THE JOURNAL OF COMPARATIVE NEUROLOGY 250 364-376 (1986)

Optic Fibers Follow Aberrant Pathways From Rotated Eyes in Xenopus Zaeuis

PHILIP GRANT AND POKAY M. MA Department of Biology and Institute of Neuroscience, University of Oregon, Eugene,

Oregon 97403

ABSTRACT The rotated eye paradigm has been a major experimental test of the

neuronal specificity model for the development of ordered retinotectal con- nections in amphibians. In most studies, however, no optic fiber pathways were traced from rotated eyes and correlated with visuotectal projections. As an initial approach to this question, optic fibers from eyes rotated at different embryonic stages were traced with 3H-proline autoradiography. Three experimental series were prepared: (1) in situ eye rotations, (2) iso- chronic transplants of eyes rotated between embryos at the same stage, and (3) heterochronic transplants of eyes rotated between embryos at different stages. Single or multiple optic fiber pathways developing from rotated eyes are identified by their sites of entry and trajectory in the brain. These include a normal chiasmatic (CH) pathway, and three aberrant pathways, identified as trigeminal (TR), diencephalic (DI), and oculomotor (OC). The latter three enter the brain ipsilaterally, some crossing contralaterally via commissural pathways. Depending on stage and type of operation, TR path- ways develop in 50-100% of the animals, while CH pathways are more common after rotation at stage 21122. The surgical procedure affects the initial trajectory of fibers from the retina, perturbs guidance cues in the surrounding orbit, and determines the patterns of optic pathways that de- velop. In most cases, optic fibers follow motor (oculomotor) or sensory (tri- geminal) nerves, usually the first fibers encountered near the orbit by axonal pioneers exiting the retina. Evidently, optic fibers exhibit no pathway selec- tivity; any axon serves as a guidance cue. Tecta are innervated in about 50% of the cases, usually by fibers following abnormal trajectories from CH and OC pathways. The results suggest that the development of ordered visuotec- tal projections from rotated eyes is a complex process that may be indepen- dent of the trajectory of fiber arrival. Unless pathways and visuotectal maps are directly compared in each animal, however, the question remains open because we still do not know which anomalous pathways, if any, correlate with ordered projections.

Key words: retinotectal, frog, development, autoradiography

Pathway selectivity as well as target selectivity may play Fiber pathways, however, may be important during de- a role in ordering retinotectal connections in lower verte- velopment of visuotectal projections from rotated embry- brates (Attardi and Sperry, '63; Horder and Martin, '78; onic eyes, the classic experimental paradigm responsible Straznicky et al., '79). Regenerating optic fibers, however, for the neuronal specificity hypothesis (Sperry, '45). The following aberrant trajectories to the tectum, still give rise to normal visuotectal projections (Gaze, '59; Hibbard, '67; Sharma, '72, '73; Beazley and Lamb, '79). Evidently target selectivity, not pathway selectivity, is the final arbiter in ordering retinotectal connections of regenerating optic fibers.

Accepted March 18, 1986. Pokay M. Ma's present address is Dept. Neurobiology, Harvard

Medical School, 25 Shattuck St., Boston, MA 02115.

0 1986 ALAN R. LISS, INC.

ABERRANT OPTIC PATHWAYS FROM ROTATED EYES 365

issue is controversial, since in one laboratory, visuotectal recording from eyes rotated at early embryonic stages sug- gests sequential and independent specification of retinal axes (Jacobson, '68), while these observations are uncon- firmed in other laboratories (Gaze et al., '79; Sharma and Hollflield, 'SO). To explain these contradictory results, differences in the surgical procedure or in the operating medium used have been invoked (Gaze et al., '79). Unfor- tunately, in none of these experiments were optic fiber pathways traced from rotated eyes, though Gaze et al. ('79) mention briefly that many pathways from rotated eyes are aberrant. Anomalous ipsilateral projections were also ob- tained aRer eye rotations in Rana (Jacobson and Hirsch, '731, suggesting that, in contrast to regenerating optic fi- bers, the trajectory of fiber pathways may be more critical to development of visuotectal projections from rotated eyes.

It is, therefore, important to correlate the optic pathway from rotated eyes with visuotectal projections. As a prelim- inary approach to this question, optic fiber pathways from rotated eyes in Xenopus tadpoles were traced by 3H-proline autoradiography. No evidence of pathway selectivity was found; optic fibers may follow several different normal and or aberrant nonoptic pathways to the brain, often arriving in tecta with trajectories 180" inverted from normal, single, or multiple pathways. Pathway development is affected most by the surgical procedure and the stage of the opera- tion. How aberrant patterns of tectal innervation affect development of the visuotectal projection remains to be determined. Since the usual visuotectal projection from ro- tated eyes is an ordered but inverted map (Gaze et al., '80; Sharma and Hollyfleld, '801, our results suggest that, as in optic nerve regeneration, the spatial ordering of the retino- tectal projection is independent of the trajectory of fiber arrival in the tectum. A more critical evaluation of the relationship between fiber trajectories and ordering of the visuotectal projection, however, calls for experiments in which the physiologically recorded map is correlated di- rectly with the anatomically traced pathway in each ani- mal. Such studies are currently in progress.

MATERIALS AND METHODS Eggs and embryos were obtained from Xenopzu females

by the usual procedures of gonadotrophin-induced mating. The embryos were allowed to develop to the appropriate stages for surgery. ARer surgery in full-strength Stein- berg's solution, embryos were placed in 10% Steinberg's solution to develop at 22-24°C and killed at different tad- pole stages.

Surgical procedures Three series of operations were carried out: Series Z-in situ rotations. Embryos at stages 21-32

(Nieuwkoop and Faber, '56) were anesthetized in tricaine methane sulfonate (MS 222) and the entire left eye anlagen was removed from its orbit, rotated 180", replaced, and covered with a small glass bridge to heal. After healing, embryos developed to tadpole stages before being sampled for fixation. Control embryos were operated in the same manner except that operated eye primordia were replaced in the orbit with normal orientation.

Series ZZ-isochronic transplantations. Pairs cf embryos at identical stages were anesthetized as above and the left eye anlagen of the host embryo was removed and replaced with a 180" rotated left eye primordium from a donor em- bryo. Control embryos were operated in the same manner with donor grafts replaced in normal orientation.

Series ZZZ-hetemchronic transplantations. Younger do- nor left eye grafts were transplanted with 180" rotation into the left orbit of older host embryos. In a few cases, older donor eye anlagen were also transplanted into orbits of younger host embryos. Host and donor control embryos of different stages were treated in the same manner except for the normal orientation of the transplant in the host orbit.

Autoradiograp h y Tadpoles or metamorphosed juveniles were anesthetized

and 3H-proline (10-5-10-3 p1 of 27 Ci/mmol stock, New England Nuclear) was injected into the left eye, usually at the peripheral dorsal margin. The animals were kept at least 24 hours before fixation in aqueous Bouin's solution. They were washed, dehydrated, and embedded in paraffin. Ten-micron transverse serial sections were prepared, dried on slides, dipped in Kodak NTB2 emulsion (1:l with dis- tilled water), and exposed at 4°C for 1-2 weeks. Slides were developed in Kodak D-19 developer at room temperature, counterstained with toluidine blue, and mounted in Per- mount.

Autoradiographs were photographed in dark- or bright- field on Pan X film by using a Nikon photo-attachment on a Zeiss microscope. Serial section reconstructions were prepared from camera lucida drawings made of every fifth or tenth section.

Silver staining of optic fibers The initial orientation and trajectory of optic fibers after

exiting the rotated retina were determined by rotating eye primordia of stage 32 embryos, sampling every 2 hours, and fixing in Bouin's. The embryos were dehydrated, embedded in paraffin, serially sectioned at 10 pm in the transverse plane, mounted on slides, and stained with a modified silver stain (Rowell, '63).

RESULTS Chiasmatic pathways (CH) are designated as normal if

all optic fibers from the retina arrive at the chiasm and follow the normal contralateral trajectory to the tectum (Fig. 1A). Aberrant optic pathways appear either as single pathways or as a branch of multiple pathways and are identified by their initial point of entry into the brain. The trigeminal ("R) pathway is the most common; optic fibers enter the ipsilateral trigeminal ganglion, following branches of cranial nerve V to the hindbrain, entering at the trigeminal root (Fig. 1D). Optic fibers in the dience- phalic pathway @I) enter any ectopic region of the ipsilat- era1 diencephalon (Fig. 1B). Fibers in the oculomotor pathway (OC) follow an oculomotor nerve from the orbit and enter ipsilateral ventral midbrain at the root of cranial nerve III (Fig. 1C). An example of each pathway is shown below.

The CH pathway Since most optic fiber pathways are traced in tadpoles at

stages 51-54, a reconstruction of a normal CH pathway at stage 54/55 is shown in Figure 2. Only the contralateral pathway is evident a t this stage; ipsilateral fibers are too few to be labeled by the procedure (Grant and Ma, unpub- lished). Except for this difference and the absence of the rostra1 visual neuropil, the contralateral pathway at this stage resembles that of the juvenile @vine, '80; Grant and Ma, unpublished).

366 P. GRANT AM) P.M. MA

Fig. 1. Sites of entry of optic pathways. Transverse sections. Unlabeled arrows point to sites of optic nerve entry into brain. A. Stage 52/53, chias- matic (CH) pathway, with fibers entering normally at chiasm. B. Stage 53, diencephalic (DI) pathway with fibers entering ventrolaterally in caudal ipsilateral diencephalon. C. Stage 52/53, oculornotor (OC) pathway. Fibers

enter root of cranial nerve I11 and cross midline. D. Stage 51/52, trigerninal (TR) pathway with fibers entering hindbrain at root of cranial nerve V. NB = neuropil of Bellonci; PF = pretectal field; U = uncinate. Scale bars = 100 fim.

The TR pathway enters the optic foramen and courses dorsallv and caudally The TR pathway often appears as a single pathway (Fig.

3). Optic fibers course dorsally and caudally from the retina without entering the optic foramen and follow a rostral branch of the trigeminal complex (ophthalmic?), joining the trigeminal ganglion ventral and lateral to the tectum (Fig. 4). Together with trigemfinal fibers, optic fibers enter the hindbrain at the root of cranial nerve V and descend to the tail in a spinal dorsolateral trunk, the descending tract of the trigeminus (Fuller and Ebbesson, '73).

It is not known whether any optic fibers make any syn- aptic connections anywhere in the trigeminal pathway, but optic fibers do persist in this pathway, at least up to 8 months postmetamorphosis.

The DI pathway DI pathways generally appear as a branch of multiple

pathways (Fig. 5A,B). In this case, a large CH branch (bl) enters a t the chiasm and projects normally into the contra- lateral optic tract including the basal optic neuropil @ON) azld innervates most of the tectum. The DI branch 032)

until it plunges into the lateral wall of thecaudal ipsilat- era1 diencephalon. (Fig. 5A, b2). From here, most fibers turn caudally, following superficial and deep ectopic trajectories in the optic tract, innervating ipsilateral pretectal and un- cinate neuropils, as far as dorsolateraI tectum (Fig. 5B). Fibers also enter the ipsilateral basal optic tract and run caudally to the BON.

Rostrally directed fibers in this pathway follow aberrant trajectories from their site of entry and partially innervate more rostral neuropils such as the neuropil of Bellonci (NB) and the corpus geniculatum thalamicum (CGT) with trajec- tories totally reversed from normal (Fig. 5A).

In only one case did fibers in a DI pathway cross to innervate contralateral tectum and thalamic neuropils.

The oculomotor (OC) pathway OC pathways are complex branches of multiple pathways

that usually innervate contralateral and ipsilateral visual neuropils (Fig. 6). In this series of transverse sections, por- tions of the OC pathway are shown, labeled bl. Although

ABERRANT OPTIC PATHWAYS FROM ROTATED EYES

c-r

367

A

B Fig. 2. Camera lucida reconstruction of radioautographs of transverse

sections of stage 54/55 tadpole brain showing normal CH pathway. AOT = axial optic tract; BON = basal optic neuropil; BOT = basal optic trad; CGT

the sections are in rostrocaudal sequence, it is best to start at the site of entry at the oculomotor root in Figure 6D. The bl branch has projected caudally in the head, following oculomotor fibers to the root of cranial nerve I11 (bl) where they take several trajectories. Some fibers rise directly up the lateral edge of the tectum from the point of entry and innervate ipsilateral tectum. Other optic fibers cross to the contralateral side in a commissural bundle on the ventral edge of the midbrain (unlabeled arrows Fig. 6D,E) and rise along the lateral edge to innervate contralateral tectum caudally and rostrally. Other fibers rise on the lateral edge from the point of entry and innervate ipsilateral tectum.

Fibers coursing rostrally from contralateral tectum spread through the contralateral pretectal field, the uncinate, the BON and the basal optic tract (BOT), and the NB (in the order 6C,B,A). In rostral diencephalon, fibers descend along the lateral edge of the optic tract and cross to the ipsilateral side in the suprachiasmatic ventral decussation (arrows in Fig. 6A) where they turn caudally and dorsally to innervate

= corpus geniculatum thalamicum; CH = chiasm. MOT = marginal optic tract; NB = neuropil of BelIonci; PF = pretectal field; RT = rostral tectum. U = uncinate neuropil. Arrow labeled r c is rostrocaudal axis.

ipsilateral NB, BOT, BON, and uncinate (Fig. 6A-C). Some of these fibers continue caudally on the ipsilateral side, probably intermingling with rostrally directed fibers com- ing from the ipsilateral tectum (Fig. 6C). This one OC branch sends fibers to almost all neuropils, which enter each neuropil with anomalous trajectories, often 180" re- versed from normal.

The b2 TR branch enters at the Vth root and some fibers cross the midline on the lateral ventral edge of the hind- brain (arrows Fig. 6F).

To study how these aberrant pathways arise, pathways in three series of rotated eye experiments were studied.

Series I-in situ rotations One-hundred fifty experimental and 43 control embryos,

operated at different stages, yield 107 experimental and 40 control tadpoles with traceable optic pathways (Table 1).

Among control tadpoles, 93% have ventral fissures with

368 P. GRANT AND P.M. MA

X U

Fig. 3. Trigeminal (TR) optic pathway. Camera lucida reconstruction of transverse sections of stage 51/52 tadpole brain whose leR eye was rotated at stage 26. Branch bl joins ipsilateral trigeminal ganglion Crc) and follows trigeminal fibers to enter the hindbrain at the root of cranial nerve V.

Fig. 4. Radioautograph of horizontal section of stage 56/57 tadpole brain whose left eye was rotated from a stage 35/36 donor into a stage 32 host orbit. Large arrows point to TR pathway entering trigemlnal ganglion (TG). Small arrows point to OC pathway inside cranium (C) coursing proximal to blood vessel (BV) toward root of cranial nerve 111.

normal CH pathways, indicating that surgery has no effect on development of the normal pathway. Of three abnormal control tadpoles, two exhibit a CH pathway together with a TR branch.

Pathway patterns among the few tadpoles with eyes ro- tated at stage 21/22 seem to differ from those operated at later stages. Half the tadpoles in the younger group with D or I fissures have normal pathways while only 7-9% of older stages possess a normal CH branch. In addition, tad- poles developing from older embryos display more TR and OC pathways.

Most stage 26-32 tadpoles with V fissures also exhibit aberrant pathways (60-73%), with the majority showing TR and OC branches. This is somewhat surprising in view of the control patterns. Evidently, fissure orientation does not always correlate with pathway development after eye rotations in older embryos.

Series I1 rotations-isochronic transplantations The few results obtained in this series are presented in

Table 2. Nine control tadpoles (75%) with ventral fissures display normal pathways while three are aberrant, includ- ing two CH branches. Four controls with D fissures show one normal and three aberrant pathways. It is uncertain exactly how such rotated eyes arise unless the animals were mislabeled or the surgery was unusually traumatic.

Three experimental tadpoles developing after rotation at stage 21/22 possess V fissures but only one normal pathway is seen. Both tadpoles with abnormal pathways exhibit a CH branch, however.

ABERRANT OPTIC PATHWAYS FROM ROTATED EYES 369

A

. CONTR

IPS1

8 Fig. 5. Camera lucida reconstruction of a multiple pathway in stage 52/

53 tadpole (stage 26 donor leR eye rotated into stage 39 host orbit) that includes a DI branch @2). In 5A, a CH branch @I) follows a normal contra- lateral trajectory into diencephalon and tectum (5B). The DI pathway, branch b2, courses caudally and dorsally in the head (5A) before entering

lateral ipsilateral diencephalon (A,B). From this site, fibers turn dorsally and caudally into ipsilateral caudal thalamic neuropils, BON, and dorsolat- era1 tectum (5B). Other fibers turn rostrally into an aberrant dorsolateral pathway to more rostra1 thalamic neuropils (5A). Symbols as in Figure 2.

No normal pathways develop in the 14 tadpoles from stage 26-32 rotations including two with V fissures. Only two aberrant pathways include a CH branch. As in series I, the TR pathway predominates and is seen in about 70% of the cases.

Series I11 rotations-heterochronic transplants Heterochronic transplants may reveal whether fiber tra-

jectories are affected by stage-specific factors acting in the donor retina or in the host orbit and optic pathway. In this series, donors are stages 21/22, 26/28, and 30132, and de- pending on donor stage, hosts are stages 26, 32, 35136, 371 38, or 39 (Table 3). Control operations are similar. Of the few old to young transplants, stage 32 is the host for stage 37138 donor eyes. Since host age had no effect on pathway development, all data are evaluated in terms of donor stage.

Six of the eight control transplants show normal path- ways, including two tadpoles with D fissures. CH branches are present in the two animals with aberrant pathways.

Stage-specific patterns are more apparent in this group. A total of 16 pathways from stage 21122 donors were traced in hosts of different ages. Of six normal pathways, three are found in tadpoles with V fissures and three in tadpoles with D fissures. Four of the ten aberrant pathways also include a CH branch while only three TR branches (30%) are seen compared to the more than 90% TR pathways possessed by tadpoles with eyes from older donors in this series.

The higher frequency of CH pathways developing from hosts with stage 21122 transplants suggests that conditions in the donor eye graft and not in the host orbit determine pathway development.

370 P. GRANT AND P.M. MA

Fig. 6 . Radioautographs of transverse sections of stage 52/53 tadpole brain showing oculomotor (OC) pathway (bl). Heterochronic transplant of the stage 37/38 donor left eye into a stage 32 host orbit. All labeled fibers in each section (except 6F) derived from the original bl branch. A. Section 260 pm caudal to chiasm (no CH branch present). Arrows point to fibers in supraoptic ventral decussation crossing from contralateral side (left) to ipsilateral optic tract. Note that contralateral NB is more heavily labeled than ipsilateral NB, which receives the crossing fibers. B. Section 240 pm caudal to A. OC branch (bl) coursing caudally in cranium toward root of cranial nerve I11 on ipsilateral side. Note aberrant bl fibers on contralateral side (large arrow) with rostra1 trajectory, and labeling in contra and ipsi BOT as well as ipsi uncinate W). C. Section 60 pm caudal to B. Contralat-

era1 tract labeled in lateral edge (fibers coursing rostrally in commissura transversal). BON labeled ipsilaterally with diffuse labeling in pretectal field. D. Caudal (100 pm) to C. bl branch enters root of cranial nerve 111. Fibers then cross the midline in ventral commissure (arrows). Diffuse deep labeling rises to innervate contralateral tectum 0. Fibers also rise ipsilat- erally and innervate ipsi tectum. E. Caudal (160 pm) to D. Many more fibers from OC branch have coursed caudally, some crossing the midline in ventral edge of midbrain to contralateral tectum (arrows). F. Caudal (150 pm) to E. bz branch ClR) having entered trigeminal root, descends in hindbrain and some fibers cross midline at ventral edge to contralateral hindbrain (arrows). All magnifications same as Figure 1B.

ABERRANT omrc PATHWAYS FROM ROTATED EYES 371

TABLE 1. Series I1

Total Stage operations

D and I fissures 21/22 7

26/28 59

31/32 41

V fissures 21/22

26/28

Pathway types Total

pathways N A

6 3 3

54 5 49

30 2 28

150) (50)

(9) (91)

(7) (93)

1 1 0

5 2 3 (100)

(40) 160)

Aberrant pathways CH DI OC TR

1 2 2

2 3 12 28 (71 (11) (43) (100)

1 2 3 (33) (67) (100)

31/32 11 3 8 2 1 5 8

Controls 43 43 40 3 2 1 (27) (73) (25) (131 (631 (100)

(93) (7) (67) (33)

'Numbers in parentheses are percentages. Percentages of N and A are based on total number of fissures. Those for all others are based on total number of A pathways. Here and throughout Tables 1-8 N. normal pathway; A, aberrant pathways, single or multiple, CH, chiasmatic; DI, diencephalic; OC, oculomotor; TR, trigeminal. D and I fissures, dorsal and intermediate fissures (between 30 and 160"); V, ventral fissures.

TABLE 2. Series 11'

Total Stage operations

D and I fissures 21/22 3 26/28 5

31/32 9

V fissures 21/22

26/28

31/32

Controls 16

Total pathways

-

4

8

3

1

1

D 4

v 12

Pathway types

N A

1 2 (33) (67) 0 1

0 1

1 3 (25) (75) 9 3 (75) (25)

(100)

(100)

Aberrant pathways CH DI OC TR

1 1 3 (25) (25) (75)

1 2 2 5 (13) (25) (25) (63)

2 1 (100) (501

1 1 (100) (100)

1 (100)

2 1 (67) (331

1 2 2 (67) (331 (67) (671

'See footnote to Table 1

TABLE 3. Series 111'

Total Pathway types Aberrant pathways Donor Total stage operations pathways N A CH DI OC TR

D and I fissures 21/22 16

26/32 24

35/39 11

V fissures 21/22

26/28

Controls 8

13 3 10 4

21 0 21 1

11 0 11

(23) (77) (40)

(100) (5)

(100)

3 3 0 (100)

3 0 3 (100)

D 2 2 0 (100)

V 6 4 2 2 (67) (33) (1001

4 2 3 (40) (201 (30) 3 8 20 (14) (38) (95)

10 2 8 (18) (731 (91)

1 3 (33) (100)

1 (50)

'See footnote to Table 1.

372 P. GRANT AND P.M. MA

TABLE 4. Tadpoles With D and I Fissures ARer Eye Rotation Possessing Normal or Aberrant Optic Pathways Projecting to Both Tecta'

Contra'

Stage Total N A Ipsi'

21/22 20 6 9 1

26/28 79 5 29 15 (30) (45) (5)

(6) (37) (19) 30135 49 2 15 12

(4) 131) 124)

'Combined data for all series. Numbers in parentheses are percentages of total 'Contra, numbers of pathways innervating contralateral tectum. %psi, number of pathways innervating ipsilateral tectum.

TABLE 5. Relationship Between Stage of Rotation and Orientation of Fissures'

Fissure orientation

Stage Total D I V

21/22 27 12 8 7

26/28 88 54 25 9

31/35 61 32 17 12

(45) (30) (25)

(61) (28) (10)

(52) (28) (20)

'Numbers in parentheses are percentages of total. Combined data from all series. D, dorsal fissure; I, intermediate (between 30 and 160") ; V, ventral fissure.

Among 24 tadpoles developing from hosts receiving stage 26-32 grafts, 20 with D or I fissures have aberrant optic pathways with TR branches. Almost 40% of these also have an OC branch that is primarily responsible for most tectal innervation. Only one CH branch appears in the entire series. Even the few animals with V fissures display aber- rant TR branches.

In the final series, stage 35-39 donor eyes placed in stage 32 orbits result in 11 tadpoles with abnormal fissures. No normal pathways are seen. Ten of the 11 exhibit multiple pathways with TR branches, 73% of which also possess an OC branch.

Tectal projection patterns What proportion of tadpoles with rotated eyes possess

normal or aberrant optic pathways projecting to contralat- era1 tectum? The data from all series are combined in Table 4.

Stage-specific patterns become more apparent in these data. Tadpoles from stage 21/22 eye rotations show a much higher proportion of tectal innervation, about 75% (includ- ing 30% normal CH pathways), compared to only 3543% of tadpoles developing from later stage rotations. Moreover, only 4-6% of the latter possess normal CH pathways. Only 5% of tadpoles from stage 21122 rotations exhibit aberrant ipsilateral pathways while 24% of the older groups contain such aberrant pathways.

The OC pathway is the principal source of tectal inner- vation (including ipsilateral tectum) in tadpoles operated at older stages. For example, in series 11, seven of nine stage 21/22 animals show tectal innervation via the CH branch; the remaining animals show innervation from the OC branch. Of 16 stage 26-35 animals in series I11 with contra- lateral tectal innervation, all except one are innervated via the OC branch.

Orientation of choroid fissure and development of the optic pathway

In the normal eye, fibers emerge from a V fissure, are oriented toward the optic stalk, and enter the CH pathway. Fibers emerging from a rotated eye exit from a D fissure and are oriented dorsally in the orbit, away from the stalk. One might predict that orientation of the fissure would correlate with optic fiber pathway development: V fissures correlating with normal pathways and D or I fissures with aberrant pathways. The limited data in each series seem to be inconsistent with this prediction. To examine this fur- ther, data from all series are combined.

First, the relationship between stage of rotation and ori- entation of the fissure is examined (Table 5). Polarization of the fissure in urodeles and the chick is stage dependent; rotation of an eye anlagen at early stages usually results in a normally oriented fissure while later rotations yield D or I fissures and sometimes two fissures (Beckwith, '27; Sato, '33; Stone, '66; Goldberg, '76). Our data, however, show only modest, probably insignificant, stage differences in fissure orientation and suggest that fissure determina- tion may be completed by stage 21/22 in Xenopus. The few ventral fissures could have arisen by derotation of the eye anlagen within the orbit during healing as has been sug- gested for the chick (Goldberg, '76).

Stage differences become more apparent when fissure orientation is correlated with pathway development (Table 6). For most stages, a V fissure correlates with a higher frequency of normal pathways. Among tadpoles from stage 21/22 rotations, about 70% with V fissures posssess normal pathways compared to 32% with D and I fissures. Among animals from later stage rotations, only 20-25% with V fissures show normal pathways compared to 4-6% with D or I fissures. Nevertheless 75% of tadpoles with V fissures developing from embryos operated at late stages display aberrant pathways, which suggests that stage of rotation overrides any effect of fissure orientation.

Surgical procedure and pathway development The surgical procedures of two individuals (P.G. and

P.M.M.) were compared in series I operations (stages 21-32) carried out in Holtfreter and Steinberg solutions.

The results shown in Table 7 reveal significant differ- ences in pathway development among animals prepared by each investigator. First, choroid fissure orientations differ; P.M.M. operations yield fewer animals with D fissures (33% vs. 56% for P.G.) and a much greater proportion of ventral fissures (40% to 9%). Optic nerves emerge at about the same frequency in both groups but more neuromas develop after PG operations. Contralateral tectal innervation patterns in animals with D fissures also differ; P.M.M.-operated ani- mals show 63% innervation while P.G.-operated animals exhibit only 13% successful innervation. This correlates with the observation that 50% of P.M.M.-operated tadpoles with D fissures display a CH pathway compared to only 11% of P.G.-operated animals. Most tectal innervation in PG-operated animals is via the OC pathway. TR pathways also predominate in P.G.-operated animals with 89% of tadpoles with dorsal fissures showing a TR pathway com- pared to 56% of P.M.-operated tadpoles.

The operating solutions have no effect; patterns are simi- lar in both solutions in each operated group.

Since P.M.M. carried out all series of operations, the re- sults shown in Table 7 (in situ rotations) can be compared

ABERRANT OPTIC PATHWAYS FROM ROTATED EYES 373

to his isochronic and heterochronic transplantation results (Tables 2,3) . For example, only five of a total of 17 tadpoles with D and I fissures in Table 2 (29%) show innervation of contralateral tectum, compared to 46% seen in Table 7. Heterochronic transplants in older embryos also yield a lower frequency of contralateral tectal innervation, with most derived from the OC pathway, rather than the CH pathway as seen in Table 7. P.M.M. in situ rotations yield a much higher proportion of successful tectal innervation than transplant operations.

Evidently, optic pathway development after eye rotation depends upon the surgical procedure. Since the general operating procedure employed by each individual is basi- cally similar, we can only suggest that subtle procedural differences are sufficient to bias the developmental outcome.

Origin of the TR pathway Why does an anomalous TR pathway develop with such

high frequency after eye rotation? To determine the initial trajectory of optic fibers as they exit the retina, we fixed embryos every 2 hours after eye rotation at stage 32. At this stage, the profundis region of the developing trigemi- nal ganglion is situated dorsal and caudal to the optic vesicle (KnoufY, '27). The ganglion is large and develop- mentally more mature than the retina; it already contains numerous nerve fibers in or entering the ganglion. In Fig- ure 7A, a transverse section caudal to the eye of a stage 41 embryo, 4 hours postsurgery, is shown. An optic nerve stump exits the retina dorsally, oriented toward the trigem- inal ganglion. The distance between the choroid fissure in the retina and the trigeminal ganglion is slightly more

than 50 pm. In the next caudal section (Fig. 7B), the optic nerve is seen in the trigeminal ganglion (TG).

The close topographic relationship between the optic nerve head in a rotated eye primordium and the trigeminal gan- glion after eye rotation suggests that fiber bundles in or entering the trigeminal complex (ophthalmic branch?) are contacted as soon as retinal fibers exit the retina. All reti- nal fibers emerging later in development probably follow these first fibers into the trigeminal ganglion and root.

DISCUSSION Perhaps the most important factor affecting optic fiber

development from rotated embryonic eyes is the surgical procedure itself. Two investigators, using the same operat- ing procedure, produce tadpoles with different optic path- way patterns. The differences are subtle and are expressed only after eye rotation since control operations performed by both individuals yield similar results. Moreover, differ- ent eye rotation paradigms (in situ and transplantation) performed by the same individual also yield different path- way patterns.

Mechanical perturbations of eye primordia after rotation seem to override such factors as stage, or orientation of choroid fissure. The surgery biases the initial trajectory of pioneering fibers exiting the retina and defines their topo- graphic relationships to adjacent nerves that may serve as guidance cues. Because all later-arising retinal fibers follow these pioneers, these early events determine all subsequent patterns of optic pathway development.

Such factors as the size and depth of the wound and its effect on healing, or the extent of orbital and adjacent tissue

TABLE 6. Relationship Between Fissure Orientation and Optic Pathway Development'

Aberrant pathways Stage Total N A CH DI oc TR

21/22 19 6 13

26128 79 5 74

30135 49 2 47

(32) (68)

(6) (94)

(4) 196) V fissure 21/22 7 5 2

(22) (78)

(25) (75) 30135 12 3 9

5 (36) 9 (12) 3 (6)

6 (43) 12 (16) 7 (15)

2 5 (14) (36) 22 63 (30) (85) 22 43 (47) (90)

'Numbers in parentheses are percentages. Percentages of N and A are based on total number of fissures. Those for all others are based on totat number of A pathways. Combined data for all series. N, normal pathway; A, aberrant pathways, single or multiple.

TABLE 7. Surgical Procedure Comparison'

Percentages Name Total Fissure ON Neuroma contra IDsi CH DI OC TR

P.M.M. 52 D17 94 31 63 31 50 19 13 56 I 14 80 18 27 27 18 27 9 64

25 50 V21 76 56 P.G. 81 D45 84 50 13 3 11 3 29 89

I 28 93 42 42 19 15 4 38 73 V 8 88 57 57 14 43 14 71

69 6 69

'Stages of operation 21-32. Total, total number of operations. Fissure, number of fissures with orientations shown. All other numbers are percentages of total fissures. ON, percentage of optic nerves present; Neuroma, percentage showing neuroma near retina; contra, innervation contralateral tectum; ipsi, innervation ipsilateral tectum. All others are pathways and include singles and multiples.

374 P. GRANT AND P.M. MA

Fig. 7. Silver-stained transverse sections of stage 41 embryo 24 hours after its leR eye was rotated 180" at stage 32. Sections are caudal to the eye in the region of trigeminal ganglion (TG). A. Last section through eye (El showing pigmented epithelium and optic nerve (ON) emerging with a dorsal

trajectory. MB = midbrain. Arrow labeled d shows dorsal-ventral axis. B. Next 10-pm section caudal to A, showing optic nerve entering trigeminal ganglion (arrow).

damage, or the amount of optic stalk deflection after sur- gery, may affect the outgrowth and initial orientation of the first fibers to exit a rotated retina. If the procedure consistently damages the primordium or the orbit in a specific manner, one pathway may be favored over another. Destroying the adjacent TR ganglion, for example, may divert dorsally directed optic fibers toward oculomotor fi- bers. No systematic effort to evaluate these subtle effects was made, however.

The stage of rotation also affects the surgical outcome. Rotation of an eye primordium a t stage 21/22 results in more CH and fewer TR or OC pathways than rotation at later stages. This property seems to be intrinsic to the eye since age of the host orbit in heterochronic transplantation experiments has no effect on pathway development.

Several factors may explain these stage-dependent differ- ences. Stage 21/22 optic vesicles lack fibers while stage 26- 32 retinas possess fibers, some crossing the chiasm to con- tralateral diencephalon at the time of surgery (Cima and Grant, '80; Grant and Rubin, '80a; Holt, '84). Surgery at stage 21/22 induces no optic fiber trauma; pioneering fibers appear after surgery. Instead, the more precocious oculo- motor or trigeminal fibers may be damaged or destroyed and in their absence, pioneering optic fibers may be more prone to enter the CH pathway as they emerge. TR and OC fibers in older embryos may be less vulnerable to surgical

damage, perhaps because many more fiber bundles are present.

Surgery at later stages damages pioneering fibers extend- ing into the optic stalk. Their proximal cut ends may be mechanically misdirected or displaced by the healing pro- cess. As fibers regenerate, numerous branching collaterals are produced that may be diverted into several pathways in response to alternative guidance cues.

Surgery also overcomes any cues from the choroid fissure. Normally, retinal fibers converge on the ventral fissure and are funneled into the optic stalk leading to the chiasm. Almost all control tadpoles with ventral fissures possess a normal CH pathway. On the other hand, most experimental tadpoles with V fissures possess aberrant pathways after eye rotation at late embryonic stages. Anomalous pathways in animals with V fissures may arise if exiting optic fibers contact trigeminal or oculomotor fibers before or during derotation of the operated eye. After derotation, all fibers arising later will follow these displaced pioneers to estab- lish TR and OC pathways in spite of a ventral fissure. Derotation of eye anlage during healing was a common occurrence in the chick embryo (Goldberg, '76).

For the most part, tadpoles with D or I fissures exhibit aberrant pathways, except for a few operated at stage 21/ 22 with normal CH pathways. In these latter cases, damage to oculomotor or trigeminal fiber cues has already been

ABERRANT OPTIC PATHWAYS FROM ROTATED EYES 375

TABLE 8. Tectal Innervation

Animal Total Tectal (stages) operations maps Percentage Reference

Xenopus 52 12 23 Jacobson ('68)

Xenopus 73 53 7 3 Sharma and Hollyfield ('80)

Xenopus 77 39 51 Gaze et al. ('80)

28-35

24-32

22-30

offered as one explanation. On the other hand, fibers may actively "seek out'' the appropriate pathway, following che- mospecific cues in the orbit or optic stalk. These cues may be transient, sufficient to guide the initial pioneers, only to disappear at later stages, after pioneers have entered the optic pathway. This could explain the few CH pathways after rotation at late stages. Unfortunately, the old to young (stage 21/22) heterochronic experiments designed to test this were technically unsuccessful.

We see that some of the variations and apparent contra- dictions in reported visuotectal projections from rotated eyes may be explained, in part, by subtle differences in surgical procedures employed in different laboratories.

Fiber following and the problem of pathway selectivity

The rule of fiber following, or fasciculation, explains bun- dle formation in the retina (Grant and Rubin, '80a,b) and assembly of fibers in the optic nerve and tract (Horder and Martin, '78; Bunt and Horder, '80; Bodick and Levinthal, '80; Cima and Grant, '82). Growth cones of ganglion cell axons, however, show no preferences for other optic fibers as guidance cues; any axon acts as a preferred substrate for migration. Any accessible nonoptic fibers in or adjacent to the orbit, (e.g., trigeminal and oculomotor) are contacted by the first retinal fibers exiting rotated eyes and all optic fibers arising later follow these initial trajectories, assem- bling into a mixed nerve to the brain.

Optic fibers follow various nonoptic pathways caudally from the site of an auditory vesicle or rostrally in the spinal cord from eye primorida placed ectopically on the flank of an embryo (Constantine-Paton and Capranica, '76; Con- stantine-Paton, '78; Katz and Lasek, '78, '79). Contrary to Katz and Lasek's suggestion, however, that optic fibers only follow sensory fiber bundles, we find them following motor fibers in the oculomotor nerve and sensory fibers (ophthalmic?) in the trigeminal complex. The apparent pref- erence shown by optic fibers for sensory tracts in the spinal cord (Katz and Lasek, '78, '79) may simply reflect the fact that embryonic sensory fibers in the cord are the first to be contacted by optic fibers exiting an ectopically placed eye primordium.

A properly oriented tissue terrain seems to be essential for the guidance of regenerating optic fibers into appfopri- ate pathways (Bohn et al., '82). Displaced conhective tissue elements near the retina can impede and misdirect regen- erating fibers to aberrant pathways along blood vessels, muscle fibers, or branches of cranial nerves. Growth cones may follow such cues by contact guidance mechanisms (Weiss, '61).

The heterochronic transplant results suggest that factors in the eye primordium and not the surrounding orbit seem to be most important in pathway development. In all cases,

the initial trajectory of fibers exiting the retina determines the first guidance cue encountered.

Fibers entering the brain ectopically, particularly those in ipsilateral DI and OC pathways, tend to innervate tha- lamic and basal optic neuropils, often with anomalous tra- jectories. Since optic fibers from the unoperated contralateral eye occupy these regions prior to the arrival of ectopic fibers, they will serve in the brain as preferred pathways to visual neuropils. In spite of their aberrant trajectories to and within the brain, ectopically entering fibers often encounter the correct neuropils.

Optic fiber pathways and development of the retinotectal projection

Rotation of embryonic eye primordia results in develop- ment of inverted, ordered visuotectal projections (Jacobson, '68; Jacobson and Hirsch, '73; Gaze et al., '79; Sharma and Hollyfield, '80). Regenerating optic fibers, following aber- rant pathways (e.g., oculomotor), project normally to the tectum, and if the eye is rotated, they also give rise to inverted projections (Gaze, '59; Beazley and Lamb, '79). In view of our results, it is likely that many inverted projec- tions reported in the literature correlate with aberrant pathways. In fact, Jacobson and Hirsch ('73) report that 41% of late-stage Rana embryos with rotated eyes develop anomalous ipsilateral visuotectal projections, which is in agreement with the frequency of ipsilateral pathways in our results.

The extent of tectal innervation from rotated eyes, as determined by tracing optic pathways, compares favorably with mapping data in the literature (Table 8). As can be seen, tectal innervation was demonstrated in 23% of the cases in one laboratory (Jacobson, '68), while the most suc- cessful results are the 73% contralateral maps reported by Sharma and Hollyfield ('80). Our own data, limited entirely to tadpoles with D and I fissures, vary from 25 to 90% contralateral tectal innervation (depending on stage and series), which are in good agreement.

Since pathways were not traced and correlated with vi- suotectal maps in any of the studies cited (including our own), we still do not know how many maps from rotated eyes reflect aberrant pathways. If, however, operative pro- cedures in other laboratories yield pathway patterns com- parable to our own, then many visuotectal maps from animals with rotated eyes may arise from aberrant multi- ple pathways. Such pathways often yield complex innerva- tion patterns of the type shown in Figures 5 and 6, and may explain, in part, the 41% anomalous compound maps re- ported by Gaze et al. ('79).

During development, as in regeneration, optic fibers with aberrant trajectories from rotated eyes may organize into inverted visuotectal maps. The data are consistent with the hypothesis that ordering of the retinotectal projection is

376 P. GRANT AND P.M. MA

independent of the pathways taken by fibers to reach the tectum. On the other hand, the high frequency of ordered, but inverted, projections reported by some laboratories after eye rotation (e.g., Sharma and Hollyfield, '80) may be due to subtle differences in operative procedures that induce a high proportion of normal chiasmatic pathways. Ordered and inverted retinotectal projections are also obtained after rotating the presumptive embryonic tectum (Chung and Cooke, '79). In those cases in which fiber pathways could be traced and correlated with visuotectal maps, the direction of fiber arrival usually corresponded with the orientation of tectal histogenesis. It is uncertain, however, whether the polarity of fiber arrival is involved in ordering the projec- tion since other factors, such as fiber-fiber interactions or activity-dependent mechanisms between pre- and postsyn- aptic cells, may be primarily responsible for organizing the map (Schmidt, '85). To unequivocally eliminate any rela- tionship between pathway trajectory and the ordering of the retinotectal projection from rotated eyes, visuotectal maps should be compared to the anatomically traced path- way in each animal. It would be of interest to determine, for example, which anomalous pathways invariably corre- late with inverted but ordered projections and which do not.

ACKNOWLEDGMENTS This work was supported by NIH grant EY 02642

awarded to P. Grant by the National Eye Institute, Na- tional Institutes of Health. We wish to thank Harry How- ard and Carol Cogswell for photographic assistance.

LITERATURE CITED Attardi, D.G., and R.W. Sperry (1963) Preferential selection of central path-

ways by regenerating optic fibers. Exp. Neurol. 7:46-64. Beazley, L.D., and A.H. Lamb (1979) Re-routed axons in Xenopus tadpoles

form normal visuotectal projections. Brain Res. 179t373-378. Beckwith, C.J. (1927) The effect of the extirpation of the lens rudiment on

the development of the eye in Amblystoma punctatum with special reference to the choroid fissure. J. Exp. Zool. 49:217-259.

Bodick, N., and C. Levinthal (1980) Growing optic nerves follow neighbors during embryogenesis. Roc. Natl. Acad. Sci. USA 77:4374-4378.

Bohn, R.C., P.J. Reier, and E.B. Sourbeer (1982) Axonal interactions with connective tissue and glial substrata during optic nerve regeneration in Xenopus larvae and adults. Am. J. Anat. 165397-419.

Bunt, S., and T.J. Horder (1983) Evidence for an orderly arrangement of optic fibers within the optic nerves of the major non-mammalian verte- brate classes. J. Comp. Neurol. 213394-114.

Chung, S-H., and J. Cooke (1978) Observations on the formation of the brain and of nerve connections following embryonic manipulation of the am- phibian neural tube. Proc. R. SOC. Lond. [Biol.] 201r335-373.

Cima, C., and P. Grant (1980) Ontogeny of the retina and optic nerve in Xenopus laeuis: Ultrastructural evidence of early ganglion cell differen- tiation. Dev. Biol. 78.229-237.

Constantine-Paton, M. (1978) Central projection of anuran optic nerves penetrating hindbrain or spinal cord regions of the neural tube. Brain Res. 158:31-43.

Constantine-Paton, M., and R.P. Capranica (1976) Axonal guidance of devel- oping optic nerves in the frog. 11. Anatomy of the projection from trans- loeated eyes in the leopard frog (Ranapipiens). J. Comp. Neurol. 17Or17- 32.

Fuller, P.M., and S.O.E. Ebbesson (1973) Central projections of the trigemi-

nal nerve in the bull frog (Rana catesbiarza). 3. Comp. Neurol. 152t193- 200.

Gaze, R.M. (1959) Regeneration of the optic nerve in Xenopus laeuis. Q. J. Exp. Physiol. 44290-308.

Gaze, R.M., M.J. Keating, A. Ostberg, and S.-H. Chung (1979) The relation- ship between retinal and tectal growth in larval Xenopus: Implications for the development of the retinotectal projection. J. Embryol. Exp. Morphol. 53:103-143.

Goldberg, S. (1976) Polarization of the avian retina. Ocular transplantation studies. J. Comp. Neurol. 168r379-392.

Grant, P., and E. Rubin (198Oa) Ontogeny of the retina and optic nerve in Xenopus lueuis. 11. Ontogeny of the optic fiber pattern in the retina. J. Comp. Neurol. 189671-698.

Grant, P., and E. Rubin (198Ob) Disruption of optic fibre growth following eye rotation in Xenopus laeuis embryos. Nature 287:845-848.

Hibbard, E. (1967) Visual recovery following regeneration of the optic nerve root in Xenopus. Exp. Neurol. 19r350-356.

Horder, T.J., and K.A.C. Martin (1978) Morphogenetics as an alternative to chemospecificity in the formation of nerve connections. In A.S.G. Curtis (ed): Cell-Cell Recognition. Cambridge: Cambridge University Press, pp.275-385.

Holt, C.E. (1984) Does timing of axon outgrowth influence initial retinotec- tal topography in Xenopus? J. Neurosci. 4:1130-1152.

Jacobson, M. (1968) Development of neuronal specification in retinal gan- glion cells in Xenopus. Dev. Biol. 17:202-218.

Jacobson, M. (1968) Development of neuronal specification in retinal gan- glion cells in Xenopus. Dev. Biol. 17~202-218.

Jacobson, M., and H.V.B. Hirsch (1973) Development and maintenance of connectivity in the visual system of the frog. I. The effects of eye rotation and visual deprivation. Brain Res. 49t47-65.

Katz, M.J., and R.J. Lasek (1978) Eyes transplanted to tadpole tails send axons rostrally in two spinal cord tracts. Science 199:202-204.

Katz, M.J., and R.J. Lasek (1979) Substrate pathways which guide axons in Xenopus embryos. J. Comp. Neurol. 183~817-832.

Knouf€, R.A. (1927) The origin of the cranial ganglion of Rana. J. Comp. Neurol. 44.259-361.

Levine, R.L. (1980) An autoradiographic study of the retinal projection in Xenopus lueuis with comparisons to Rana J. Comp. Neurol. I89:l-29.

Nieuwkoop, P.D., and J. Faber (1956) Normal Table of Xenopus lueuu (Daudin). Amsterdam: North Holland.

Rowell, C.H.F. (1963) A general method for silvering invertebrate central nervous systems. J. Microsc. Soc. 104381-87.

Sato, T. (1922) Ueber die determination der fetal Augenspalts bei Biton taeniatus. Arch. Entw. Mech. Org. 128:342-377.

Schmidt, J.T. (1985) Formation of retinotopic connections: Selective stabili- zation by an activity-dependent mechanism. Cell. Mol. Neurobiol. 5:65- 84.

Sharma, S.C. (1972) Retinotectal connections of a heterotopic eye. Nature New Biol. ,238286-287.

Sharma, S.C. (1973) Anomalous retinal projections after removal of contra- lateral optic tectum in adult goldfish. Exp. Neurol. 41:661-669.

Sharma, S.C., and Hollyfield, J.G. (1980) Specification of retinotectal con- nections during development of the toad Xenopus Zaeuis. J. Embryol. Exp. Morphol. 5577-92.

Sperry, R.W. (1945) The problem of central nervous reorganization after nerve regeneration and muscle transposition. Rev. Biol. 20:311-369.

Stone, L.S. (1966) Development, polarization and regeneration of the ventral iris cleft (remnant of choroid fissure) and protractor lentis muscle in urodele eyes. J. Exp. Zool. 161:95-108.

Straznicky, C., R.M. Gaze, and T.J. Horder (1979) Selection of the appropri- ate medial branch of the optic tract by fibres of ventral retinal origin during development and in regeneration: An autoradiographic study in Xenopus. J. Embryol. Exp. Morphol. 26:523-542.

Weiss, P. (1961) Guiding principles in cell locomotion and cell aggregation. Exp. Cell Res. [Suppl.] 8:260-281.