The establishment of correct neuronal circuitry is crucial for proper function of the vertebrate nervous system. During development of the vertebrate hindbrain, the early but transient subdivision of the neuroepithelium into rhombomeres 1 2 lays the scaffold for the development of the complex nervous system. Studies have focused on identifying the intercellular signals and transcription factors involved in defining and maintaining the cell identity of individual rhombomeres 3 4 5 6 . Less is known about the mechanisms that regulate cell movements important for the patterning and morphogenesis of the hindbrain.

The vestibular nucleus (VN) is generated as four main neuronal clusters, each spanning several rhombomeres but differing in the rostrocaudal span along the hindbrain 7 8 9 10 . Radiographic birth-dating indicated an orderly progression led by neurons of the lateral VN, then the superior VN and inferior VN, and finally the medial VN, with peaks respectively on embryonic day (E)12.5 to E14.5 in the rat 7 . Afferent projections from these nuclei to major vestibulo-motor-related targets were observable as early as E11.5 (mouse) and E12.5 (rat) 7 8 9 10 . Once the pioneer afferents have been initiated, the advancing growth cones navigate the 'local' environment for positional and directional cues in the determination of a path to their target. Little is known about molecules involved in the pathway navigation. Studies on other systems indicated involvement of the Eph receptor tyrosine kinases and their membrane-bound ligand, ephrins, in growth cone negotiation with rhombomere boundaries 11 12 13 . The chondroitin sulfate (CS) proteoglycans (CSPGs) are strategically located in the extracellular matrix of the developing brain to influence cell-matrix adhesion and neurite outgrowth. In the chick brain, expression of CS was found to peak at the rhombomeric stage of hindbrain development 14 . Despite reports of CSPGs in the developing brain, their role in fiber projections in early embryos when pioneer axons are negotiating the path to reach their targets remains largely unexplored.

In this study, we are the first to report live treatment of the hindbrain matrix of rat embryos (E12.5 to E14.5) with chondroitinase ABC (Ch^ABC) for 1 day in culture during which the course of commissural projections is perturbed by enzymatic depletion of CS moieties. Following the treatment, we employed the lipophilic dye DiI to track contralateral projections from the VN in fixed hindbrains. The consequent increases in projection distance and number of unfasciculated fibers as well as the deviated courses of unfasciculated fibers revealed that axon-restrictiveness of CS moieties in the early hindbrain matrix controls the timing and abundance of axons that end at the contralateral VN target.

In the present study, our results show that CS moieties in the rat hindbrain matrix (E12.5_{(+1 DIV)} and E13.5_{(+1 DIV)} ) are important for controlling commissural projection of VN neurons at a time when few, if any, fiber tracts have charted out their course in the early hindbrain.

During the development of the hindbrain, segmentation of the neuroepithelium into rhombomeres is important in laying out the framework for correct neuronal patterning 2 . Projection of VN commissures during this period when few fiber tracts exist and the coincidental abundance of CSPGs in the hindbrain matrix suggest a role for this molecule in regulating the commissural projection. Studies of the embryonic chick brain showed CSPG accumulation in the extracellular matrix of rhombomere boundaries 17 18 . The CS moiety has been considered important in restricting cell clusters and preventing them from intermingling with clusters of different cell lineages and thus in maintaining identities of cell clusters in the hindbrain. With the use of immunostaining for the CS-56 epitope and the 2B6 stub epitope following Ch^ABC treatment of the hindbrain matrix, we showed that the CS moieties are localized in the extracellular matrix of rhombomeric segments as well as the boundaries of rhombomeres (Figure 3A,E). Cells and their projections can traverse this CS-rich region in the rhombomeres. So, does the restrictive role of CS in rhombomeres apply in such circumstances?

The CS moiety has been implicated in the control of neurite growth and extension. Culture of chick retinal ganglion cells on nitrocellulose filters that have been implanted in a lesioned cerebral cortex to capture CSPGs of the glial scar showed that treatment of the filter with Ch^ABC resulted in cultures bearing longer neurites 19 . Treatment of chiasmatic brain slice cultures (E13 to E15, mouse) with Ch^ABC resulted in misrouting of retinal ganglion cell projections at the optic chiasma 20 . In the present study, we showed that treatment of hindbrain matrix with Ch^ABC in 1-day cultures of rat embryos sped up projection along the commissural route toward the contralateral VN. This effect was evident in both E12.5_{(+1 DIV)} and E13.5_{(+1 DIV)} embryos - in the former, projections reached further by 800 μm along the commissural route, and in the latter, projections ended up in regions beyond the contralateral VN (Figure 8). Our results suggest that CS moieties prevailing in the early hindbrain matrix play an axon-restrictive role in controlling the speed of projection of the VN commissures such that the terminals can recognize the targets on arrival.

In this work, we found that VN commissural fibers were less fasciculated in hindbrains that had been treated with Ch^ABC than those of controls treated with PBS. For assessment of the extent of fasciculation of the VN commissural fibers in 100-μm steps across the hindbrain, we chose fiber diameters of > 5 μm as an arbitrary indication of fasciculation and those ≤5 μm as an indication of non-fasciculation. This cutoff makes it possible for exhaustive assessment of all DiI-tracked fibers at the light microscopic level. Assessment of fibers of ≤5 μm in close-up view revealed that these included three to five single projections that, at times, were separate from each other and, at other times, remained indiscernible. To decipher these would require viewing at the electron microscopic level. This latter approach can only reveal a small portion of the examined area each time and is impractical for assessment of all the labeled VN commissures. Our observation of increase in unfasciculated fibers with Ch^ABC treatment of the hindbrain suggests that axon-restrictive CS moieties in the hindbrain matrix induced the late outgrowths or follower projections to join the track of pioneers and thus to fasciculate with each other.

Earlier studies considered CSPG as an entity and did not attempt to distinguish between roles of the CS moiety and those of the core proteins. Thus, such CSPGs as neurocan and phosphacan were found to mediate axonal fasciculation during development of the hippocampal circuitry in vivo 21 . In cultures of hippocampal neurons, dorsal root ganglia or habenula nucleus neurons, neurite extension into a CSPG substratum exhibited fasciculation as well 21 22 23 . These provide evidence for axonal fasciculation as a means to modulate the ability of the growth cone to navigate through restrictive environments or regulate axonal pathfinding during circuit formation.

Our results provide an in vivo correlate to the in vitro findings of 24 that the CS moieties of neural proteoglycans such as neurocan and phosphacan have roles in the modulation of neuronal adhesion and neurite growth by binding to neural cell adhesion molecules like neuron-glia cell adhesion molecule (Ng-CAM) and neural cell adhesion molecule (N-CAM). Treatment of the hindbrain matrix with Ch^ABC when the projecting fibers are in the growing mode not only diminished matrix restriction of cues and guideposts, but can likely interfere with the modulation of molecular interactions involving the CAMs at close axonal contacts. The CAMs on neurons have sites for homophilic binding with cell attached CAMs 25 26 27 , and other sites for binding with neurocan and phosphacan, both the glycosylated form and the Ch^ABC-treated counterpart 28 29 . Direct binding of neurons with the native or Ch^ABC-treated neurocan and phosphacan via Ng-CAM and N-CAM was demonstrated 28 29 . We reason that the CS moieties are uniquely poised to fine-tune the balance between adhesion and anti-adhesion for growing axons to navigate along pre-existing axonal trajectories.

The strong CS-56 immunopositivity remaining at the ventricular surface despite the proximity to the infused Ch^ABC suggests limited access of the enzyme to CS moieties on the ventricular surface. It is likely that the CSPGs were protected from enzymatic digestion via interaction with other extracellular matrix molecules. In one of the other experiments in our lab, we have shown that the release of semaphorin3A, which binds to CS moieties in the central nervous system 30 , could only be achieved when the samples were treated with both Ch^ABC and heparitinases, but not with Ch^ABC alone (unpublished data). These results support that by interaction with other extracellular matrix molecules, the CS moiety is protected from the action of Ch^ABC and presents as residual immunoreactivity to CS-56.

There is a concern that the differences we observed in axonal trajectories with and without Ch^ABC treatment is due to increased apoptosis in the embryos, in particular the higher mortality rate among E14.5 embryos in culture (Figure 2). Given that CSPGs bind various chemokines and growth factors (personal communication), Ch^ABC cleavage of the CS moiety may result in loss of these factors and therefore increased incidence of cell death. In order to verify that the results we reported are due to Ch^ABC cleavage of the CS moiety but not other cellular processes, such as apoptosis, we monitored the development of the embryos by their heartbeat and their growth in size after the ex vivo culture. We did not observe any differences in size between the PBS- and Ch^ABC-treated embryos. Embryos with slow heartbeats or reduced size were discarded at the end of the culture and were not included for DiI tracking of commissural projections from the VN. Moreover, cross-sections of these embryos have been immunostained with NF165 and results showed that the numbers of neurites were similar in both cases (results not shown). Admittedly, these results cannot provide definitive proof that all subpopulations of neurons are equally protected. More experiments have to be performed to reveal the contribution of CSPGs to the survival of subpopulations of neurons.

CS is a well known inhibitory molecule in the nervous system. The high abundance of CSPGs in the glial scar that forms after nervous tissue injury suggests their role in limiting regeneration 31 32 . With the removal of CS by Ch^ABC treatment of the glial scar, axonal transgression of the scar was observed 33 34 35 36 37 38 39 . This raises the question of how the projections negotiate their way through the CS-rich environment. With rat dorsal root ganglia in culture on aggrecan-laminin substrata, inhibition of neurite extension by the CS moiety of aggrecan was attributed to suppression of integrin signaling and that the inhibition could be reversed by integrin activation 40 . Alternatively, chick dorsal root ganglia culture treated with versican V0/V1 forms revealed concentration-dependent neurite responses that ranged from rapid retraction and deflection to defasciculation and growth arrest 41 . Our work is the first to demonstrate the role of CS moieties in the early hindbrain matrix in controlling the pattern of projection of VN commissures for timely arrival at the contralateral target. Further study on various CS isoforms may give new insights into mechanisms by which the CS moiety can fine-tune adhesion versus anti-adhesion signaling at the growth terminal to allow navigation along axonal trajectories in early hindbrain development.

The authors declare that they have no competing interests.

Ch^ABC: chondroitinase ABC; CAM: cell adhesion molecule; CS: chondroitin sulfate; CSPG: chondroitin sulfate proteoglycan; Dulbecco's modified Eagle's Medium; E: embryonic day; N-CAM: Neural cell adhesion molecule; Ng-CAM: neuron-glia cell adhesion molecule; PBS: phosphate-buffered saline; VN: vestibular nucleus.

JCFK performed chondroitinase treatment of rat embryos in culture, analyzed mmunohistochemical results and DiI-tracked fiber projections, and drafted the manuscript. YLY and WKL carried out embryo culture studies, including DiI tracking of commissural projections in the embryonic hindbrain. FXZ performed initial fiber-tracking experiments that standardized the DiI application site in the embryonic hindbrain. JWF participated in the conception of the study and interpretation of chondroitinase activity in the hindbrain. YSC participated in planning of the experiments, analysis and interpretation of fiber projections, and the writing of the manuscript. DKYS conceived of the study, designed and coordinated the experiments, and wrote the manuscript. All authors read and approved the final manuscript.