The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

Precocious pathfinding: retinal axons can navigate in an axonless brain.

The developing axons of retinal ganglion cells follow a stereotyped trajectory through the diencephalon to the optic tectum. In Xenopus, this trajectory closely parallels that of a preexisting fiber tract, the tract of the postoptic commissure (TPOC). This tract comprises part of the early CNS scaffold and has been proposed to play a critical role in guiding the later growing optic axons. We have tested this possibility using heterochronic and xenoplastic transplants of eye primordia to force optic axons to enter the brain before scaffold tracts have arisen in the forebrain. We show that optic axons can navigate appropriately in the absence of the TPOC or any other axons, indicating that axonal pathfinding cues are present in the axonless neuroepithelial sheet. We suggest that molecularly distinct heterogeneities within the neuroepithelium are used for pathfinding by early and late developing axons alike in normal development.     

A specific brain tract guides follower growth cones in two regions of the zebrafish brain

Neurons of the nucleus of the posterior commissure (nuc PC), an identifiable cluster of neurons in the embryonic zebrafish brain, project growth cones ventrally along the posterior commissure to the anterior tegmentum where the PC intersects two longitudinal tracts, the tract of the postoptic commissure (TPOC) and the medial longitudinal fasciculus (MLF). Once at the intersection, nuc PC growth cones turn posteriorly onto the TPOC in the dorsal tegmentum and follow it to the hindbrain. Previously we showed that in the absence of the TPOC, nuc PC growth cones often extended along aberrant path ways suggesting that fasciculation, that is, contact with TPOC axons is an important factor in guiding growth cones along their normal pathway. However, a significant number of nuc PC growth cones also followed their normal pathway suggesting that cues associated with the dorsolateral tegmentum, independent of the TPOC, can also guide nuc PC growth cones. We have now confirmed using electron microscopy that nuc PC growth cones fasciculate with axons in the TPOC. In the absence of the TPOC, the nuc PC growth cones that extend along their normal pathway do so in contact with dorsolateral neuroepithelial cells. This suggests that cues associated with these cells can also guide the nuc PC growth cones. Furthermore, in the absence of the TPOC axons, these growth cones now inappropriately turn onto axons that normally intersect the TPOC near the border of the midbrain and hindbrain, that is, at a second intersection of tracts. This suggests that fasciculation with TPOC axons may also guide nuc PC growth cones in this second region of the brain. © 1992 John Wiley & Sons, Inc.     

A specific brain tract guides follower growth cones in two regions of the zebrafish brain.

Neurons of the nucleus of the posterior commissure (nuc PC), an identifiable cluster of neurons in the embryonic zebrafish brain, project growth cones ventrally along the posterior commissure to the anterior tegmentum where the PC intersects two longitudinal tracts, the tract of the postoptic commissure (TPOC) and the medial longitudinal fasciculus (MLF). Once at the intersection, nuc PC growth cones turn posteriorly onto the TPOC in the dorsal tegmentum and follow it to the hindbrain. Previously we showed that in the absence of the TPOC, nuc PC growth cones often extended along aberrant pathways suggesting that fasciculation, that is, contact with TPOC axons is an important factor in guiding growth cones along their normal pathway. However, a significant number of nuc PC growth cones also followed their normal pathway suggesting that cues associated with the dorsolateral tegmentum, independent of the TPOC, can also guide nuc PC growth cones. We have now confirmed using electron microscopy that nuc PC growth cones fasciculate with axons in the TPOC. In the absence of the TPOC, the nuc PC growth cones that extend along their normal pathway do so in contact with dorsolateral neuroepithelial cells. This suggests that cues associated with these cells can also guide the nuc PC growth cones. Furthermore, in the absence of the TPOC axons, these growth cones now inappropriately turn onto axons that normally intersect the TPOC near the border of the midbrain and hindbrain, that is, at a second intersection of tracts. This suggests that fasciculation with TPOC axons may also guide nuc PC growth cones in this second region of the brain.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

C. elegans dystroglycan coordinates responsiveness of follower axons to dorsal/ventral and anterior/posterior guidance cues

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle‐shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN‐1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn‐1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC‐6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN‐1 in unc‐6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN‐1 does not mediate follower/pioneer adhesion. Instead, DGN‐1 appears to block premature responsiveness of follower axons to a preanal ganglion‐directed guidance cue, which mediates ventral‐to‐anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N‐terminal DGN‐1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Robo-Slit interactions regulate longitudinal axon pathfinding in the embryonic vertebrate brain

The early network of axons in the embryonic brain provides connectivity between functionally distinct regions of the nervous system. While many of the molecular interactions driving commissural pathway formation have been deciphered, the mechanisms underlying the development of longitudinal tracts remain unclear. We have identified here a role for the Roundabout (Robo) family of axon guidance receptors in the positioning of longitudinally projecting axons along the dorsoventral axis in the embryonic zebrafish forebrain. Using a loss-of-function approach, we established that Robo family members exhibit complementary functions in the tract of the postoptic commissure (TPOC), the major longitudinal tract in the forebrain. Robo2 acted initially to split the TPOC into discrete fascicles upon entering a broad domain of Slit1a expression in the ventrocaudal diencephalon. In contrast, Robo1 and Robo3 restricted the extent of defasciculation of the TPOC. In this way, the complementary roles of Robo family members balance levels of fasciculation and defasciculation along this trajectory. These results demonstrate a key role for Robo-Slit signaling in vertebrate longitudinal axon guidance and highlight the importance of context-specific guidance cues during navigation within complex pathways.     

Fascicle switching generates a chiasmal neuroarchitecture in the embryonic central body of the grasshopper Schistocerca gregaria

The central body is a prominent neuropilar structure in the midbrain of the grasshopper and is characterized by a fan-shaped array of fiber columns, which are part of a chiasmal system linking anterior and posterior commissures. These columns are established during embryogenesis and comprise axons from cell clusters in the pars intercerebralis, which project to the central body via the so-called w, x, y, z tracts. Up to mid-embryogenesis the primary axon scaffold in both the brain and ventral nerve cord comprises a simple orthogonal arrangement of commissural and longitudinal fiber pathways. No chiasmata are present and this pattern is maintained during subsequent development of the ventral nerve cord. In the midbrain, individual axons entering the commissural system from each of the w, x, y, z tracts after mid-embryogenesis (55%) are seen to systematically de-fasciculate from an anterior commissure and re-fasciculate with another more posterior commissure en route across the midline, a feature we call "fascicle switching". Since the w, x, y, z tracts are bilaterally symmetrical, fascicle switching generates chiasmata at stereotypic locations across the midbrain. Choice points for leaving and entering fascicles mark the anterior and posterior positions of each future column. As the midbrain neuropil expands, the anterior and posterior groups of commissures condense, so that the chiasmata spanning the widening gap between them become progressively more orthogonally oriented. A columnar neuroarchitecture resembling that of the adult central body is already apparent at 70% of embryogenesis.     

Central projections of the pineal complex in the silver lamprey Ichthyomyzon unicuspis

Summary The central projections of the pineal complex of the silver lamprey Ichthyomyzon unicuspis were studied by injection of horseradish peroxidase. The pineal tract courses caudally along the left side of the habenular commissure, and a few fibers penetrate the brain through the caudalmost portion of this commissure. Most of the fibers, however, continue caudally and enter the brain through the posterior commissure. The pineal tract projects bilaterally to the subcomissural organ, the superficial and periventricular pretectum, the posterior tubercular nucleus, the dorsal and ventral thalamus, the dorsal hypothalamus, the optic tectum, the torus semicircularis, the midbrain tegmentum, and the oculomotor nucleus. A few fibers decussate in the tubercular commissure, but the course of these decussate fibers could not be followed owing to the bilateral nature of the projections. No retrogradely labeled cells were found in the brain. With the exception of the projections to the optic tectum and torus semicircularis, the pineal projections in the silver lamprey are similar to those reported in other anamniote vertebrates.     

Development of the anterior commissure in the opossum: Midline extracellular space and glia coincide with early axon decussation

While the anterior commissure has been shown to be an important route of information transfer in the forebrain, relatively little is known about its anatomical development. Glial substrates and extracellular spaces have been associated with the maturation of other large-fiber tracts, such as the corpus callosum and retinofugal pathway. The present study examined early stages in the maturation of the commissure in the gray short-tailed opossum, Monodelphis domestica. Monodelphis offspring are born after a short 14-day gestation, and, unlike in rats and mice, the anterior commissure develops entirely during the postnatal period. A number of techniques were employed: the carbocyanine dye Dil was used to label early axons in the region, semithin plastic sections were used to examine the extracellular environment of the developing commissure, and immunocytochemistry for glial fibrillary acidic protein (GFAP) was used to characterize glial components. Results suggest that the first commissural fibers that cross the midline pass through a region of large extracellular spaces and may use GFAP-immunoreactive cells and processes as guides during their midline decussation. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 403–414, 1997.     

DCC plays a role in navigation of forebrain axons across the ventral midbrain commissure in embryonic xenopus

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain.     

The relationship of acetylcholinesterase activity to optic fiber projections in the tiger salamander Ambystoma tigrinum

In the tiger salamnder the distribution of optic fibers, as revealed by the Fink-Heimer method, is compared with the localization of acetylcholinesterase, as revealed by histochemical methods. AChE activity coincides with optic nerve axons in the optic fiber layer of the retina, in the optic nerve, in the optic tracts and in every optic projection. Reginons of optic fiber terminals show heavier activity than optic fibers of passage. Comparison with other vertebrates is also made.     

Analysis of a repulsive axon guidance molecule, draxin, on ventrally directed axon projection in chick early embryonic midbrain

The mesencephalic V neurons and tectobulbar axons in chick embryo project over long distances that appear during the early development of the chick optic tectum. The mesencephalic V neuron and tectobulbar axonal growth begin at Hamburger and Hamilton stage 14 and stage 18, respectively. Both fibers proceed downward from the dorsal to the ventral side of the lateral wall of the optic tectum and then turn caudally and join the medial longitudinal fasciculus. Their axons appear in the most superficial layer of the tectum at early stages and do not cross the dorsal midline of the tectum. Here, we report the role of draxin, a recently identified axon guidance protein, in the formation of the ventrally directed tectum axonal tracts in chicken embryo. draxin is expressed in a high dorsal to low ventral gradient in chick optic tectum. In vitro experiments show that draxin repels neurite outgrowth from dorsal tectum explants. In vivo overexpression resulted in inhibition or misrouting of axon growth in the tectum. Therefore, draxin may be an important member of the collection of repulsive guidance molecules that regulate the formation of the ventrally directed tectum axon tracts.     

Oriented extracellular channels and axonal guidance in the embryonic chick retina

Transmission electron microscopy and serial reconstruction of lum sections were used to determine whether aligned extracellular channels precede the outgrowth of optic fibers in the embryonic chick retina. At stage 16, just prior to the migration of optic axons toward the optic stalk, extracellular spaces bounded by neuroepithelial cell processes, in the superficial (vitread) region of the retina, were aligned toward the optic stalk. The optic axons subsequently entered and grew within these spaces. After formation of the ganglion cell fiber layer (GCFL), the growth cones of new optic axons entered the most vitread portion of that layer. Hypertonic fixatives caused shrinkage of cell processes, resulting in intercellular separation. However, growth cone filopodia retained close contacts with neighboring glial cell endfeet and with optic axons in these solutions. This suggests that growth cones may be adherent to these structures. Transmission electron microscopy in conjunction with the use of hypertonic solutions may become a useful technique for assaying intercellular adhesivity.     

The paraventriculo-infundibular corticotropin releasing factor (CRF) pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats

The immunocytochemical localization of corticotropin releasing factor (CRF)-containing pathways projecting from the paraventricular nucleus (PVN) to the external layer of the median eminence (ME) in long-term hypophysectomized or adrenalectomized rats is described. Immunocytochemistry was followed by silver intensification of the diaminobenzidine end-product. In comparison with untreated control rats, both hypophysectomy and adrenalectomy resulted in a dramatic increase in immunostaining of the CRF-containing perikarya and fibers, particularly those originating from the PVN and terminating in the ME. The staining was more intense in adrenalectomized than in hypophysectomized rats. The CRF-positive fibers emerging from the PVN form a medial, an intermediate and a lateral fiber pathway. The lateral and intermediate CRF tracts leave the dorsolateral part of the PVN and course laterally and medially of the fornix, respectively, then ventrally toward the optic tract. Just dorsal to the optic tract they turn in caudal direction and run parallel with and very close to the basal surface of the hypothalamus; individual fibers then turn medially to terminate in the external layer of the ME. Only a few fibers originate from the medial-ventral part of the PVN (medial pathway). These fibers run in ventral direction along the walls of the 3rd ventricle and terminate in the ME. Thus the majority of CRF fibers, similarly to other peptidergic systems, reach the medial basal hypothalamus from the anterolateral direction.     

Antiserum to an eye-specific protein identifies photoreceptor and circadian pacemaker neuron projections in Aplysia

The marine gastropod Aplysia has a circadian clock in each eye that generates a circadian rhythm of optic nerve activity. The axons of pacemaker neurons carry the rhythmic activity to the brain where it can be recorded from various ganglionic connectives as it is distributed throughout the CNS. We had previously identified an eye-specific 48-kD protein using an antiserum, anti-S, that recognizes the period gene product of Drosophila. We have now obtained two partial amino acid sequences of the 48-kD protein and raised a polyclonal antiserum using a synthetic peptide with the amino acid sequence of one of them. The antiserum recognizes a family of spots of M_r 47–48 kD and P_i 5.9–6.0 on 2D immunoblots of eye proteins. The immunoblot staining intensity does not exhibit a circadian rhythm. Used in immunocytochemistry, the antiserum recognizes fibers in the optic nerve and retinal neuropil, pacemaker neurons, certain photoreceptors, and the photoreceptor rhabdom layer. It stains the optic nerve fibers and optic fiber terminals in the cerebral optic ganglion and recognizes the cerebral optic tracts, putative synaptic exchange areas, and optic tract projections from the cerebral ganglion into various head nerves and interganglionic connectives. The function of the 48-kD protein is not known but it could be involved in the maintenance or regulation of the retinal afferent pathways, including the pacemaker neuron axons, known from previous axonal transport and electrical recording studies to be the circadian output pathway. © 1993 John Wiley & Sons, Inc.     

Projections of the optic tectum and the mesencephalic nucleus of the trigeminal nerve in the tegu lizard (Tupinambis nigropunctatus)

Summary Fibers undergoing Wallerian degeneration following tectal lesions were demonstrated with the Nauta and Fink-Heimer methods and traced to their termination. Four of the five distinct fiber paths originating in the optic tectum appear related to vision, while one is related to the mesencephalic nucleus of the trigeminus. The latter component of the tectal efferents distributes fibers to 1) the main sensory nucleus of the trigeminus, 2) the motor nucleus of the trigeminus, 3) the nucleus of tractus solitarius, and 4) the intermediate gray of the cervical spinal cord.The principal ascending bundle projects to the nucleus rotundus, three components of the ventral geniculate nucleus and the nucleus ventromedialis anterior ipsilaterally, before it crosses in the supraoptic commissure and terminates in the contralateral nucleus rotundus, ventral geniculate nucleus and a hitherto unnamed region dorsal to the nucleus of the posterior accessory optic tract.Fibers leaving the tectum dorso-medially terminate in the posterodorsal nucleus ipsilaterally and the stratum griseum periventriculare of the contralateral tectum. The descending fiber paths terminate in medial reticular cell groups and the rostral spinal cord contralaterally and in the torus and the lateral reticular regions ipsilaterally. The ipsilateral fascicle also issues fibers to the magnocellular nucleus isthmi.     

Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin

Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin-N-hydroxysuccinimidester, biotin-epsilon-aminocaproic acid-N-hydroxysuccinimidester, and desthiobiotin-N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5-E5.5) to 89% (stage 29 to 35; E6-E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9-E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.     

A developmental and ultrastructural study of the optic chiasma in Xenopus

The structure of the optic chiasma in Xenopus tadpoles has been investigated by light and electron microscopy. Where the optic nerve approaches the chiasma, a tongue of cells protrudes from the periventricular cell mass into the dorsal part of the nerve. Glial processes from this tongue of cells ensheath fascicles of optic axons as they enter the brain. Coincident with this partitioning, the annular arrangement of axons in the optic nerve changes to the laminar organization of the optic tract. Beyond the site of this rearrangement, all newly growing axons accumulate in the ventral-most part of the nerve and pass into the region between the periventricular cells and pia which we have called the 'bridge'. This region is characterized by a loose meshwork of glial cell processes, intercellular spaces and the presence of both optic and nonoptic axons. In the bridge, putative growth cones of retinal ganglion cell axons are found in the intercellular spaces in contact with both the glia and with other axons. The newly growing axons from each eye cross in the bridge at the midline and pass into the superficial layers of the contralateral optic tracts. As the system continues to grow, previous generations of axon, which initially crossed in the existing bridge, are displaced dorsally and caudally, forming the deeper layers of the chiasma. At their point of crossing in the deeper layers, these fascicles of axons from each eye interweave in an intimate fashion. There is no glial segregation of the older axons as they interweave within the chiasma.     

Stereotyped and variable growth of redirected Mauthner axons

To assay the axon tract organizing capabilities of different regions of the vertebrate CNS, Mauthner axons were redirected by grafting supernumerary hindbrains in Xenopus embryos. The 63 redirected Mauthner axons thus produced included donor axons projecting into the host CNS and host axons that grew through the graft or that were redirected in the host CNS. Two major phenomena were observed. Caudal to the optic chiasm, the Mauthner axons followed a single ipsilateral stereotyped route—the basal substrate pathway—extending in the ventral and ventrolateral marginal zone from the diencephalon to the caudal spinal cord. In contrast, rostral to the optic chiasm, these same Mauthner axons followed variable ipsilateral and contralateral routes. Even pairs of Mauthner axons entering the optic chiasm side-by-side eventually followed different routes in normal forebrains. The contrasting behaviors of the Mauthner axons growing in the rostral diencephalon and telencephalon and of the same Mauthner axons growing elsewhere suggest that there are differences in the effective guidance cues between these two regions of the developing brain. This is consistent with other types of neuroanatomical and neuroembryological evidence indicating a fundamental division between the rostral and the caudal diencephalon.