Trajectories of axons in ectopic VIIIth nerves

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BIOLOGY

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BRIEF NOTES Trajectories of Axons in Ectopic Vlllth Nerves MARTHA Department

CONSTANTINE-PATON

of Biology, Princeton

University,

Princeton,

Received July 30, 1982; accepted in l;evised form

New Jersey 08544

December 2.2, 1982

Transplantation of the aeousticolateral placode to the evacuated eye position in embryos of the frog Rana pipiens has been used to force axons of the ViIIth cranial nerve to penetrate the diencephalon. These ectopic axons establish a growth trajectory that is strikingly similar to their normal growth trajectory within the medulla oblongata despite the fact that no other axons within the diencephalon normally follow this route. The result is discussed in terms of the “blueprint” and substrates pathway hypotheses which have been advanced to explain the initial development of axon tracts within the central nervous system. INTtiODUCTION

The mechanisms responsible for the stereotyped development of axon pathways continue to engender de= bate despite concentrated attention since the beginning of this century. The chronologically oldest chemotropic and galvanotropic theories of axon growth require aci tion at great distances (Weiss, 1955). These have been widely criticized in favor of passive “timing and outgrowth” models (Rager and von Oeynhausen, 1979; Nornes et aL, 1980) and mechanical guidance (Weiss, 1955). Nevertheless demonstrations of pathway selectivity during axon development (Straznicky et al., 1979; Lance-Jones and Landmesser, 1980) coupled with in vitro evidence of substrate preferences (Letourneau, 1975; Bonhoeffer and Huf, 1980) and of chemotropic responses to NGF (Levi-Montalcini et ab, 1978; Gundersen and Barrett, 1980) suggest that vertebrate axonal growth cones can actively discriminate between chemicai characteristics of their environment. Similar conclusions have been reached in several invertebrate systems where selective cellular interactions between “pioneer” axons and the microenvironment have been implicated in the development of characteristic neuronai projections (Bate, 1976; Macagno, 1978; Goodman et ah, 1981; Edwards et ciL, 1981). However, questions about how these cues are read by the first axons, or how substrate differences and selective substrate preferences become distributed still remain unanswered. Earlier studies have addressed the rules governing stereotyped axon growth using transplantations of eye primordia or Mauthner neurons to abnormal locations relative to the neural axis in amphibian embryos (Constantine-Paton and Capranica, 1976; Constantine-Paton, 1978; Giorgi and Van der Loos, 1978;Jacobson, 1978; Katz and Lasek, 1979, 1981; Harris, 1980). Subsequent

anatomical tracing and reconstruction of the pathways established by these misplaced axons have revealed striking consistencies in their growth patterns. Regardless of whe-re along the neural axis, optic nerve axons penetrate the central nervous system they grow adjacent to the glia limitans and establish a longitudinally oriented optic tract in a position corresponding to the dorsolateral quadrant of the primitive neural tube. This same growth behavior is characteristic of retinal ganglion eeli axons growing unperturbed within the diencephalon, suggesting that normal optic tract development is not dependent upon cues unique to forebrain regions of the neural tube (Constantine-Paton and Capranica, 1976; Constantine-Paton, 1978; Katz and Lasek, 1979; Harris, 1980). Similar conclusions have been reached from work with embryonically misplaced Mauthner neurons. Axons from these motor “interneurons” whether growing normally or after transplantation extend longitudinally in ventral (or “motor”) regions of the primitive neural tube (Katz and Laseki 1981). In shorti all of these observations support the idea that a relatively small number of cues distributed similarly throughout the neural prirnordium may be sufficient to specify a wide range of projections within the developing CNS (Constantine-Paton, 1978; Jacobson, 1978; Katz and Lasek, 1979, 1981; Harris, 1980). The critical requirement is that the different sets of axons respond selectively to these cues (Constantine-Paton, 1978; Katz and Lasek, 1979, 1981). Unfortunately the optic tract and Mauthner cell experiments are complicated by the fact that the pathways established by ectopic axons frequently overlap the sensory or motor tracts that are native to the CNS region in which the misplaced fibers are forced to grow. It is consequently possible that ectopic axons are responding to passive channeling by normal axon bundles

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and, if this is the case, the resemblance of these ectopic mitted reconstruction of the polarity with which the pathways to the same pathways in their normal posi- ectopic labyrinth developed. Only the basilar papilla, tion would be coincidental rather than indicative of a the smallest of the frog’s eight labyrinthine sensory generalized system for CNS axon guidance. organs (Birkmann, 1940, Witschi, 1949) was absent in To test this possibility the acousticolateral placode the majority (n = 1’7) of the animals examined. of Rana p+%ens embryos (Knouff, 1935) has been emOur limited observations on fixation of inner ear pobryonically implanted in the vacated eye position and larity indicate that it occurs in the early primordium the growth of VIIIth nerve axons which subsequently as initially reported by Harrison (1936). Of the 14 anextend from the membraneous labyrinth into the di- imals with left ear grafts adjacent to the right side of encephalon has been examined. This particular transthe diencephalon 9 had sufficient numbers of clearly plantation paradigm overcomes previous interpretive identifiable papillae and cristae to establish A-P axis difficulties because the growth pattern of VIIIth nerve orientation. All showed the expected 180” inversion fibers within the medulla oblongata, though stereo- along this axis. The ectopic labyrinths of the remaining typed, is completely unlike the growth trajectory of ei- 12 animals showed the expected, normal, A-P axis orither the optic tract or any other defined axon bundle entation. Eighth nerve ganglion cells are derived from the menative to diencephalic forebrain regions (Frontera, 1952; dial wall of the otic capsule (Knouff, 1927;Yntema, 1937), Scalia et ah, 1968). thus all transplanted inner ears had some sensory ganglion cells associated with them and all preparations METHODS stained with the Holmes method showed distal proThe observations presented in this report were ob- cessesof VIIIth nerve ganglion cells in close association tained from analysis of serial lo-pm sections taken from with the base of the hair cells (Constantine-Paton, 1976). 9 tadpoles and 17 postmetamorphic embryonically ma- However in 13 cases either the ganglion cell bodies nipulated Rana pipiens. The dorsal skull and brain of themselves or their central processes fused with the these animals were dissected and fixed in 10% buffered normal trigeminal ganglion. None of these ganglia had Formalin or Susa’s fluid. After decalcification the tissue central processes which penetrated the diencephalon. was embedded in paraffin and stained by the KluverThe pattern of entry of the 13 ectopic VIIIth nerves Barrer method or a modified Holmes silver procedure which penetrated the diencephalon is shown in the cam(Humason, 196’7).These animals were derived from an era lucidae tracings of Fig. 1. In all except one of these initially much larger group of over 200 tadpoles that cases the VIIIth nerve axons extended dorsally from a had been subjected to the embryonic placode transrelatively ventrally placed ganglion and penetrated the plants but either failed to survive to analysis or failed lateral region of the diencephalon. Although not obto show differentiation of ectopic inner ears. vious in all the camera lucidae tracings of sections at The procedures used for embryonic microsurgery have the point of entry, all of these projections could be folbeen detailed previously (Constantine-Paton and Ca- lowed into the periventricular cellular regions of the pranica, 1976). Briefly, the left acousticolateralis plac- frog’s dorsal thalamus. One animal (Fig. lm) had an ode of a Shumway stage 1’7-19Rana p@iem (Shumway, ectopic VIIIth ganglion which developed in an unusually 1940) was removed along with overlying ectoderm and posterior and ventral position. The central processes of placed into the position from which either the left or this ganglion grew dorsolaterally adjacent to the hythe right eye of the same embryo had been previously pothalamus and penetrated the ventral thalamic gray evacuated. The operation was performed with minimal matter. In all 13 animals the pattern of entry into the rotation around the dorsoventral axis of the placode so brain and the behavior of axons immediately upon penthat implantation into the left eye position correctly etration were remarkably similar to the growth pattern aligned the placodes’ presumptive anterior-posterior (Aof the normal frog VIIIth nerve when it enters the CNS P) axis, and implantations into the right eye site re- at the level of the medulla oblongata (Larsell, 1934; sulted in inversions of that axis. Boord et aZ., 1971; Gregory, 1972). This similarity is illustrated in Figs. 2a and d. In the normal tadpole (Fig. RESULTS 2a) VIIIth nerve axons extend dorsally beside the wall The ectopic inner ears were generally distorted in of the neural tube and actually penetrate the medulla oblongata in the ventralmost regions of the alar plate overall shape and in the alignment of the semicircular canals. However, the distinctive morphology of the which can be distinguished as the region of neural tube acoustic and vestibular sensory organs (Geisler et aZ., lying dorsal to the lateral sulcus (Is). The ectopic VIIIth 1964; Wever, 1973) and their positions relative to one nerve axons (Fig. 2d) also extend central processes adanother (Birkmann, 1940; Witschi, 1949) usually per- jacent to the neural tube but in this case they penetrate

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tions of the diencephalon before disappearing but unfortunately the nonselective impregnation provided by the Holmes silver nitrate stain did not permit the question of inappropriate VIIIth nerve synapses to be explored in this material. Axons of the VIIIth and optic nerves develop over the same embryonic and larval periods (Larsell, 1934; Witchi, 1949; Currie and Cowan, 1975). However, as the inset of Fig. 2d demonstrates, ectopic VIIIth nerve axons are not deflected from their pattern of growth despite the fact that they cross the fine calibar retinal ganglion cell axons of the optic tract at nearly right angles. Thus passive mechanical channeling of ectopic VIIIth nerve growth cones by the retinal ganglion cell axons native to the diencephalon is not occurring in these ear transplant preparations.

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FIG. 1. Camera lucida tracings of single transverse sections showing the manner in which VIIIth nerve axons from transplanted inner ears penetrate the diencephalon. Each tracing is from a separate animal but the tracings are arranged in a rostral-caudal sequence. The Roman numeral to the lower left of each figure indicates the type of embryonic transplant performed. I, a left ear primordium implant to the position of the left eye; II, a right ear primordium implant to the position of the left eye. telen., telencephalon; o.n., optic nerve; o.t., optic tectum.

the alar plate of the diencephalon. Within the CNS both normal and transplanted VIIIth nerve axons traverse the white matter and grow among the somata lying in the vicinity of their entry points. Upon entering the medulla oblongata the central processes of normal VIIIth nerve ganglion make synaptic contacts within the dorsal acoustic nucleus and the magnocellular vestibular nucleus (Larsell, 1934; Boord et ab, 19’71; Gregory, 1972). These neuron populations are situated in the dorsolateral gray matter adjacent to the point of VIIIth nerve entry. Ectopic VIIIth nerve axons could be followed for variable distances through serial sec-

The present observations indicate that axons of the VIIIth cranial nerve growing ectopically within the diencephalon can establish a growth trajectory like that of normal VIIIth nerve axons within the medulla oblongata. It is significant that no other axons within the abnormally penetrated forebrain regions of the neural tube exhibit a growth trajectory that is anything like that of the VIIIth nerve. Nevertheless the trajectories of all ectopic VIIIth nerve bundles were so similar to each other that they could be immediately identified after even the most casual examination of the tissue. Apparently the ectopic growth cones were consistently able to respond in a highly selective fashion to cues that were present in the diencephalon but normally not used in the same way by axons native to that region. These cues must be stereotypically aligned relative to the major axis of the alar and basilar regions of the tube. However, neither the present observations nor any of the other data available at present allows the nature or the distribution of these cues to be specified more completely. Singer et al. (1979) have, for example, outlined a “blueprint” hypothesis in which the growth trajectories of the major CNS axonal tracts are believed to be determined initially by mechanicochemical “pathways” in the glial support cells. When this hypothesis is examined in light of the present results, it must either be discarded or extended to imply that “blueprints” of pathways are present even in regions of the neural tube where that blueprint would never normally be used. In this context, the label “blueprint” would seem to be a misnomer. An alternative, substrate pathway hypothesis has been proposed by Katz and Lasek on the basis of experiments with ectopic optic nerves and Mauthner cells (Katz and Lasek, 1979, 1981). They suggest that the embryonic neural plate has established in it a finite

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FIG. 2. Comparisons of normal and ectogic VIIIth nerve penetration into the central nervous system of &?&a pipiens tadpoles. (a, b) Photomicrogcaphs of a transverse silver-stained section through the hindbrain of normal Taylor and Rollros stage III (Taylor and Kollros, 1946) tadpole showing the patterns of VIIIth nerve growth established by axons penetrating the medulla oblangata from an unperturbed inner earl 6%d) Photomicrwrapbs from transverse sections through the diencephalon of a Tiylor and ICollros stage XVIII tadpole who had a Fight inner ear primordium implanted into the left eye position at Shumwsy embryonic stage 17. An enlargement of ectopic VIIIth nerve axons coursing among the periventricular gray matter of the diencepbalon is presented in c and the inset to d illustrates that the trajectories of large caliber VIII& nerve axons are not deflected as they cross the finer &COWof the optic tract at nearly right angles. ganglion, the cell bodies of the acousticoveatibular nerve. Is.-lateral sulcus; n? VIII! the central processes of the acousticovestibular nerve; on., right optic nerve. (a-d) Scale bar: 100 hrn; Inset scale bar: 200 pm.

number of well-defined pathways which can be followed selectively by a given axon population depending upon some rather general early embryonic specifkation of those azons such as whether they are motor or sensory in function, According to this scheme all sensory axons follow a dorsolateral substrate pathway in the alar plate while motor axons follow a ventral basal plate substrate

pathway. Unfortunately this hypothesis also fails to account for the present results, Neither in their normal position nor in an ectopie position in the diencephalon do the sensory axons of the VWh cranial nerve follow a dorsolateral pathway characteristic of the major longitudinally running sensory tracts of the CNS. An additiosal dorsoradiat pathway distributed in all neural

BRIEF NOTES

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WITSCHI,E. (1949). The larval ear of the frog and its transformation during metamorphosis. Z&t. Naturforschung 46, 230-242. YNTEMA,C. L. (1937). An experimental study of the origin of the cells which constitute the VIIth and VIIIth ganglia nerves in the embryo J. Exp. ZooL 75, 75-105. of Amblystoma pun&turn