye (double ventral) to the left tectumin Xenopus VV 8.

In general the nasal portion of the field projection follows prediction butthe orientation of the rows of positions in the temporal field progressivelydeparts from the dorsoventral, so that the field rows projecting to the mostcaudal tectum run in the nasotemporal direction rather than dorsoventral ly.In addition the field projection to caudal tectal areas does not show reduplica-tion of field positions to one tectal site. The absence of field reduplication tothe caudal one-third of the tectum is seen more clearly in Fig. 7. The sequentialarrangement of the field rows in the temporal field is such as to indicate that

9-2

132 K. STRAZNICKY, R. M. GAZE AND M.J.KEATING

the original ventral retina (dorsal field) is 'predominating' over the transplantedventral retina. In no case did we observe the opposite effect.

Of the 18 animals in which the transplanted half-retina (or retina occupyingthis dorsal position) was shown to project to the tectum, 12 gave a projectionpattern closely approximating to that in Fig. 5. The other six animals showeda generally similar pattern with some modifications. Thus four animals didshow reduplication of field positions in the projection to more caudal tectum(Fig. 6). Even in Fig. 6 it may be seen that the most caudal tectal row does notshow field reduplication but this is probably not significant because the ex-pected double field positions would be so close as to be inseparable. Theremaining two animals showed an additional variation from the pattern ofFig. 5 in that the ' cartwheeling' of field row orientation was seen not only inthe temporal field but also in the nasal field (Fig. 7). There was, however, nosign in the nasal field of the dominance of one or other half-retina.

The second group (four animals) comprised those experiments in which thefield projection arose from only the dorsal field (original ventral retina). Thetransplanted half-retina in the dorsal position did not appear to connect withthe contralateral optic tectum. Two of these four animals showed a patternequivalent to the projection from the dorsal field in Fig. 5. Thus in the nasalfield the rows run ventrodorsally but in the temporal field the rows curl roundto run nasotemporally (Fig. 8). The other two animals of this group showed'cartwheeling' in both nasal and temporal fields (Fig. 9).

DISCUSSION

The response of the retinotectal system to the surgical creation of relativesize-disparities between the retina and the tectum can tell us something of themechanisms responsible for the ordering of neuronal connexions in this system.Recently, therefore, there has been a number of studies using this experimentaldesign. Each study has produced results which are fairly consistent but theconclusions emerging from the various studies have not been uniform. Someexperiments indicate a 'plasticity 'in the tectal site at which retinal axonswill terminate following tectal or retinal lesions, while other experiments yieldresults showing a form of fixed 'place-specificity' in that retinal neurones con-nect at the same tectal loci as they would have done if the size disparity had notbeen created. Even worse, there is no obvious reason for the partitioning of theexperimental situation into those yielding one type of result or the other. Thus'fixed place specificities' has been the conclusion of some studies involvingretinal ablations in adult goldfish (Attardi & Sperry, 1963) and embryonic chick(DeLong & Coulombre, 1965,1967) and after tectal lesions in post-metamorphicXenopus (Straznicky, 1973). The spreading, compression or translation of con-nexions has been described after the construction of compound double-nasalor double-temporal eyes in Xenopus (Gaze et al. 1963, 1965), after tectal

Retinotectai projection in Xenopus 133

Fig. 10 (A). The retinal (not field) projection to the tectum in normal Xenopusshowing the disposition of the horizontal meridian. (B) The retinal projection tothe tectum that would be expected from a double ventral eye on the assumption thatthe normal retinotopic organization was preserved, and no spreading occurred.That part of the tectum lateral to the projection of the horizontal meridian shouldgive no responses as indicated by the cross-hatched area. (C) The retinal pro-jection to the tectum as recorded in animals with a double ventral eye. The tectumgives responses right out to the lateral edge and the projection of the horizontalmeridian is displaced laterally.

134 K. STRAZNICKY, R. M. GAZE AND M.J.KEATING

lesions in adult goldfish (Gaze & Sharma, 1970; Yoon, 1971-1972; Sharma,1972 a, b) and following retinal lesions in goldfish (Horder, 1971; Yoon, 1973).

In normal Xenopus the dorsal surface of the tectum receives input from ventralretina (dorsal field) and a considerable part of the dorsal retina (ventral field).The tectal projection of the horizontal meridian of the field therefore runsrostrocaudally across the tectal surface, some distance (several hundred microns)from the lateral edge (Fig. 10 A). If the construction of a VV eye results in acompound eye possessing neuronal specificities found normally only in ventralretina and if the projection from such a VV eye preserves the topography of thenormal projection, then those tectal positions lateral to the normal projection ofthe horizontal meridian should give no response (Fig. 10B). In fact the pro-jections from a VV eye in all cases extends right up to the most lateral part ofthe tectum, with an accompanying displacement laterally of the projection ofthe horizontal meridian (Fig. IOC). It seems, therefore, that the half-retinaederiving from both the original and transplanted ventral retinal primordia (orretinal tissue occupying the latter dorsal position, whatever its origin) havespread their connexions across a greater extent of the tectum than wouldnormally be innervated by ventral retina. We do not know the nature of thefield projection to the most infero-lateral aspects of the tectum because thispart of the brain is inaccessible to the electrode under our conditions of experi-ment. We can say, however, that the 'spreading' of the projection in the case ofa VV eye is non-linear in that there is a much expanded representation of thecentral field compared with the peripheral field (Figs. 7-9). Since there are noobvious consistent gaps in the visual field projection to the tectum in theseexperimental animals we would guess that, if there is indeed a retinal input tothe inaccessible lateral tectal areas, it would also arise from the central retina,which in VV eyes appropriates an increased tectal area.

In NN and TT eyes the spreading of the projection from each half-retina wasdistributed evenly along the rostrocaudal tectal axis (Gaze et ah 1963, 1965).We have discussed this phenomenon in detail in a previous paper (Straznickyet al. 1971) in which we considered three possible mechanisms underlying thespreading. We argued that the spreading was more likely to be due to a plasticityin fibre connexions than to some form of regulation of the embryonic retinawhereby each half-retina somehow reconstitutes the range of neuronal specificityfound in a normal eye. We concluded, therefore, that each half-retinal anlagegenerated, during growth subsequent to the operation, retinal ganglion cellswith neuronal specificities appropriate only to that half-retina. Surgical manipu-lation of retinal fragments at stage 32 did not appear, in the cases of NN andTT eyes, to alter radically the specificities of the fragments eventually produced.

The 'cartwheeling' effect in the rows of temporal (and sometimes nasal) fieldpositions in W projections was a completely unexpected finding in the presentexperiments and it must cast some doubt on the validity of our earlier conclu-sions. This cartwheeling effect is sufficiently different from the expected pattern

Retinotectalprojection in Xenopus 135

to warrant a critical examination of the hypotheses on which our predictionswere based. The pattern we expected to find showed vertically running rowsof field positions across the visual field, arranged in a mirror-reduplicationabout the horizontal meridian. This prediction was based on the followingthree assumptions: first, that the polarization of the retina is about two ortho-gonal axes, nasotemporal and dorsoventral; secondly, that the mechanismsresponsible for this axial polarization operate independently about the twoaxes; and thirdly, that the surgical operation on the developing retina, since ittakes place after the establishment of retinal polarity, does not alter the polarityof the fragments of the reconstituted eye.

Goodwin (1971) has carried out a theoretical analysis of some of the resultsreported in this paper, in fact concentrating on the type of pattern seen in Fig. 5.He concluded that the pattern was explicable if one postulated that the tworetinal axes were not truly orthogonal, because of an interaction between theaxis-generating mechanisms. This conclusion is plausible; it does not, however,account for the apparent predominance of the original ventral retina over thetransplanted ventral (now dorsal) retina described in the results. Goodwin'spredictions display mirror symmetry about the horizontal meridian and thissymmetry was not usually observed in the projection from the temporal fieldto the caudal tectum. It is interesting to note that the pattern predicted byGoodwin for the projection from a VV eye, on the basis of two non-interactingorthogonal gradients, showed the 'cartwheeling' phenomenon in both nasaland temporal field rows. This pattern was found in four animals (Figs. 7-9).The reasons for this variation in results is not apparent to us.

Goodwin thus questioned two of the three assumptions described above, theorthogonality of the axes and the independence of the mechanisms generatingthe axes. If we now question the third assumption, we can say from the presentresults and from the observations of Hunt & Jacobson (1973) that this assump-tion is certainly invalid, at least under the circumstances of these experiments.The abnormal orientation of the field rows from the temporal field of VV eyesindicates that the polarity of nasal retina in these eyes is different from that ofthe nasal retina which would have developed from the two ventral fragments ifthey had been left in situ. This alteration of the polarity of transplanted frag-ments in compound eyes may perhaps be shown even more dramatically in theexperiments of Hunt & Jacobson (1973). If we assume, in these compoundeye experiments, that the 'transplanted' retina did indeed derive from thetransplanted retinal primordium rather than by regeneration from the originalhalf-retina (such enantiomorphic twinning, as described by Harrison (1921) inlimb-bud development, has been seen in surgically produced half-eyes undercomparable circumstances - Feldman & Gaze, unpublished), then the polarity ofthe transplanted fragment can undergo complete reversal of both nasotemporaland dorsoventral axes.

Thus surgical interference with the developing retina may lead to alterations

136 K. STRAZNICKY, R. M. GAZE AND M. J. KEATING

in the polarity of the retina deriving from the reconstituted fragments. Thismeans, unfortunately, that experiments involving the construction of compoundeyes cannot at the moment tell us much of the rules governing the formation ofretinotectal connexions. The rationale of such experiments has involved theassumption that the operation did not interfere with the range of neuronalspecificities produced by the reconstituted fragments. We now find that notonly this assumption is unjustified, but further, we cannot even assume that thepolarity of such subsequently generated neuronal specificity will be unaltered.

If one accepts that selective neuronal connexions reflect the acquisition, duringdevelopment, of neuronal specificities and that these are imposed upon neuronalpopulations by polarized fields (Sperry, 1943,1944.1945), then the value of com-pound eyes will lie in what they can tell us of this process of polarization. Whenthis latter process is more completely understood then the retinotectal pro-jections formed by compound eyes may tell us something more definitive aboutthe rules by which polarized neuronal arrays interconnect. Results alreadyobtained indicate that the polarity of subdivisions of the retina is not irreversiblydetermined at stage 32. They may also indicate that the axial polarization whichin the intact eye appeared to be laid down orthogonally and independently(Szekely, 1954; Jacobson, 1968) are in fact neither exactly orthogonal norindependent.

The work described in this paper was carried out in the Department of Physiology,University of Edinburgh, during the tenure by K. Straznicky of a Wellcome ResearchFellowship.

REFERENCES

ATTARDI, D. G. & SPERRY, R. W. (1963). Preferential selection of central pathways byregenerating optic fibres. Expl Neurol. 7, 46-64.

DELONG, G. R. & COULOMBRE, A. J. (1965). Development of the retinotectal projection inthe chick embryo. Expl Neurol. 13, 351-363.

DELONG, G. R. & COULOMBRE, A. J. (1967). The specificity of retinotectal connectionsstudied by retinal grafts onto the optic tectum in chick embryos. DevlBiol. 16, 513-531.

GAZE, R. M. (1970). The Formation of Nerve Connections. London: Academic Press.GAZE, R. M., JACOBSON, M. & SZEKELY, G. (1963). The retinotectal projection in Xenopus

with compound eyes. / . Physiol, Lond. 165, 484-499.GAZE, R. M., JACOBSON, M. & SZEKELY, G. (1965). On the formation of connections by

compound eyes in Xenopus. J. Physiol., Lond. 176, 409-417.GAZE, R. M. & SHARMA, S. C. (1970). Axial differences in the reinnervation of the goldfish

optic tectum by regenerating optic nerve fibres. Expl Brain Res. 10, 171-181.GAZE, R. M., KEATING, M. J. & STRAZNICKY, K. (1971). The retinotectal projection from a

double-ventral compound eye in Xenopus. J. Physiol., Lond. 214, 37-38P.GAZE, R. M., KEATING, M. J., SZEKELY, G. & BEAZLEY, L. (1970). Binocular interaction in

the formation of specific intertectal neuronal connections. Proc. R. Soc, Lond. B 175, 107—147.

GOODWIN, B. C. (197.1). An analysis of the retinotectal projection of the amphibian visualsystem. Lectures on Mathematics in the Life Sciences, 3, 77-98. Providence: AmericanMathematical Society.

HARRISON, R. G. (1921). On relations of symmetry in transplanted limbs. / . exp. Zool. 32,1-136.

Retinotectal projection in Xenopus 137HORDER, T. J. (197.1). Retention, by fish optic nerve fibres regenerating to new terminal sites

in the tectum, of 'chemospecific' affinity for their original sites. J. Physiol., Lond. 216, 53-55 P.

HUNT, R. K. & JACOBSON, M. (1973). Neuronal locus specificity: Altered pattern of spatialdeployment in fused fragments of embryonic Xenopus eyes. Science, N.Y. 180, 509-511.

JACOBSON, M. (1968). Development of neuronal specificity in retinal ganglion cells of XenopusDevi Biol. 17,202-218.

NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table of Xenopus laevis (Daudin). Amster-dam: North Holland Publishing Company.

SHARMA, S. C. (1972a). Redistribution of visual projections in optic tecta of adult goldfish.Proc. natn. Acad. Sci. U.S.A. 69, 2637-2639.

SHARMA, S. C. (19726). Reformation of retinotectal projections after various tectal ablationsin adult goldfish. Expl Neurol. 34, 171-182.

SPERRY, R. W. (1943). Visuomotor coordination in the newt {Triturus viridescens) afterregeneration of the optic nerve. /. comp. Neurol. 79, 33-55.

SPERRY, R. W. (1944). Optic nerve regeneration with return of vision in anurans. /. Neuro-physiol. 7, 57-70.

SPERRY, R. W. (1945). Restoration of vision after crossing of optic nerves and after contra-lateral transplantation of eye. / . Neurophysiol. 8, 15-28.

STONE, L. S. (1944). Functional polarization in retinal development and its reestablishmentin regenerating retinae of grafted eyes. Proc. Soc. exp. Biol. Med. 57, 13-14.

STRAZNICKY, K. (1973). The formation of the optic fibre projection after partial tectalremoval in Xenopus. J. Embryol. exp. Morph. 29, 397-409.

STRAZNICKY, K., GAZE, R. M. & KEATING, ML J. (1971). The retinotectal projections afteruncrossing the optic chiasma in Xenopus with one compound eye. / . Embryol. exp. Morph.26, 523-542.

SZEKELY, G. (1954). Zur Ausbildung der lokalen funktionellen Spezifitat der Retina. Actabiol. Acad. Sci. hung. 5, 157-167.

YOON, M. (1971). Reorganization of retinotectal projection following surgical operations onthe optic tectum in goldfish. Expl Neurol. 33, 395-411.

YOON, M. (1972). Reversibility of the reorganization of retinotectal projection in goldfish.Expl Neurol. 35, 565-577.

YOON, M. (1973). Transposition of the visual projection from the nasal hemiretina onto theforeign rostral zone of the optic tectum in goldfish. Expl Neurol. 37, 451-462.

{Received 22 May 1973)