Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE‐ and CGE‐derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT‐PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE‐ and CGE‐derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 647–672, 2013     

The corpus callosum as an evolutionary innovation

The corpus callosum (CC) is the major interhemispheric fibre bundle in the eutherian brain and has been described as a true evolutionary innovation. This paper reviews the current literature with regard to functional, developmental and genetic concepts that may help elucidate the evolutionary origin of this structure. It has been suggested that the CC arose in the eutherian brain as a more direct and, therefore, more effective system for the interhemispheric integration of topographically organized sensory cortices than the anterior commissure (AC) and hippocampal commissure (HC) already present in nonplacental mammals. It can also be argued, however, that the ability of the CC to integrate the newly evolving motor cortices of placental mammals may have played a role in the evolutionary fixation of this structure. Investigations into the developmental mechanism involved in the formation of the CC and their underlying patterns of gene expression make it possible to formulate a tentative hypothesis about the evolutionary origin of this commissure. This paper suggests that changes in the developmental patterns of the expression of certain regulatory genes may have allowed a first group of callosal pioneering axons to cross the cortical midline. These pioneering fibres may have used the axons of the HC to find their way across the midline. Additional callosal fibres may then have fasciculated with these pioneers. Once the CC had formed in this way, more complex systems of axonal guidance may have evolved over time, thus enabling a gradual increase in the size and complexity of the CC.     

Multiple Slits regulate the development of midline glial populations and the corpus callosum

The Slit molecules are chemorepulsive ligands that regulate axon guidance at the midline of both vertebrates and invertebrates. In mammals, there are three Slit genes, but only Slit2 has been studied in any detail with regard to mammalian brain commissure formation. Here, we sought to understand the relative contributions that Slit proteins make to the formation of the largest brain commissure, the corpus callosum. Slit ligands bind Robo receptors, and previous studies have shown that Robo1(-/-) mice have defects in corpus callosum development. However, whether the Slit genes signal exclusively through Robo1 during callosal formation is unclear. To investigate this, we compared the development of the corpus callosum in both Slit2(-/-) and Robo1(-/-) mice using diffusion magnetic resonance imaging. This analysis demonstrated similarities in the phenotypes of these mice, but crucially also highlighted subtle differences, particularly with regard to the guidance of post-crossing axons. Analysis of single mutations in Slit family members revealed corpus callosum defects (but not complete agenesis) in 100% of Slit2(-/-) mice and 30% of Slit3(-/-) mice, whereas 100% of Slit1(-/-); Slit2(-/-) mice displayed complete agenesis of the corpus callosum. These results revealed a role for Slit1 in corpus callosum development, and demonstrated that Slit2 was necessary but not sufficient for midline crossing in vivo. However, co-culture experiments utilising Robo1(-/-) tissue versus Slit2 expressing cell blocks demonstrated that Slit2 was sufficient for the guidance activity mediated by Robo1 in pre-crossing neocortical axons. This suggested that Slit1 and Slit3 might also be involved in regulating other mechanisms that allow the corpus callosum to form, such as the establishment of midline glial populations. Investigation of this revealed defects in the development and dorso-ventral positioning of the indusium griseum glia in multiple Slit mutants. These findings indicate that Slits regulate callosal development via both classical chemorepulsive mechanisms, and via a novel role in mediating the correct positioning of midline glial populations. Finally, our data also indicate that some of the roles of Slit proteins at the midline may be independent of Robo signalling, suggestive of additional receptors regulating Slit signalling during development.     

Axon fasciculation and differences in midline kinetics between pioneer and follower axons within commissural fascicles

Early neuronal scaffold development studies suggest that initial neurons and their axons serve as guides for later neurons and their processes. Although this arrangement might aid axon navigation, the specific consequence(s) of such interactions are unknown in vivo. We follow forebrain commissure formation in living zebrafish embryos using timelapse fluorescence microscopy to examine quantitatively commissural axon kinetics at the midline: a place where axon interactions might be important. Although it is commonly accepted that commissural axons slow down at the midline, our data show this is only true for leader axons. Follower axons do not show this behavior. However, when the leading axon is ablated, follower axons change their midline kinetics and behave as leaders. Similarly, contralateral leader axons change their midline kinetics when they grow along the opposite leading axon across the midline. These data suggest a simple model where the level of growth cone exposure to midline cues and presence of other axons as a substrate shape the midline kinetics of commissural axons.     

Recombinant inbreeding in mice reveals thresholds in embryonic corpus callosum development

The inbred strains BALB/cWah1 and 129P1/ReJ both show incomplete penetrance for absent corpus callosum (CC); about 14% of adult mice have no CC at all. Their F(1) hybrid offspring are normal, which proves that the strains differ at two or more loci pertinent to absent CC. Twenty-three recombinant inbred lines were bred from the F(2) cross of BALB/c and 129, and several of these expressed a novel and severe phenotype after only three or four generations of inbreeding - total absence of the CC and severe reduction of the hippocampal commissure (HC) in every adult animal. As inbreeding progressed, intermediate sizes of the CC and the HC remained quite rare. This striking phenotypic distribution in adults arose from developmental thresholds in the embryo. CC axons normally cross to the opposite hemisphere via a tissue bridge in the septal region at midline, where the HC forms before CC axons arrive. The primary defect in callosal agenesis in the BALB/c and 129 strains is severe retardation of fusion of the hemispheres in the septal region, and failure to form a CC is secondary to this defect. The putative CC axons arrive at midline at the correct time and place in all groups, but in certain genotypes, the bridge is not yet present. The relative timing of axon growth and delay of the septal bridge create a narrow critical period for forming a normal brain.     

Organization of a midline proliferative cluster in the embryonic brain of the grasshopper

We have studied a group of midline cells in the embryonic brain of the grasshopper by using immunocytochemical and intracellular dye injection techniques. This cluster of midline cells differentiates between the pars intercerebralis lobes of the protocerebrum during early embryogenesis, and is composed of putative midline progenitors as well as neuronal and glial cells. Annulin immunoreactive glial processes surround the borders of the midline cell cluster and also form a network of processes extending from there to the borders of proliferative clusters in the brain hemispheres. Among the cells that derive from the midline cluster are two bilaterally symmetrical pairs of identified primary commissure pioneer neurons. By navigating along the glial bound borders of the midline proliferative cluster, the axons of these pioneers establish an initial axonal bridge across the brain midline. This analysis identifies a glial-bound midline proliferative cluster in the brain and shows that neuronal and glial cells of this cluster are closely associated with neurons pioneering the primary brain commissure. Comparable features of midline cells in the ventral ganglia and similarities to other proliferative clusters in the brain hemispheres are discussed.     

Transient cellular structures in developing corpus callosum of the human brain

The corpus callosum connects two cerebral hemispheres as the most voluminous fiber system in the human brain. The developing callosal fibers originate from immature pyramidal neurons, grow through complex pathways and cross the midline using different substrates in transient fetal structures. We analyzed cellular structures in the human corpus callosum on postmortem brains from the age of 18 weeks post conception to adult, using glial fibrillary acidic protein, neuron-specific nuclear protein, and chondroitin sulphate immunocytochemistry. We found the presence of transient cellular structures, callosal septa, which divide major fiber bundles and ventrally merge with subcallosal zone forming grooves for callosal axons. The callosal septa are composed of glial fibrillary acidic protein reactive meshwork, neurones and the chondroitin sulphate immunoreactive extracellular matrix. The developmental window of prominence of the callosal septa is between 18-34 weeks post conception which corresponds to the period of most intensive growth of callosal axons in human. During the early postnatal period the callosal septa become thinner and shorter, lose their neuronal and chondroitin sulphate content. In conclusion, transient expression of neuronal, glial and extracellular, growing substrate in the callosal septa, as septa itself, indicates their role in guidance during intensive growth of callosal fibers in the human brain. These findings shed some light on the complex morphogenetic events during the growth of the corpus callosum and represent normative parameters necessary for studies of structural plasticity after perinatal lesions.     

Anatomy and physiology of neurons composing the commissural ring nerve of the cricket, Acheta domesticus

The commissural ring nerve (RN) of the cricket Acheta domesticus links together the two cercal motor nerves of the terminal abdominal ganglion. It contains the axons of almost 100 neurons including two bilateral clusters of eight to 13 ventrolateral neurons and approximately 75 neurons with midline somata within the terminal abdominal ganglion. The ventrolateral neurons have an ipsilateral dendritic arborization within the dorsal neuropil of the ganglion and their axons use the RN as a commissure in order to enter the contralateral nerves of the tenth ganglionic neuromere. In contrast, most midline neurons have bifurcating axons projecting bilaterally into the neuropil of the ganglion as well as into the RN where they often branch extensively before entering the contralateral tenth nerves. Most RN neurons have small, non-spiking somata with spike initiation zones distant from the soma. Many midline neurons also produce double-peaked spikes in their somata, indicative of multiple spike initiation zones. Spontaneous neuronal activity recorded extracellularly from the RN reveals several units, some with variable firing patterns, but none responding to sensory stimuli. The RN is primarily composed of small (50 nm diameter) axon profiles with a few large (0.5-1 microm diameter) profiles. Occasionally, profiles of nerve terminals containing primarily small clear vesicles and a few large dense vesicles are observed. These vesicles can sometimes be clustered about an active zone. We conclude that the primary function of the RN is to serve as a peripheral nerve commissure and that its role as a neurohemal organ is negligible. J. Exp. Zool. 286:350-366, 2000.Copyright 2000 Wiley-Liss, Inc.     

Frizzled-3a and slit2 genetically interact to modulate midline axon crossing in the telencephalon

The anterior commissure forms the first axon connections between the two sides of the embryonic telencephalon. We investigated the role of the transmembrane receptor Frizzled-3a in the development of this commissure using zebrafish as an experimental model. Knock down of Frizzled-3a resulted in complete loss of the anterior commissure. This defect was accompanied by a loss of the glial bridge, expansion of the slit2 expression domain and perturbation of the midline telencephalic-diencephalic boundary. Blocking Slit2 activity following knock down of Frizzled-3a effectively rescued the anterior commissure defect which suggested that Frizzled-3a was indirectly controlling the growth of axons across the rostral midline. We have shown here that Frizzled-3a is essential for normal development of the commissural plate and that loss-of-function causes Slit2-dependent defects in axon midline crossing in the embryonic vertebrate forebrain. These data supports a model whereby Wnt signaling through Frizzled-3a attenuates expression of Slit2 in the rostral midline of the forebrain. The absence of Slit2 facilitates the formation of a midline bridge of glial cells which is used as a substrate for commissural axons. In the absence of this platform of glia, commissural axons fail to cross the rostral midline of the forebrain.     

Tsukushi is required for anterior commissure formation in mouse brain

The anterior commissure (AC) is one of the important commissure projections in the brain that conveys information from one side of the nervous system to the other. During development, the axons from the anterior AC (aAC) and the posterior AC (pAC) course in the same dorsoventral plane and converge into a common fascicle for midline crossing. Previously, we reported that Tsukushi (TSK), a member of the secreted small leucine rich repeat proteoglycan family, functions as a key coordinator of multiple pathways outside of cells through the regulation of an extracellular signaling network. Here, we show evidence that TSK is critical for the formation of the AC. In mice lacking TSK, the aAC and the pAC axons fail to cross the midline, leading to an almost total absence of the AC in adult mice. DiI labeling indicated that the aAC axons grew out from the anterior olfactory nucleus and migrated along normal pathways but never crossed the midline. Therefore, we have uncovered a crucial role for TSK for AC formation in the mouse brain.     

Abelson tyrosine kinase is required to transduce midline repulsive cues

Tyrosine phosphorylation-dependent signaling cascades play key roles in determining the formation of an axon pathway. The cytoplasmic Abelson tyrosine kinase participate in several signaling pathways that orchestrate both growth cone advance and steering in response to guidance cues. Here, a genetic approach is used to evaluate the role for Abelson in growth cones during a decision to cross or not to cross the Drosophila embryonic midline. Our data indicate that both loss- and gain-of-function conditions for Abl cause neurons within the pCC/MP2 pathway to project across the midline incorrectly. The frequency of abnormal crossovers is enhanced by mutations in the genes encoding the midline repellent, Slit, or its receptor, Roundabout. In comm mutants, where repulsive signals remain elevated, increasing or decreasing Abl activity partially rescues commissure formation. Thus, both too much and too little Abl activity causes axons to cross the midline inappropriately, indicating that Abl plays a critical role in transducing midline repulsive cues. How Abl functions in this role is not yet clear, but we suggest that Abl may help regulate cytoskeletal dynamics underlying a growth cone's response to midline cues.     

EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure

The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor‐mediated signaling contribute to ACp and CC axon guidance. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 405–420, 2016     

Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS

Whereas considerable progress has been made in understanding the molecular mechanisms of axon guidance across the midline, it is still unclear how the axonal trajectories of longitudinal pioneer neurons, which never cross the midline, are established. Here we show that longitudinal glia of the embryonic Drosophila CNS direct formation of pioneer axon pathways. By ablation and analysis of glial cells missing mutants, we demonstrate that glia are required for two kinds of processes. Firstly, glia are required for growth cone guidance, although this requirement is not absolute. We show that the route of extending growth cones is rich in neuronal cell bodies and glia, and also in long processes from both these cell types. Interactions between neurons, glia and their long processes orient extending growth cones. Secondly, glia direct the fasciculation and defasciculation of axons, which pattern the pioneer pathways. Together these events are essential for the selective fasciculation of follower axons along the longitudinal pathways.     

Development of midline cell types and commissural axon tracts requires Fgfr1 in the cerebrum

The adult cerebral hemispheres are connected to each other by specialized midline cell types and by three axonal tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. Many steps are required for these tracts to form, including early patterning and later axon pathfinding steps. Here, the requirement for FGF signaling in forming midline cell types and commissural axon tracts of the cerebral hemispheres is examined. Fgfr1, but not Fgfr3, is found to be essential for establishing all three commissural tracts. In an Fgfr1 mutant, commissural neurons are present and initially project their axons, but these fail to cross the midline that separates the hemispheres. Moreover, midline patterning defects are observed in the mutant. These defects include the loss of the septum and three specialized glial cell types, the indusium griseum glia, midline zipper glia, and glial wedge. Our findings demonstrate that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures. In addition, analysis of the Fgfr1 heterozygous mutant, in which midline patterning is normal but commissural defects still occur, suggests that at least two distinct FGF-dependent mechanisms underlie the formation of the cerebral commissures.     

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 midline of the Drosophila central nervous system: a model for the genetic analysis of cell fate, cell migration, and growth cone guidance

A row of mesectodermal cells separates the two lateral neurogenic regions in the Drosophila embryo and generates a discrete set of glia and neurons. Most CNS growth cones initially head straight toward the midline, suggesting that these midline cells play a key role in the formation of the axon commissures. We have used antibodies that stain the first axons, beta-galactosidase enhancer trap lines that selectively stain the different midline cells, and electron microscopic studies to elucidate the cells and interactions that mediate the normal formation of the two major commissures in each segment. This analysis has led to a model that proposes a series of sequential cell interactions controlling the development of the axon commissures. A genetic test of this model has utilized a number of mutations that, by either eliminating or altering the differentiation of various midline cells, perturb the development of the axon commissures in a predictable fashion.