Midbrain dopaminergic axons are guided longitudinally through the diencephalon by Slit/Robo signals

Dopaminergic neurons from the ventral mesencephalon/diencephalon (mesodiencephalon) form vital pathways constituting the majority of the brain's dopamine systems. Mesodiencephalic dopaminergic (mdDA) neurons extend longitudinal projections anteriorly through the diencephalon, ascending toward forebrain targets. The mechanisms by which mdDA axons initially navigate through the diencephalon are poorly understood. Recently the Slit family of secreted axon guidance proteins, and their Robo receptors, have been identified as important guides for descending longitudinal axons. To test the potential roles of Slit/Robo guidance in ascending trajectories, we examined tyrosine hydroxylase-positive (TH+) projections from mdDA neurons in mutant mouse embryos. We found that mdDA axons grow out of and parallel to Slit-positive ventral regions within the diencephalon, and that subsets of the mdDA axons likely express Robo1 and possibly also Robo2. Slit2 was able to directly inhibit TH axon outgrowth in explant co-culture assays. The mdDA axons made significant pathfinding errors in Slit1/2 and Robo1/2 knockout mice, including spreading out in the diencephalon to form a wider tract. The wider tract resulted from a combination of invasion of the ventral midline, consistent with Slit repulsion, but also axons wandering dorsally, away from the ventral midline. Aberrant dorsal trajectories were prominent in Robo1 and Robo1/2 knockout mice, suggesting that an aspect of Robo receptor function is Slit-independent. These results indicate that Slit/Robo signaling is critical during the initial establishment of dopaminergic pathways, with roles in the dorsoventral positioning and precise pathfinding of these ascending longitudinal axons.     

Robos are required for the correct targeting of retinal ganglion cell axons in the visual pathway of the brain

Axonal projections from the retina to the brain are regulated by molecules including the Slit family of ligands [Thompson, H., Barker, D., Camand, O., Erskine, L., 2006a. Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract. Dev. Biol. 296, 476–484, Thompson, H., Camand, O., Barker, D., Erskine, L., 2006b. Slit proteins regulate distinct aspects of retinal ganglion cell axon guidance within dorsal and ventral retina. J. Neurosci. 26, 8082–8091]. However, the roles of Slit receptors in mammals, (termed Robos), have not been investigated in visual system development. Here we examined Robo1 and 2 mutant mice and found that Robos regulate the correct targeting of retinal ganglion cell (RGC) axons along the entire visual projection. We noted aberrant projections of RGC axons into the cerebral cortex, an area not normally targeted by RGC axons. The optic chiasm was expanded along the rostro-caudal axis (similar to Slit mutant mice, Plump, A.S., Erskine, L., Sabatier, C., Brose, K., Epstein, C.J., Goodman, C.S., Mason, C.A., Tessier-Lavigne, M., 2002. Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33, 219–232), with ectopic crossing points, and some axons projecting caudally toward the corticospinal tract. Further, we found that axons exuberantly projected into the diencephalon. These defects were more pronounced in Robo2 than Robo1 knockout animals, implicating Robo2 as the predominant Robo receptor in visual system development.     

Robo2-Slit and Dcc-Netrin1 Coordinate Neuron Axonal Pathfinding within the Embryonic Axon Tracts

In the embryonic vertebrate brain, early born neurons establish highly stereotyped embryonic axonal tracts along which the neuronal interconnections form. To understand the mechanism underlying neuron axonal pathfinding within the embryonic scaffold of axon tracts, we studied zebrafish anterior dorsal telencephalic (ADt) neuron development. While previous studies suggest the ADt neuronal axons extend along a commissural tract [anterior commissure (AC)] and a descending ipsilateral tract [supraoptic tract (SOT)], it is unclear whether individual ADt neuronal axons choose specific projection paths at the intersection between the AC and the SOT. We labeled individual ADt neurons using a forebrain-specific promoter to drive expression of fluorescent proteins. We found the ADt axonal projection patterns were heterogeneous and correlated with their soma positions. Our results suggest that cell intrinsic differences along the dorsal ventral axis of the telencephalon regulate the axonal projection choices. Next, we determined that the guidance receptors roundabout2 (Robo2) and deleted in colorectal cancer (Dcc) were differentially expressed in the ADt neurons. We showed that knocking down Robo2 function by injecting antisense morpholino oligonucleotides abolished the ipsilateral SOT originating from the ADt neurons. Knocking down Dcc function did not prevent formation of the AC and the SOT. In contrast, the AC was specifically reduced when Netrin1 function was knocked down. Further mechanistic studies suggested that Robo2 responded to the repellent Slit signals and suppressed the attractive Netrin signals. These findings demonstrate how Robo2-Slit and Dcc-Netrin coordinate the axonal projection choices of the developing neurons in the vertebrate forebrain.     

Regionally specific expression of L1 and sialylated NCAM in the retinofugal pathway of mouse embryos

We have examined expression of L1 and the polysialic acid-associated form of the neural cell adhesion molecule (PSA-NCAM) in mouse embryos during the major period of axon growth in the retinofugal pathway to determine whether they are expressed in patterns that relate to the changes in axon organization in the pathway. Immunostaining for L1 and PSA-NCAM was found on all axons in the retina and the optic stalk. In the chiasm, while L1 immunoreactivity remained high on the axons, PSA-NCAM staining was obviously reduced. At the threshold of the optic tract, L1 immunoreactivity was maintained only in a subpopulation of axons, whereas PSA-NCAM staining was dramatically elevated in axons at the caudal part of the tract. Further investigations of the tract showed that both L1 and PSA-NCAM were preferentially expressed on the dorsal but not ventral optic axons, indicating a regionally specific change of both adhesion molecules on the axons at the chiasm-tract junction. Moreover, intense PSA-NCAM expression was also observed in the tract of postoptic commissure (TPOC), which lies immediately caudal to the optic tract. Immunohistochemical and retrograde tracing studies showed that these PSA-NCAM-positive axons arose from a population of cells rostral to the CD44-positive chiasmatic neurons. These findings indicate that, in addition to the chiasmatic neurons, these PSA-NCAM-positive diencephalic cells also contribute axons to the TPOC. These early generated commissural axons together with the regionally specific pattern of cell adhesion molecule expression on the optic axons may control formation of the partial retinotopic axon order in the optic tract through homophilic or heterophilic interactions that involve PSA-NCAM.     

Coexpression of high-voltage-activated ion channels Kv3.4 and Cav1.2 in pioneer axons during pathfinding in the developing rat forebrain

Precise axon pathfinding is crucial for establishment of the initial neuronal network during development. Pioneer axons navigate without the help of preexisting axons and pave the way for follower axons that project later. Voltage‐gated ion channels make up the intrinsic electrical activity of pioneer axons and regulate axon pathfinding. To elucidate which channel molecules are present in pioneer axons, immunohistochemical analysis was performed to examine 14 voltage‐gated ion channels (Kv1.1–Kv1.3, Kv3.1–Kv3.4, Kv4.3, Cav1.2, Cav1.3, Cav2.2, Nav1.2, Nav1.6, and Nav1.9) in nine axonal tracts in the developing rat forebrain, including the optic nerve, corpus callosum, corticofugal fibers, thalamocortical axons, lateral olfactory tract, hippocamposeptal projection, anterior commissure, hippocampal commissure, and medial longitudinal fasciculus. We found A‐type K⁺ channel Kv3.4 in both pioneer axons and early follower axons and L‐type Ca²⁺ channel Cav1.2 in pioneer axons and early and late follower axons. Spatially, Kv3.4 and Cav1.2 were colocalized with markers of pioneer neurons and pioneer axons, such as deleted in colorectal cancer (DCC), in most fiber tracts examined. Temporally, Kv3.4 and Cav1.2 were expressed abundantly in most fiber tracts during axon pathfinding but were downregulated beginning in synaptogenesis. By contrast, delayed rectifier Kv channels (e.g., Kv1.1) and Nav channels (e.g., Nav1.2) were absent from these fiber tracts (except for the corpus callosum) during pathfinding of pioneer axons. These data suggest that Kv3.4 and Cav1.2, two high‐voltage‐activated ion channels, may act together to control Ca²⁺‐dependent electrical activity of pioneer axons and play important roles during axon pathfinding. J. Comp. Neurol. 520:3650–3672, 2012. © 2012 Wiley Periodicals, Inc.     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.     

Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system

The Robo family of molecules is important for axon guidance across the midline during central nervous system (CNS) development in invertebrates and vertebrates. Here we describe the patterns of Robo protein expression in the developing mouse CNS from embryonic day (E) 9.5 to postnatal day (P) 4, as determined by immunohistochemical labeling with an antibody (S3) raised against a common epitope present in the Robo ectodomain of Robos 1 and 2. In the spinal cord, midline-crossing axons are initially (at E11) S3-positive. At later times, midline Robo expression disappears, but is strongly upregulated in longitudinally running postcrossing axons. It is also strongly expressed in noncrossing longitudinal axons. Differential expression of Robo along axons was also found in axons cultured from E14 spinal cord. These findings resemble those from the Drosophila ventral nerve cord and indicate that in vertebrates a low level of Robo expression occurs in the initial crossing of the midline, while a high level of expression in the postcrossing fibers prevents recrossing. Likewise, Robo-positive ipsilateral axons are prevented from crossing at all. However, in the brain different rules appear to apply. Most commissural axons including those of the corpus callosum are strongly S3-positive along their whole length from their time of formation to postnatal life, but some have more complex age-dependent expression patterns. S3 labeling of the optic pathway is also complex, being initially strong in the retinal ganglion cells, optic tract, and chiasma but thereafter being lost except in a proportion of postchiasmal axons. The corticospinal tract is strongly positive throughout its course at all stages examined, including its decussation, formed at about P2 in the central part of the medulla oblongata.     

The adaptor protein Nck2 mediates Slit1-induced changes in cortical neuron morphology

Slits are multifunctional guidance cues, capable of triggering neurite repulsion, extension, or branching, depending on cell type and developmental context. While the Robo family of Slit receptors is a well-established mediator of axon repulsion, a role for Robos in Slit-mediated neurite growth and branching is not well defined, and the signaling molecules that link Robo to the cytoskeletal changes that drive neurite outgrowth are not well characterized in vertebrates. We show that Slit stimulates cortical dendrite branching, and we report that Slit also triggers a robust increase in the length of cortical axons in vitro. Moreover, neurons derived from Robo1; Robo2 deficient mice do not display an increase in neurite length, indicating that endogenous Robos mediate Slit's growth-promoting effects on both axons and dendrites. We also demonstrate that the SH2/SH3 adaptor proteins Nck1 and Nck2 bind to Robo via an atypical SH3-mediated mechanism. Furthermore, we show that only Nck2 is required for the Slit-induced changes in cortical neuron morphology in vitro. These findings indicate a specific role for Nck2 in linking Robo activation to the cytoskeleton rearrangements that shape cortical neuron morphology.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Slit2/Robo1 signaling in glioma migration and invasion

Slit2/Robo1 is a conserved ligand-receptor system, which greatly affects the distribution, migration, axon guidance and branching of neuron cells. Slit2 and its transmembrane receptor Robo1 have different distribution patterns in gliomas. The expression of Slit2 is at very low levels in pilocytic astrocytoma, fibrillary astrocytoma and glioblastoma, while Robo1 is highly expressed in different grades of gliomas at both mRNA and protein levels. Acquisition of insidious invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways. Although the specific mechanisms of tumor-suppressive effect of Slit2/Robo1 have not been elucidated, it has been proved that Slit2/Robo1 signaling inhibits glioma cell migration and invasion by inactivation of Cdc42-GTP. With the research development on the molecular mechanisms of Slit2/Robo1 signaling in glioma invasion and migration, Slit2/Robo1 signaling may become a potential target for glioma prevention and treatment.     

Robo3.1A suppresses Slit‐mediated repulsion by triggering degradation of Robo2

Slits and Robos control the midline crossing of commissural axons, which are not sensitive to the midline repellent Slit before crossing but gain Slit responsiveness to exit the midline and avoid recrossing. Robo3.1A promotes midline crossing of commissural axons by suppressing the axonal responsiveness to the midline repellent Slit, but the underlying mechanism remains unclear. By using a cell surface binding assay and immunoprecipitation, we observed that Robo3.1A did not bind Slit on its own but prevented the specific binding of Slit to the cell surface when it was coexpressed with its close homologue Robo1 or Robo2 (Robo1/2), which are known to mediate the Slit repulsion. Cotransfection with Robo3.1A significantly reduced the protein level of Robo2 in HEK293 cells, and overexpression of Robo3.1A also significantly decreased Robo2 protein level in cerebellar granule cells. Downregulation of endogenous Robo3 by specific small interference RNA (siRNA) significantly increased Robo1 protein level, Slit binding to the cell surface was significantly elevated, and Slit‐triggered growth cone collapse appeared after downregulation of Robo3 in cultured cortical neurons. Immunocytochemical staining showed that Robo2 and Robo3 colocalized in intracellular vesicles positive for the marker of late endosomes and lysosomes, but not trans‐Golgi apparatus and early endosomes. Thus Robo3.1A may prevent the Slit responsiveness by recruiting Robo1/2 into a late endosome‐ and lysosome‐dependent degradation pathway. © 2014 Wiley Periodicals, Inc.     

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig-1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit-responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up-regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain.     

Chick wing innervation. III. Formation of axon collaterals in developing peripheral nerves

Axon navigation during vertebrate limb innervation has been shown to be associated with position-dependent changes in size and complexity of the axon growth cones, and sometimes with bifurcation of terminal growth cones and axon branching (Hollyday and Morgan-Carr, companion paper). We have further examined axon branching and asked whether it extends to the projection of collaterals to different nerves. Injections of horseradish peroxidase or DiI were made into individual peripheral nerves in the wings of chick embryos at stages 28–35, and the trajectories of solidly labeled axons were traced proximally from the injection site in tissue sections. During stages when the peripheral nerves were first forming in the shoulder region, collaterals of retrogradely labeled axons were frequently observed to project into uninjected nerves proximal to the injection site. These two-nerve collaterals were formed by a small percentage of axons in a high percentage of the embryos studied and could occur in both motor and sensory axons. Two-nerve collateral projections were observed between nerves separated along both the proximodistal and anteroposterior axes of the limb, but they were limited in spatial extent to nerves supplying adjacent limb regions and were never seen between nerves projecting to widely disparate regions of the limb. Collaterals were not seen at the plexus projecting to both dorsal and ventral pathways. The apparent frequency of two-nerve collaterals was found to decline progressively from stage 28–29 to stage 32; no two-nerve collaterals were seen in the proximal wing at stage 33 and older. The mechanism of their elimination is presently unknown. These observations suggest that some axon branching seen during outgrowth is sufficiently divergent to result in axon collaterals which project to two different peripheral nerves. Presumably, two-nerve collaterals reflect both the neuron's ability to branch and some imprecision in the axonal guidance mechanisms. Together these give rise to minor errors in projection which are subsequently removed. © 1995 Wiley-Liss, Inc.     

Heparan sulfate proteoglycan expression in the optic chiasm of mouse embryos.

Previous studies have demonstrated that heparan sulfate (HS) proteoglycans (PGs) regulate neurite outgrowth through binding to a variety of cell surface molecules, extracellular matrix proteins, and growth factors. The present study investigated the possible involvement of HS-PGs in retinal axon growth by examining its expression in the retinofugal pathway of mouse embryos by using a monoclonal antibody against the HS epitope. Immunoreactive HS was first detected in all regions of the retina at embryonic day (E) 11. The staining was gradually lost in the central regions and restricted to the retinal periphery at later developmental stages (E12--E16). Prominent staining for HS was consistently found in the retinal fiber layer and at the optic disk, indicating a possible supportive role of HS-PGs in axon growth in the retina. At the ventral diencephalon, immunostaining for HS was first detected at E12, before arrival of any retinal axons. The staining matched closely the neurons that are immunopositive for the stage-specific embryonic antigen 1 (SSEA-1). At E13 to E16, when axons are actively exploring their paths across the chiasm, immunoreactivity for HS was particularly intense at the midline. This characteristic expression pattern suggests a role for HS-PGs in defining the path of early axons in the chiasm and in regulating development of axon divergence at the midline. Furthermore, HS immunoreactivity is substantially reduced at regions flanking both sides of the midline, which coincides spatially to the position of actin-rich growth cones from subpial surface to the deep regions of the optic axon layer at the chiasm. Moreover, at the threshold of the optic tract, immunoreactive HS was localized to deep parts of the fiber layer. These findings indicate that changes in age-related fiber order in the optic chiasm and optic tract of mouse embryos are possibly regulated by a spatially restricted expression of HS-PGs.     

Evidence for the existence of two Robo3 isoforms with divergent biochemical properties

Robo3 is a member of the roundabout (Robo) family of proteins that plays a key role in axon guidance and cell migration in the developing nervous system. Recent studies have shown that Robo3 plays a crucial role in controlling axon guidance at the midline of the CNS. Here we describe and compare two human Robo3 isoforms, Robo3A and Robo3B, which differ by the insertion of 26 amino acids at the N-terminus, and these forms appear to be evolutionary conserved. We investigated the bioactivity of these isoforms and show that they have different binding properties to Slit, and that orthologs of these forms are expressed in the mouse embryo. In addition, we show that, like other members of the Robo family, Robo3 can bind homophilically, but it is also capable of binding heterophilically to Robo1 and NCAM. We propose that these properties of Robo3 may contribute to its function at the midline of the CNS.     

Timing and origin of the first cortical axons to project through the corpus callosum and the subsequent emergence of callosal projection cells in mouse

A precise knowledge of the timing and origin of the first cortical axons to project through the corpus callosum (CC) and of the subsequent emergence of callosal projection cells is essential for understanding the early ontogeny of this commissure. By using a series of mouse embryos and fetuses of the hybrid cross B6D2F₂/J weighing from 0.36 g to 1.0 g (embryonic day E15.75–E17.25), we examined the spatial and temporal distribution of callosal projection cells by inserting crystals of the lipophilic dye (DiI: 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate) into the contralateral white matter just lateral to the midsagittal plane. Around 0.4 g or E15.8, retrogradely labeled cells were found restricted to a discrete cluster continuously distributed from the most ventral part of presumptive cingulate cortex to the hippocampus. During subsequent development, however, the tangential distribution of these labeled cells in ventromedial cortex did not extend further dorsally, and in fetuses where the CC became distinct from the hippocampal commissure (HC), labeled axons of cells in the ventral cingulate cortex were observed to intersect the callosal pathway and merge with labeled axons of the HC derived from cells in the hippocampus. The first cortical axons through the CC crossed the midline at about 0.64 g or E16.4, and these axons originated from a scattered neuronal population in the dorsal to lateral part of the presumptive frontal cortex. The earliest callosal cells were consistently located in the cortical plate and showed an immature bipolar appearance, displaying an ovoid- or pearl-shaped perikaryon with an apical dendrite coursing in a zig-zagging manner toward the pial surface and a slender axon directed toward the underlying white matter. Callosal projection cells spread progressively with development across the tangential extent of the cerebral cortex in both lateral-to-medial and rostral-to-caudal directions. In any cortical region, the first labeled cells appeared in the cortical plate and their number in the subplate was insignificant compared to that in the cortical plate. Thus, these results clarify that the CC is pioneered by frontal cortical plate cells, and the subsequent ontogeny of callosal projection cells proceeds according to the gradient of cortical maturation. J. Comp. Neurol. 400:197–206, 1998. © 1998 Wiley-Liss, Inc.     

Induction of aberrant functional afferents to the chick cochlear nucleus

Surgical extirpation of the otocyst on embryonic day (E) 3 in chick embryos prevents formation of the cochlear nerve and results in development of an aberrant axonal projection from the contralateral cochlear nucleus (nucleus magnocellularis, NM) to the deafferented NM. We have studied the morphology of this projection using horseradish peroxidase injections in NM axons and light and electron microscopy. The ability of the projection to activate its target neurons synaptically was assessed by means of extracellular microelectrode recording from in vitro preparations of the chick brainstem. The aberrant projection arises as a vertically directed branch from the contralaterally traveling NM axon at the medial border of nucleus laminaris (NL). This axonal branch forms boutonal endings that may terminate anywhere in NM but are most common in its ventral and medial regions. In our experiments, this projection is not seen on the unoperated side of experimental animals or in normal controls from E11 onward but is found on the operated sides of all experimental animals, including those with bilateral removal of the otocysts. The aberrant projection persists at least from E11 through hatching and has essentially identical features in unilaterally and bilaterally lesioned animals. The endings of the aberrant projection are boutonal in form and, in the electron microscope, exhibit all of the elements associated with normal synapses. Electrophysiological studies confirm that stimulation of the aberrant axons can elicit postsynaptic responses in NM and suggest that these synapses use an excitatory amino acid neurotransmitter.     

Pretarget sorting of retinocollicular axons in the mouse

The map of the retina onto the optic tectum is a highly conserved feature of the vertebrate visual system; the mechanism by which this mapping is accomplished during development is a long-standing problem of neurobiology. The early suggestion by Roger Sperry that the map is formed through interactions between retinal ganglion cell axons and target cells within the tectum has gained significant experimental support and widespread acceptance. Nonetheless, reports in a variety of species indicate that some aspects of retinotopic order exist within the optic tract, leading to the suggestion that this "preordering" of retinal axons may play a role in the formation of the mature tectal map. A satisfactory account of pretarget order must provide the mechanism by which such axon order develops. Insofar as this mechanism must ultimately be determined genetically, the mouse suggests itself as the natural species in which to pursue these studies. Quantitative and repeatable methods are required to assess the contribution of candidate genes in mouse models. For these reasons, we have undertaken a quantitative study of the degree of retinotopic order within the optic tract and nerve of wild-type mice both before and after the development of the retinotectal map. Our methods are based on tract tracing using lipophilic dyes, and our results indicate that there is a reestablishment of dorsoventral but not nasotemporal retinal order when the axons pass through the chiasm and that this order is maintained throughout the subsequent tract. Furthermore, this dorsoventral retinotopic order is well established by the day after birth, long before the final target zone is discernible within the tectum. We conclude that pretarget sorting of axons according to origin along the dorsoventral axis of the retina is both spatially and chronologically appropriate to contribute to the formation of the retinotectal map, and we suggest that these methods be used to search for the molecular basis of such order by using available mouse genetic models.