Crossing the floor plate triggers sharp turning of commissural axons.

During development of the vertebrate CNS, commissural axons initially grow circumferentially toward the ventral midline floor plate. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. Although recent studies have unraveled the mechanisms that control navigation of these axons along the circumferential axis, those that result in the transition from circumferential to longitudinal trajectory remain unknown. Here, we examined whether an interaction with the floor plate is a prerequisite for the initiation of trajectory transition of commissural axons, using in vitro preparations of the rat metencephalon. We found that commissural axons in the metencephalon, once having crossed the floor plate, turned sharply to grow longitudinally. In contrast, axons extending in floor plate-deleted preparations, continued to grow circumferentially, ignoring the hypothetical turning point. These results suggest that a prior interaction of commissural axons with floor plate cells is a key step for these axons to activate a navigation program required for their change in axonal trajectory from the circumferential to the longitudinal axis.

Local nonpermissive and oriented permissive cues guide vestibular axons to the cerebellum.

Information that originates from peripheral sensory organs is conveyed by axons of cephalic sensory cranial ganglia connecting the sensory organs to appropriate central targets in the brain. Thus, the establishment of correct axonal projections by sensory afferents is one of the most important issues in neural development. Previously, we examined the development of the vestibular nerve that originates from the VIIIth ganglion using a flat whole-mount preparation of the rat hindbrain and developed an in vitro, culture preparation that can recapitulate vestibular nerve development (Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M., Heizmann, C. W. and Murakami, F. (2000) J. Comp. Neurol. 417, 491-500). Both in vivo and in vitro, the ascending branch of the VIIIth ganglion projecting to the cerebellum reaches the base of the cerebellar primordium and starts to splay out towards the rhombic lip, apparently avoiding the ventral metencephalon. We now examine the nature of cues that guide vestibulocerebellar axons by applying various manipulations to the flat whole-mount in vitro preparation. Our observations suggest that local nonpermissive cues and oriented cues play a pivotal role in the guidance of vestibular axons to their central target.

Dorsal spinal cord inhibits oligodendrocyte development.

Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development.

Local directional cues control growth polarity of dopaminergic axons along the rostrocaudal axis.

The vertebrate CNS is composed of a variety of longitudinal axonal tracts extending rostrally and caudally. Although recent studies have demonstrated that chemoattraction and chemorepulsion play key roles in axon guidance along the circumferential axis in the neural tube of the vertebrate, mechanisms of axonal elongation along the longitudinal axis, and most importantly, what determines rostrocaudal polarity of axonal growth, remains unknown. Here, we examined the mechanism that guides midbrain dopaminergic axons rostrally, using flat whole-mount preparations of embryonic rat brain both in vivo and in vitro. At embryonic day 11 (E11) and early stage E12, dopaminergic neurons in the ventral midbrain extended short axons dorsally. By middle stage E12, these axons had increased in number, some deflecting rostrally and others caudally. At E13, almost all axons showed rostrally oriented growth heading toward the forebrain targets. In in vitro whole-mount preparations prepared from an E12 embryo and cultured for 24 hr, these axons showed rostrally oriented growth, but when they were forced to grow on substratum of reversed rostrocaudal polarity, they turned abruptly and grew following the polarity of the reversed midbrain substratum. These results suggest that local directional cues in the midbrain guide these axons rostrally and support the idea that substratum-associated polarized cues play an important role in axon guidance along the longitudinal axis.

Afferents of cranial sensory ganglia pathfind to their target independent of the site of entry into the hindbrain.

In vertebrates, sensory neurons interconnect a variety of peripheral tissues and central targets, conveying sensory information from different types of sensory receptors to appropriate second-order neurons in the central nervous system (CNS). To explore the possibility that the different rhombomere environments where sensory neurons enter into the hindbrain affect the pathfinding capability of growth cones, we studied the development of the VIIIth ganglion afferent both in vivo and in vitro. We focused on the vestibular nerve because it is the only cranial nerve projecting to the cerebellum, allowing for ready identification from its pattern of projection. Embryonic rat brain was cut along the dorsal midline and, with the VIIIth and Vth ganglia still attached, flat mounted and visualized with antibodies specific for sensory ganglia. Axons reached the cerebellar primordium at embryonic day (E) 13, then splayed out towards the edges of the rhombic lip of rostral hindbrain. In vitro, the VIIIth ganglion showed development similar to that in vivo and innervated the cerebellum, an appropriate target, indicating that mechanisms for axon guidance and target recognition are preserved in vitro. When the VIIIth ganglion was transplanted to the position of the Vth ganglion, axons from the transplanted ganglion entered the cerebellar primordium with a trajectory characteristic of the VIIIth nerve. These results indicate that the central projection pattern of the VIIIth nerve is not affected by the environment of nerve entry into the brainstem, suggesting that axons of sensory cranial ganglion intrinsically possess the capacity to find their target correctly.

Change in chemoattractant responsiveness of developing axons at an intermediate target.

Developing axons reach their final targets as a result of a series of axonal projections to successive intermediate targets. Long-range chemoattraction by intermediate targets plays a key role in this process. Growing axons, however, do not stall at the intermediate targets, where the chemoattractant concentration is expected to be maximal. Commissural axons in the metencephalon, initially attracted by a chemoattractant released from the floor plate, were shown to lose responsiveness to the chemoattractant when they crossed the floor plate in vitro. Such changes in axon responsiveness to chemoattractants may enable developing axons to continue to navigate toward their final destinations.

Guidance of circumferentially growing axons by the floor plate in the vertebrate central nervous system.

We describe the recent progress of studies on the mechanisms of early neural patterning, focusing on the guidance of circumferentially migrating axons in the vertebrate brain. We also refer to the underlying molecular mechanisms revealed by recent studies.

Guidance of circumferentially growing axons by netrin-dependent and -independent floor plate chemotropism in the vertebrate brain.

Netrin-1, a diffusible signal secreted by floor plate cells at the ventral midline of the vertebrate CNS, can attract ventrally migrating axons and repel a subset of dorsally migrating axons in the spinal cord and rostral hindbrain in vitro. Whether netrin-1 can act as a global cue to guide all circumferentially migrating axons is, however, unknown. Here, we show that netrin-1 can attract alar plate axons that cross the floor plate along its entire rostrocaudal axis. Dorsally directed axons forming the posterior commissure are, however, repelled by the floor plate by a netrin-independent mechanism. These results suggest that netrin-1 functions as a global guidance cue for attraction to the midline. Moreover, floor plate-mediated chemorepulsion may also operate generally to direct dorsal migrations, but its molecular basis may involve both netrin-dependent and -independent mechanisms.

Floor plate chemoattracts crossed axons and chemorepels uncrossed axons in the vertebrate brain.

In the bilaterally symmetrical vertebrate CNS, all developing axons must choose between remaining on the same side of the midline or growing across it. The mechanism underlying this axonal pathfinding is, however, poorly understood. Here we demonstrate that the ventral midline floor plate (FP) chemorepels two types of ipsilaterally projecting axons, one from the alar plate and another from the basal plate in the mesencephalon. We further demonstrate that the FP chemoattracts contralaterally projecting myelencephalic as well as metencephalic axons. The FP at all axial levels displayed both chemoattractive and chemorepellent activities, suggesting that FP chemoattraction and chemorepulsion may be at work throughout the neuraxis. Chemotropic guidance by the FP may therefore play a key role in the establishment of neuronal projection laterality.

Guidance of cerebellofugal axons in the rat embryo: directed growth toward the floor plate and subsequent elongation along the longitudinal axis.

To elucidate guidance mechanisms of brain commissural axons, we examined the navigation of cerebellofugal axons. Axons were labeled by implantation of the fluorescent tracer Dil into the cerebellar plate (CP) of fixed, flat whole-mount embryonic rat brain. Axons initially grew straight toward the ventral midline floor plate (FP) in the rostral hindbrain and then, after crossing it, made a right-angled turn to grow either caudally or rostrally along the longitudinal axis. In collagen gel culture, CP axons showed directed growth toward both FP explants and heterologous cells expressing netrin-1, a FP-derived chemoattractant for spinal commissural axons. These results suggest that CP axons are guided to the midline by FP-derived chemoattractant(s) and then reoriented, possibly by another guidance cue, for longitudinal extension. Considering that the basic structures of the neural tube, including the FP, extend up to the caudal diencephalon, these results suggest that common guidance mechanisms operate for ventrally decussating commissural axons in both the brain and spinal cord.