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The corpus callosum (CC) is a major interhemispheric commissure of placental mammals. Early steps of CC formation rely on guidance strategies, such as axonal branching and collateralization. Here we analyze the time-course dynamics of axonal bifurcation during typical cortical development or in a CC dysgenesis mouse model. We use Swiss mice as a typical CC mouse model and find that axonal bifurcation rates rise in the cerebral cortex from embryonic day (E)17 and are reduced by postnatal day (P)9. Since callosal neurons populate deep and superficial cortical layers, we compare the axon bifurcation ratio between those neurons by electroporating ex vivo brains at E13 and E15, using eGFP reporter to label the newborn neurons on organotypic slices. Our results suggest that deep layer neurons bifurcate 32% more than superficial ones. To investigate axonal bifurcation in CC dysgenesis, we use BALB/c mice as a spontaneous CC dysgenesis model. BALB/c mice present a typical layer distribution of SATB2 callosal cells, despite the occurrence of callosal anomalies. However, using anterograde DiI tracing, we find that BALB/c mice display increased rates of axonal bifurcations during early and late cortical development in the medial frontal cortex. Midline guidepost cells adjacent to the medial frontal cortex are significant reduced in the CC dysgenesis mouse model. Altogether these data suggest that callosal collateral axonal exuberance is maintained in the absence of midline guidepost signaling and might facilitate aberrant connections in the CC dysgenesis mouse model.

BACKGROUND: Thalamocortical connectivity is essential for normal brain function. This important pathway is established during development, when thalamic axons extend a long distance through the forebrain before reaching the cerebral cortex. In this study, we identify a novel role for the c-Jun N-terminal Kinase (JNK) signaling pathway in guiding thalamocortical axons through intermediate target territories. RESULTS: Complete genetic removal of JNK signaling from the Distal-less 5/6 (Dlx5/6) domain in mice prevents thalamocortical axons from crossing the diencephalon-telencephalon boundary (DTB) and the internal capsule fails to form. Ventral telencephalic cells critical for thalamocortical axon extension including corridor and guidepost neurons are also disrupted. In addition, corticothalamic, striatonigral, and nigrostriatal axons fail to cross the DTB. Analyses of different JNK mutants demonstrates that thalamocortical axon pathfinding has a non-autonomous requirement for JNK signaling. CONCLUSIONS: We conclude that JNK signaling within the Dlx5/6 territory enables the construction of major axonal pathways in the developing forebrain. Further exploration of this intermediate axon guidance territory is needed to uncover mechanisms of axonal pathfinding during normal brain development and to elucidate how this vital process may be compromised in neurodevelopmental disorders.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart., It remains unclear how spatial information controls endothelial cell identity and behavior in the developing heart. Here the authors perform single cell RNA sequencing at key developmental timepoints in mice to interrogate cellular contributions to coronary vessel patterning and maturation in the epicardium.

Fetal Alcohol Spectrum Disorders (FASDs) describe a range of deficits, affecting physical, mental, cognitive, and behavioral function, arising from prenatal alcohol exposure. FASD causes widespread white matter abnormalities, with significant alterations of tracts in the cerebral cortex, cerebellum, and hippocampus. These brain regions present with white-matter volume reductions, particularly at the midline. Neural pathways herein are guided primarily by three guidance cue families: Semaphorin/Neuropilin, Netrin/DCC, and Slit/Robo. These guidance cue/receptor pairs attract and repulse axons and ensure that they reach the proper target to make functional connections. In several cases, these signals cooperate with each other and/or additional molecular partners. Effects of alcohol on guidance cue mechanisms and their associated effectors include inhibition of growth cone response to repellant cues as well as changes in gene expression. Relevant to the corpus callosum, specifically, developmental alcohol exposure alters GABAergic and glutamatergic cell populations and glial cells that serve as guidepost cells for callosal axons. In many cases, deficits seen in FASD mirror aberrancies in guidance cue/receptor signaling. We present evidence for the need for further study on how prenatal alcohol exposure affects the formation of neural connections which may underlie disrupted functional connectivity in FASD.

BackgroundSensory processing relies on projections from the thalamus to the neocortex being established during development. Information from different sensory modalities reaching the thalamus is segregated into specialized nuclei, whose neurons then send inputs to cognate cortical areas through topographically defined axonal connections.Developing thalamocortical axons (TCAs) normally approach the cortex by extending through the subpallium; here, axonal navigation is aided by distributed guidance cues and discrete cell populations, such as the corridor neurons and the internal capsule (IC) guidepost cells. In mice lacking Semaphorin-6A, axons from the dorsal lateral geniculate nucleus (dLGN) bypass the IC and extend aberrantly in the ventral subpallium. The functions normally mediated by Semaphorin-6A in this system remain unknown, but might depend on interactions with Plexin-A2 and Plexin-A4, which have been implicated in other neurodevelopmental processes.MethodsWe performed immunohistochemical and neuroanatomical analyses of thalamocortical wiring and subpallial development inSema6aandPlxna2;Plxna4null mutant mice and analyzed the expression of these genes in relevant structures.ResultsInPlxna2;Plxna4double mutants we discovered TCA pathfinding defects that mirrored those observed inSema6amutants, suggesting that Semaphorin-6A–Plexin-A2/Plexin-A4 signaling might mediate dLGN axon guidance at subpallial level.In order to understand where and when Semaphorin-6A, Plexin-A2 and Plexin-A4 may be required for proper subpallial TCA guidance, we then characterized their spatiotemporal expression dynamics during early TCA development. We observed that the thalamic neurons whose axons are misrouted in these mutants normally express Semaphorin-6A but not Plexin-A2 or Plexin-A4. By contrast, all three proteins are expressed in corridor cells and other structures in the developing basal ganglia.This could be consistent with the Plexins acting as guidance signals through Sema6A as a receptor on dLGN axons, and/or with an indirect effect on TCA guidance due to functions in morphogenesis of subpallial intermediate targets. In support of the latter possibility, we observed that in bothPlxna2;Plxna4andSema6amutants some IC guidepost cells abnormally localize in correspondence of the ventral path misrouted TCAs elongate into.ConclusionsThese findings implicate Semaphorin-6A–Plexin-A2/Plexin-A4 interactions in dLGN axon guidance and in the spatiotemporal organization of guidepost cell populations in the mammalian subpallium.

The antenna is a key sensory organ in insects. Factors which pattern its epithelium and the spacing of sensillae will play an important role in shaping its contribution to adaptive behavior. The antenna of the grasshopper S. gregaria has three major articulations: scape, pedicel, and flagellum. During postembryonic development, the flagellum lengthens as segments (so-called meristal annuli) are added at each molt. However, the five most apical annuli do not subdivide; thus, their epithelial domains must already be defined during embryogenesis. We investigated epithelial compartmentalization and its relationship to the developing primordial nervous system of the antenna by simultaneous immunolabeling against the epithelial cell surface molecule Lachesin, against neuron-specific horseradish peroxidase, and against the mitosis marker phospho-histone 3. We found that Lachesin is initially expressed in a highly ordered pattern of “rings” and a “sock” in the apical antennal epithelium of the early embryo. These expression domains appear in a stereotypic order and prefigure later articulations. Proliferative cells segregate into these developing domains and pioneer- and sensory-cell precursors were molecularly identified. Our study allows pioneer neurons, guidepost cells, and the earliest sensory cell clusters of the primordial nervous system to be allocated to their respective epithelial domain. As the apical-most five domains remain stable through subsequent development, lengthening of the flagellum must originate from more basal regions and is likely to be under the control of factors homologous to those which regulate boundary and joint formation in the antenna of Drosophila.

Celsr3 and Fzd3 regulate the development of reciprocal thalamocortical projections independently of their expression in cortical or thalamic neurons. To understand this cell non autonomous mechanism further, we tested whether Celsr3 and Fzd3 could act via Isl1-positive guidepost cells. Isl1-positive cells appear in the forebrain at embryonic day (E) 9.5-E10.5 and, from E12.5, they form 2 contingents in ventral telencephalon and prethalamus. In control mice, corticothalamic axons run in the ventral telencephalic corridor in close contact with Isl1-positive cells. When Celsr3 or Fzd3 is inactivated in Isl1-expressing cells, corticofugal fibers stall and loop in the ventral telencephalic corridor of high Isl1 expression, and thalamic axons fail to cross the diencephalon-telencephalon junction (DTJ). At E12.5, before thalamic and cortical axons emerge, pioneer projections from Isl1-positive cells cross the DTJ from both sides in control but not mutant embryos. These early projections appear to act like a bridge to guide later growing thalamic axons through the DTJ. Our data suggest that Celsr3 and Fzd3 orchestrate the formation of a scaffold of pioneer neurons and their axons. This scaffold extends from prethalamus to ventral telencephalon and subcortex, and steers reciprocal corticothalamic fibers.

In mammals, thalamic axons are guided internally towards their neocortical target by corridor (Co) neurons that act as axonal guideposts. The existence of Co-like neurons in non-mammalian species, in which thalamic axons do not grow internally, raised the possibility that Co cells might have an ancestral role. Here, we investigated the contribution of corridor (Co) cells to mature brain circuits using a combination of genetic fate-mapping and assays in mice. We unexpectedly found that Co neurons contribute to striatal-like projection neurons in the central extended amygdala. In particular, Co-like neurons participate in specific nuclei of the bed nucleus of the stria terminalis (BNST), which plays essential roles in anxiety circuits. Our study shows that Co neurons possess an evolutionary conserved role in anxiety circuits independently from an acquired guidepost function. It furthermore highlights that neurons can have multiple sequential functions during brain wiring and supports a general role of tangential migration in the building of subpallial circuits. This article is protected by copyright. All rights reserved.

International audience; Trio, a member of the Dbl family of guanine nucleotide exchange factors, activates Rac1 downstream of netrin 1/DCC signalling in axon outgrowth and guidance. Although it has been proposed that Trio also activates RhoA, the putative upstream factors remain unknown. Here, we show that Slit2 induces Trio-dependent RhoA activation, revealing a crosstalk between Slit and Trio/RhoA signalling. Consistently, we found that RhoA activity is hindered in vivo in T rio mutant mouse embryos. We next studied the development of the ventral telencephalon and thalamocortical axons, which have been previously shown to be controlled by Slit2. Remarkably, this analysis revealed that Trio knockout (KO) mice show phenotypes that bear strong similarities to the ones that have been reported in Slit2 KO mice in both guidepost corridor cells and thalamocortical axon pathfinding in the ventral telencephalon. Taken together, our results show that Trio induces RhoA activation downstream of Slit2, and support a functional role in ensuring the proper positioning of both guidepost cells and a major axonal tract. Our study indicates a novel role for Trio in Slit2 signalling and forebrain wiring, highlighting its role in multiple guidance pathways as well as in biological functions of importance for a factor involved in human brain disorders.

International audience; Agenesis of the corpus callosum (AgCC) is a frequent brain disorder found in over 80 human congenital syndromes including ciliopathies. Here, we report a severe AgCC in Ftm/Rpgrip1l knockout mouse, which provides a valuable model for Meckel–Grüber syndrome. Rpgrip1l encodes a protein of the ciliary transition zone, which is essential for ciliogenesis in several cell types in mouse including neuroepithelial cells in the developing forebrain. We show that AgCC in Rpgrip1l−/– mouse is associated with a disturbed location of guidepost cells in the dorsomedial telencephalon. This mislocalization results from early patterning defects and abnormal cortico-septal boundary (CSB) formation in the medial telencephalon. We demonstrate that all these defects primarily result from altered GLI3 processing. Indeed, AgCC, together with patterning defects and mispositioning of guidepost cells, is rescued by overexpressing in Rpgrip1l−/− embryos, the short repressor form of the GLI3 transcription factor (GLI3R), provided by the Gli3Δ699 allele. Furthermore, Gli3Δ699 also rescues AgCC in Rfx3−/− embryos deficient for the ciliogenic RFX3 transcription factor that regulates the expression of several ciliary genes. These data demonstrate that GLI3 processing is a major outcome of primary cilia function in dorsal telencephalon morphogenesis. Rescuing CC formation in two independent ciliary mutants by GLI3Δ699 highlights the crucial role of primary cilia in maintaining the proper level of GLI3R required for morphogenesis of the CC.

The corpus callosum connects cerebral hemispheres and is the largest axon tract in the mammalian brain. Callosal malformations are among the most common congenital brain anomalies and are associated with a wide range of neuropsychological deficits. Crossing of the midline by callosal axons relies on a proper midline environment that harbors guidepost cells emitting guidance cues to instruct callosal axon navigation. Little is known about what controls the formation of the midline environment. We find that two components of the Hippo pathway, the tumor suppressor Nf2 (Merlin) and the transcriptional coactivator Yap (Yap1), regulate guidepost development and expression of the guidance cue Slit2 in mouse. During normal brain development, Nf2 suppresses Yap activity in neural progenitor cells to promote guidepost cell differentiation and prevent ectopic Slit2 expression. Loss of Nf2 causes malformation of midline guideposts and Slit2 upregulation, resulting in callosal agenesis. Slit2 heterozygosity and Yap deletion both restore callosal formation in Nf2 mutants. Furthermore, selectively elevating Yap activity in midline neural progenitors is sufficient to disrupt guidepost formation, upregulate Slit2 and prevent midline crossing. The Hippo pathway is known for its role in controlling organ growth and tumorigenesis. Our study identifies a novel role of this pathway in axon guidance. Moreover, by linking axon pathfinding and neural progenitor behaviors, our results provide an example of the intricate coordination between growth and wiring during brain development.

Our understanding of axon pathfinding mechanisms has dramatically advanced thanks to the identification of guidance cues and receptors, and has been forged by the study of a limited number of model systems. Thalamocortical axons, which are essential for sensory processing and neocortical functioning, convey sensory information to the neocortex through a tightly controlled topographical interconnectivity between distinct thalamic neurons and cortical areas. Recent studies on this projection have provided mechanistic insights onto integrated processes controlling brain wiring: axons/guidepost cells interactions, building of reciprocal connections and the combinatorial activity of guidance cues. This review provides a selective overview of these novel features and stresses the interest of thalamocortical axons as an emerging model for studying axonal guidance and plasticity., Work on this topic was supported by ‘Investissements d’Avenir’ ANR-10-LABX-54 MEMO LIFE and ANR-11-IDEX-0001-02 PSL* Research University to S.G., the ANR-12-BVS4-0010-01 Corridor and INSERM to S.G. and the Spanish MINECO BFU2012-34298 and an ERC Grant ERC-2009-StG_20081210 to G.L-B. S.G. and G.L.-B. are both EMBO Young Investigators.

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.

Frutos, Cristina A. de et al., The neocortex undergoes extensive developmental growth, but how its architecture adapts to expansion remains largely unknown. Here, we investigated how early born Cajal-Retzius (CR) neurons, which regulate the assembly of cortical circuits, maintain a dense superficial distribution in the growing neocortex. We found that CR cell density is sustained by an activity-dependent importation of olfactory CR cells, which migrate into the neocortex after they have acted as axonal guidepost cells in the olfactory system. Furthermore, using mouse genetics, we showed that CR cell density severely affects the architecture of layer 1, a key site of input integration for neocortical networks, leading to an excitation/inhibition ratio imbalance. Our study reveals that neurons reenter migration several days after their initial positioning, thereby performing sequential developmental roles in olfactory cortex and neocortex. This atypical process is essential to regulate CR cell density during growth, which in turn ensures the correct wiring of neocortical circuitry. Video Abstract, We are grateful to B. Mathieu and the IBENS Imaging Facility (France BioImaging, supported by ANR-10-INBS-04, ANR-10-LABX-54 MEMO LIFE, and ANR-11-IDEX-0001-02 PSL∗ Research University, “Investments for the future”; NERF N°2011-45; FRM DGE 20111123023; and FRC Rotary International France). We also thank N. Boggetto and the Flow Cytometry platform of the ImagoSeine facility (France BioImaging, supported by ANR-10-INSB-04, “Investments for the future”). C.A.F. was supported by the IEF Marie Curie program and the Labex Memolife, “Investements for the future” (ANR-10-LABX-54 MEMO LIFE and ANR-11-IDEX-0001-02 PSL∗ Research University). G.B. was funded by Labex Memolife, FRM, and Région Ile de France. Y.A. was the recipient of a fellowship from ARC and FRM. The work in P.C. group was supported by FRM (DEQ20121126545) and work in A.P. group by grants from the ANR (ANR-2011-BSV4-023-01) and FRM (DEQ20130326521). This work was supported by grants from MRT, CNRS, INSERM, and the EURYI program to S.G. S.G. is an EMBO YIP awardee and part of the ENP program.

The vertebrate brain contains millions of neuronal and glial cells arranged in a highly organized manner forming functional neural circuits. To form these circuits during brain development, neurons extend an axon from the cell body to make connections with neurons in target brain areas, which can be a considerable distance away from the neuronal cell body. To ensure that axons accurately elongate toward the correct target field over such a distance, specific guidance cues are used to navigate the axons through their environment in a reproducible pattern of growth. These cues involve guidance molecules that can elicit attractive or repellent guidance on the extending axon and can act over long distances by secretion into extracellular space or over short distances through direct cell contact. In response to the guidance cues, the distal tip of the axon, known as the growth cone, undergoes dynamic structural changes to ensure that it is continually growing in the correct direction. In this chapter, we will discuss the range of highly conserved mechanisms and molecules involved in axon guidance, the biological changes that occur in axons during guidance, and the major assays used to measure the guidance of neuronal axons in vitro.

The cerebral cortex is the largest and most complex region of the brain. Its development is precisely regulated, first by genetic and molecular mechanisms and later by activity-dependent experience. The formation of correct cortical circuits relies upon initial patterning of the early brain into nascent functional domains, the generation of the precise number and position of neurons, and finally their assembly into functional circuits. Our current understanding of these processes in humans and animal models is based on technologies spanning cellular and molecular biology to advanced magnetic resonance imaging techniques. Here, we review our current understanding of the mechanisms regulating the development of cortical wiring, with a focus on the major interhemispheric and descending projections. Decades of work to understand these basic mechanisms are now being translated to understand the causes of human brain wiring disorders, opening new avenues for the development of therapeutics for brain wiring repair.

The olfactory system represents an excellent model for studying different aspects of the development of the nervous system ranging from neurogenesis to mechanisms of axon growth and guidance. Important findings in this field come from comparative studies. We have analyzed key events in the development of the olfactory system of the shark Scyliorhinus canicula by combining immunohistochemical and tract-tracing methods. We describe for the first time in a cartilaginous fish an early population of pioneer HuC/D-immunoreactive (ir) neurons that seemed to delaminate from the olfactory pit epithelium and migrate toward the telencephalon before the olfactory nerve was identifiable. A distinct, transient cell population, namely the migratory mass, courses later on in apposition to the developing olfactory nerve. It contains olfactory ensheathing glial (GFAP-ir) cells and HuC/D-ir neurons, some of which course toward an extrabulbar region. We also demonstrate that Pax6-ir cells coursing along the developing olfactory pathways in S. canicula are young migrating (HuC/D and DCX-ir) neurons of the migratory mass that do not form part of the terminal nerve pathway. Evidences that these Pax6 neurons originate in the olfactory epithelium are also reported. As Pax6 neurons in the olfactory epithelium show characteristics of olfactory receptor neurons, and migrating Pax6-ir neurons formed transient corridors along the course of olfactory axons at the entrance of the olfactory bulb, we propose that these neurons could play a role as guideposts for axons of olfactory receptor neurons growing toward the olfactory bulb.

Thalamocortical axons must cross a complex cellular terrain through the developing forebrain, and this terrain has to be understood for us to learn how thalamocortical axons reach their destinations. Selective fasciculation, guidepost cells and various diencephalic and telencephalic gradients have been implicated in thalamocortical guidance. As our understanding of the relevant forebrain patterns has increased, so has our knowledge of the guidance mechanisms. Our aim here is to review recent observations of cellular and molecular mechanisms related to: the growth of thalamofugal projections to the ventral telencephalon, thalamic axon avoidance of the hypothalamus and extension into the telencephalon to form the internal capsule, the crossing of the pallial-subpallial boundary, and the growth towards the cerebral cortex. We shall review current theories for the explanation of the maintenance and alteration of topographic order in the thalamocortical projections to the cortex. It is now increasingly clear that several mechanisms are involved at different stages of thalamocortical development, and each contributes substantially to the eventual outcome. Revealing the molecular and cellular mechanisms can help to link specific genes to details of actual developmental mechanisms.

14 p., 8 figures and references., How brain connectivity has evolved to integrate the mammalian-specific neocortex remains largely unknown. Here, we address how dorsal thalamic axons, which constitute the main input to the neocortex, are directed internally to their evolutionary novel target in mammals, though they follow an external path to other targets in reptiles and birds. Using comparative studies and functional experiments in chick, we show that local species-specific differences in the migration of previously identified " corridor" guidepost neurons control the opening of a mammalian thalamocortical route. Using in vivo and ex vivo experiments in mice, we further demonstrate that the midline repellent Slit2 orients migration of corridor neurons and thereby switches thalamic axons from an external to a mammalian-specific internal path. Our study reveals that subtle differences in the migration of conserved intermediate target neurons trigger large-scale changes in thalamic connectivity, and opens perspectives on Slit functions and the evolution of brain wiring., This work was supported by grants from the INSERM "Avenir" Program to S.G., the City of Paris to S.G., the ARC to S.G., the FRC to S.G., and the EURYI program to S.G.; by grants from the Spanish Ministry of Science and Innovation BFU2006-00408/BFI and BFU2009-08261 to G.L.-B., and CONSOLIDER CSD2007-00023 to G.L.-B.; and by the PAI Picasso and Acciones Integradas to S.G. and G.L.-B. F.B. was supported by a fellowship from the French Ministry of Research. P.M.-M. was supported by a FPI fellowship from the Spanish Ministry of Science and Innovation. S.G. is a EURYI Awardee.

Synapses are specialized sites of cell contact that mediate information flow between neurons and their targets. Genetic screens in the nematode C. elegans have led to the discovery of a number of molecules required for synapse patterning and assembly. Recent studies have demonstrated the importance of guidepost cells in the positioning of presynaptic sites at specific locations along the axon. Interestingly, these guideposts can promote or inhibit synapse formation, and do so by utilizing transmembrane adhesion molecules or secreted factors that act over relatively larger distances. Once the decision of where to build a presynaptic terminal has been made, key molecules are recruited to assemble synaptic vesicles and active zone proteins at that site. Multiple steps of this process are regulated by ubiquitin ligase complexes. Interestingly, some of the molecules involved in presynaptic assembly also play roles in regulating axon polarity and outgrowth, suggesting that different neurodevelopmental processes are molecularly integrated.

PMID:17392456, The function of the nervous system depends on the precision of axon wiring during development. Previous studies have demonstrated that Slits, a family of secreted chemorepellent proteins, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 or, even more so, in both Slit1 and Slit2 have defects in multiple axonal pathways, including corticofugal, thalamocortical, and callosal connections. In the spinal cord, members of the Robo family of proteins help mediate the function of Slits, but the relative contribution of these receptors to the guidance of forebrain projections remains to be determined. In the present study, we addressed the function of Robo1 and Robo2 in the guidance of forebrain projections by analyzing Robo1-, Robo2-, and Robo1;Robo2-deficient mice. Mice deficient in Robo2 and, more dramatically, in both Robo1 and Robo2, display prominent axon guidance errors in the development of corticofugal, thalamocortical, and corticocortical callosal connections. Our results demonstrate that Robo1 and Robo2 mostly cooperate to mediate the function of Slit proteins in guiding the major forebrain projections., This work was supported by grants from Spanish Government (BFU2005-04773/BMC) and the European Young Investigator (EURYI) program (O.M.) and from Association pour la Recherche sur le Cancer (A.C.).

The mammalian neocortex shows an extremely well-organized structure that underlies higher brain functions such as cognition, language, and memory. The neocortex consists of a six-layered structure, in which excitatory and inhibitory neurons form complex neural circuits in concert with glial cells. As a result of recent technological innovations in live imaging and in utero electroporation, the processes involved in neocortical development, especially the mechanism of neuronal migration, have been successively revealed. Furthermore, it has been recognized recently that defects in neuronal migration lead to brain malformations and diverse psychiatric and neurological disorders including schizophrenia, epilepsy, and autism. Accordingly, it is important to elucidate the molecular mechanism of neuronal migration in the neocortex, in order to understand not only the basic principles of brain development but also the pathological processes of these disorders. In this special issue, we attempt to cover topics ranging from the basic mechanisms of neocortical development to the malformation and evolution of the neocortex, with a special focus on neuronal migration. Radial glial cells (RGCs) are primary progenitors capable of generating various types of neurons and glial cells, which include Cajal-Retzius cells, subplate neurons, pyramidal neurons, interneurons, oligodendrocytes, and astrocytes. Thus, it is important to know how these diverse types of cells are generated from RGCs and integrated into complex neocortical circuits. Toma and Hanashima reviewed the mechanisms that regulate the changes in RGC competency and neuronal subtype transitions, focusing on the regulatory networks of various transcription factors including Foxg1. At the earlier stage of neocortical development, RGCs predominantly produce a large number of neurons, but later they change into glia-restricted progenitors. After the discovery of the importance of astrocytes in synaptic plasticity and blood flow, the mechanisms of glial development have attracted increasing interest for many neuroscientists. Tabata reviewed the mechanism controlling the production of diverse types of astrocytes and their migration behavior, demonstrating the multiple origins of glial cells in the neocortex. Neocortical circuits consist of highly interconnected excitatory glutamatergic and inhibitory GABAergic neurons, which are generated from distinct pools of RGCs. The excitatory neurons are generated from RGCs localized in the ventricular zone of the dorsal telencephalon and migrate radially toward the pial surface in an inside-out manner (radial migration). On the other hand, inhibitory neurons mainly originate from the ventral telencephalon and migrate tangentially into the neocortex (tangential migration). In spite of such different developmental origins, both excitatory and inhibitory neurons go through the multipolar stage with several minor processes in the neocortex before axon extension. Then, they undergo dramatic morphological changes to initiate axon formation, namely, neuronal polarization. Sakakibara and Hatanaka reviewed the sequential events in polarization processes of both excitatory and inhibitory neurons, and they discussed the underlying molecular mechanisms. At the multipolar stage, the excitatory neurons transiently use a multipolar migration mode, namely migration with no fixed direction, in the subventricular and intermediate zones. Then, they adopt a bipolar shape during neuronal polarization and migrate quickly toward the pial surface along RGC processes, which is called locomotion mode. Many kinds of molecules are involved in these dynamic changes in the morphology and behavior of neurons. Small GTP binding proteins belonging to the Rho family play critical roles in cytoskeletal regulation during such dynamic processes. Azzarelli et al. reviewed the roles of Rnd proteins, “atypical” Rho family members, in neuronal migration and discussed its upstream and downstream pathways. The functions of many cytoplasmic proteins including cytoskeletal components are regulated by phosphorylation and dephosphorylation processes. Ohshima focused on protein kinases, including CDK5 and JNKs, and reviewed their regulatory roles in cytoskeletal organization during multipolar-bipolar transition and radial migration. Ohtaka-Maruyama and Okado comprehensively summarized the molecular pathways involved in these developmental processes, emphasizing the importance of subplate neurons in the development and evolution of the six-layered neocortical structure. It is apparent that neuronal migration and wiring are regulated by various secreted factors such as growth factors, chemokines, and extracellular matrix molecules, although their mechanisms are poorly understood. Kondo et al. demonstrated that subplate neurons transiently express high levels of secretary proteins such as connective tissue growth factor, neuroserpin, and insulin-like growth factor binding protein 5, which may be involved in cortical circuit formation. Greenman et al. reported a novel finding that autotaxin (ENPP2), a secretary enzyme bearing lysophospholipase D activity, regulates the localization and adhesion of neural progenitor cells independent of its catalytic activity. Maeda reviewed the roles of proteoglycans in neuronal polarization and migration and discussed the possibility that extracellular matrix regulates the distribution and activity of multiple secreted factors in the developing neocortex. In addition to the long-range gradient of secreted factors, axon pathfinding is also regulated by short-range guidance cues and direct cell-cell contacts mediated by guidepost cells. Squarzoni et al. reviewed the roles of already known guideposts such as Cajal-Retzius cells for entorhinal-hippocampal axons and corridor cells for thalamocortical axons, and further proposed a new class of guidepost cells, microglia, in the cortex. Hippocampal formation has a close relationship with the neocortex both functionally and structurally, but it shows a distinct arrangement of pyramidal neurons from that of the neocortex. Hayashi et al. reviewed the differences in the migratory behaviors of neocortical and hippocampal neurons, which lead to the formation of distinct layered structures in these two cortical regions. Defects in the migration of excitatory and inhibitory neurons can lead to the various neurological and psychiatric disorders. Kato reviewed recent development in the understanding of the genetic bases of neuronal migration disorders in terms of genotype-phenotype correlations, focusing mainly on lissencepahaly. Muraki and Tanigaki discussed the possible relationship between neuronal migration defects and behavioral abnormalities relevant to schizophrenia based on studies using genetically defined animal models. The evolutionary approaches should greatly deepen our understanding of the mechanisms underlying neocortical development. Nomura et al. established the method of in ovo electroporation and ex ovo culture of reptilian embryos. Comparative studies using this method will provide significant insights into the origin of the mammalian neocortex. It is hoped that the special issue entitled “Mechanisms of Neuronal Migration during Corticogenesis” will serve as a valuable resource for many neuroscientists to promote their research perspectives. Finally, as topic editors, we would like to express our sincere appreciation to all the authors for their outstanding contributions and to all the reviewers for their insightful comments on the papers. We also thank the editorial office and the production staff for their unceasing efforts and dedication.

The striatum is a large brain nucleus with an important role in the control of movement and emotions. Medium spiny neurons (MSNs) are striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts in the forebrain develop remains poorly understood. Here, we implicate the Wnt binding receptor Frizzled3 in several uncharacterized aspects of MSN pathway formation [i.e., anterior–posterior guidance of MSN axons in the striatum and their subsequent growth into the globus pallidus (GP), an important (intermediate) target]. In Frizzled3 knock-out mice, MSN axons fail to extend along the anterior–posterior axis of the striatum, and many do not reach the GP. Wnt5a acts as an attractant for MSN axons in vitro, is expressed in a posterior high, anterior low gradient in the striatum, and Wnt5a knock-out mice phenocopy striatal anterior–posterior defects observed in Frizzled3 mutants. This suggests that Wnt5a controls anterior–posterior guidance of MSN axons through Frizzled3. Axons that reach the GP in Frizzled3 knock-out mice fail to enter this structure. Surprisingly, entry of MSN axons into the GP non–cell-autonomously requires Frizzled3, and our data suggest that GP entry may be contingent on the correct positioning of “corridor” guidepost cells for thalamocortical axons by Frizzled3. Together, these data dissect MSN pathway development and reveal (non)cell-autonomous roles for Frizzled3 in MSN axon guidance. Further, they are the first to identify a gene that provides anterior–posterior axon guidance in a large brain nucleus and link Frizzled3 to corridor cell development., This work was supported by The Netherlands Organization for Health Research and Development (ZonMW-VIDI and ZonMW-TOP), Stichting ParkinsonFonds, and the European Union (mdDANeurodev, FP7/2007–2011, Grant 222999) to R.J.P., Actions de Recherches Concerteés (ARC-10/15-026) and Fondation médicale Reine Elisabeth to F.T., and the Spanish MINECO BFU2012-34298 to G.L.-B. (who is an EMBO Young Investigator). This study was performed in part within the framework of Dutch Top Institute Pharma Project T5-207 to R.J.P.

Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures., S.M. was supported by a postdoctoral fellowship of the Fondation Pierre Mercier pour la science. A.R.Y. was a recipient of a doctoral fellowship of the Ecole des Neurosciences de Paris. The work in the laboratories of C.L. and J.-P.H. was supported by funds from Swiss National Foundation Grant no. 31003A-122550. The work in the laboratory of A.C. was supported by grants from the French State programme ‘Investissements d'Avenir’ managed by the Agence Nationale de la Recherche [LIFESENSES: ANR-10-LABX-65], the fondation pour la recherche médicale (Programme équipe FRM) and the ANR (grant ANR2011 BSV4 0091). Work in the laboratory of Y.Y. was supported by funds for Scientific Research from the MEXT, Japan and Takeda Science Foundation.

PMID:16615895, Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain., We have been supported by grants from Spanish Government BMC2002-03337, GVA GRUPOS03/053, NARSAD, the European Commission through STREP contract number 005139 (INTERDEVO), and the EURYI program to O.M.; by grants from INSERM, MENRT, ARC, and AFM to P.C.; by NIH grant NS29071 to L.R. and D.T.; and by the Picasso PAI/Programa de Acciones Integradas to O.M. and S.G. G.L.-B. is a “Ramón y Cajal” Investigator from the CSIC. F.B. was supported by a fellowship from the Académie Nationale de Médecine. S.G. is a recipient of the Human Frontier Science Program Organization CDA. O.M. is an EMBO Young Investigator, a NARSAD Young Investigator, and an EURYI Awardee.

The arealization of the mammalian cortex is believed to be controlled by a combination of intrinsic factors that are expressed in the cortex, and external signals, some of which are mediated through thalamic input. Recent studies on transgenic mice have identified families of molecules that are involved in thalamic axon growth, pathfinding and cortical target selection, and we are beginning to understand how thalamic projections impose cytoarchitectonic differentiation on the developing cortex. By unravelling these mechanisms further, we should be able to increase our understanding of the principles of cortical organization.

Axons navigate to trace stereotypic trajectories over an environment often rich in glial cells. Once axonal trajectories are defined, their structuring proceeds through multiple fasciculation and defasciculation events, to finally establish the mature bundles. Fasciculation and ensheathment also proceed in close association between axons and glial cells, and ultimately require glia. The cross-talk between axons and glia during axon guidance is manifested in: (i) axonal fasciculation and bundling, promoted by glia; (ii) growth cone guidance, as glia function as guidepost cells at choice points; (iii) glial migration patterns, which are influenced by neurons; (iv) cell survival control, which constrains position and number of both cell types; and (iv) connectivity, where an axon contacts its final target aided by glial cells. Understanding the reciprocal interactions between neurons and glia during guidance and fasciculation is absolutely necessary to implement repair of axonal trajectories upon damage. Drosophila can be used as a model system for these purposes.

Emx2 knockout mice appear to show a shift in the areal identity in the cerebral cortex of Emx2 knockout mice, which is matched with altered distribution of thalamocortical projections (Bishop et al. [2000] Science 288:344–3349; Mallamaci et al. [2000] Nat Neurosci. 3:679–686). We have examined the early establishment of these projections to understand how the altered Emx2 expression results in changes in their cortical targeting. We used carbocyanine dye tracing to visualize thalamocortical and corticofugal projections as well as immunohistochemistry for L1 and TAG-1, respective markers of the two axonal systems, in wild-type, heterozygote, and null mutant for Emx2 at embryonic (E) ages ranging from E13.5 to E18.5. These tracing studies demonstrated that, in Emx2 knockout mice, a large proportion of early thalamocortical projections were misrouted at the border between the diencephalon and telencephalon. This abnormality was associated with displaced connectivity of the internal capsule cells at the diencephalic–telencephalic junction. Interestingly, most of the aberrant thalamic projections compensated for the ventral entry to the telencephalon and still ascended to the cortex. Although this early targeting abnormality is associated with the altered Emx2 expression pattern in the cortex, it most probably occurs independently from it, and is related to earlier guidance defects at the diencephalic–telencephalic boundary. These defects might result in the altered and delayed arrival of thalamic projections to the cortex and, thus, contribute to the shifted thalamocortical matching previously observed in the Emx2 knockout mice. J. Comp. Neurol. 451:153–169, 2002. © 2002 Wiley-Liss, Inc.

The corpus callosum is a large bundle of axons that connects the right and left hemispheres of the brain. During development axons are guided across the callosum by various physical and molecular cues, such as the chemorepellent protein Slit2. Among the cells that produce guidance factors are the neural guidepost cells, which reside near the midline of the brain where the callosum forms. The scaffolding protein Nf2 (also known as Merlin) can inhibit the transcriptional coactivator Yap as part of the Hippo signaling pathway, best known for regulating cell proliferation and controlling organ size. Conditional knockout of Nf2 in the developing forebrain in mice ( Nf2 F/F ; Emx1-Cre mice) disrupts callosal development. Lavado et al. discovered that Nf2 and Yap regulated the development and function of guidepost cells. Nf2 was present in midline neural progenitor cells that give rise to guidepost cells. Although, the gross morphology of the midline was normal in Nf2 F/F ; Emx1-Cre mice, neural progenitor cells failed to retract processes attached to the midline and guidepost cells did not properly differentiate. The expression of Slit2 was increased in midline cells in Nf2 F/F ; Emx1-Cre mice. Overexpression of Slit2 in the brains of wild-type mice resulted in the accumulation of tangled axons. Heterozygous deletion of Slit2 or conditional deletion of Yap in the forebrain restored formation of the callosum in Nf2 F/F ; Emx1-Cre mice. Overexpression of Yap in midline neural progenitor cells caused callosal and guidepost cell phenotypes similar to those in Nf2 F/F ; Emx1-Cre mice. Thus, these data indicate that Nf2 inhibits the expression of Slit2 in midline cells and limits the activation of Yap, promoting formation of guidepost cells and enabling callosal axons to cross the midline. A. Lavado, M. Ware, J. Pare, X. Cao, The tumor suppressor Nf2 regulates corpus callosum development by inhibiting the transcriptional coactivator Yap. Development 141 , 4182–4193 (2014). [Abstract][Full Text]

Celsr3 and Fzd3, members of "core planar cell polarity" (PCP) genes, were shown previously to control forebrain axon guidance and wiring by acting in axons and/or guidepost cells. Here, we show that Celsr2 acts redundantly with Celsr3, and that their combined mutation mimics that of Fzd3. The phenotypes generated upon inactivation of Fzd3 in different forebrain compartments are similar to those in conditional Celsr2-3 mutants, indicating that Fzd3 and Celsr2-3 act in the same population of cells. Inactivation of Celsr2-3 or Fzd3 in thalamus does not affect forebrain wiring, and joint inactivation in cortex and thalamus adds little to cortical inactivation alone in terms of thalamocortical projections. On the other hand, joint inactivation perturbs strongly the formation of the barrel field, which is unaffected upon single cortical or thalamic inactivation, indicating a role for interactions between thalamic axons and cortical neurons in cortical arealization. Unexpectedly, forebrain wiring is normal in mice defective in Vangl1 and Vangl2, showing that, contrary to epithelial PCP, axon guidance can be Vangl independent in some contexts. Our results suggest that Celsr2-3 and Fzd3 regulate axonal navigation in the forebrain by using mechanisms different from classical epithelial PCP, and require interacting partners other than Vangl1-2 that remain to be identified.

The three-layered piriform cortex, an integral part of the olfactory system, processes odor information relayed by olfactory bulb mitral cells. Specifically, mitral cell axons form the lateral olfactory tract (LOT) by targeting lateral olfactory tract (lot) guidepost cells in the piriform cortex. While lot cells and other piriform cortical neurons share a pallial origin, the factors that specify their precise phenotypes are poorly understood. Here we show that in mouse, the proneural genesNeurog1andNeurog2are coexpressed in the ventral pallium, a progenitor pool that first gives rise to Cajal-Retzius (CR) cells, which populate layer I of all cortical domains, and later to layer II/III neurons of the piriform cortex. Using loss-of-function and gain-of-function approaches, we find thatNeurog1has a unique early role in reducing CR cell neurogenesis by temperingNeurog2's proneural activity. In addition,Neurog1andNeurog2have redundant functions in the ventral pallium, acting in two phases to first specify a CR cell fate and later to specify layer II/III piriform cortex neuronal identities. In the early phase,Neurog1andNeurog2are also required for lot cell differentiation, which we reveal are a subset of CR neurons, the loss of which prevents mitral cell axon innervation and LOT formation. Consequently, mutation ofTrp73, a CR-specific cortical gene, results in lot cell and LOT axon displacement.Neurog1andNeurog2thus have unique and redundant functions in the piriform cortex, controlling the timing of differentiation of early-born CR/lot cells and specifying the identities of later-born layer II/III neurons.

Growing axons are often guided to their final destination by intermediate targets. In the developing spinal cord and optic nerve, specialized cells at the embryonic midline act as intermediate targets for guiding commissural axons. Here we investigate whether similar intermediate targets may play a role in guiding cortical axons in the developing brain. During the development of the corpus callosum, cortical axons from one cerebral hemisphere cross the midline to reach their targets in the opposite cortical hemisphere. We have identified two early differentiating populations of midline glial cells that may act as intermediate guideposts for callosal axons. The first differentiates directly below the corpus callosum forming a wedge shaped structure (the glial wedge) and the second differentiates directly above the corpus callosum within the indusium griseum. Axons of the corpus callosum avoid both of these populationsin vivo. This finding is recapitulatedin vitroin three-dimensional collagen gels. In addition, experimental manipulations in organotypic slices show that callosal axons require the presence and correct orientation of these populations to turn toward the midline. We have also identified one possible candidate for this activity because both glial populations express the chemorepellent moleculeslit-2, and cortical axons express theslit-2receptorsrobo-1androbo-2. Furthermore,slit-2repels–suppresses cortical axon growth in three-dimensional collagen gel cocultures.

The search to understand the mechanisms regulating brain wiring has relied on biochemical purification approaches in vertebrates and genetic approaches in invertebrates to identify molecular cues and receptors for axon guidance. Here we describe a phenotype-based gene-trap screen in mice designed for the large-scale identification of genes controlling the formation of the trillions of connections in the mammalian brain. The method incorporates an axonal marker, which helps to identify cell-autonomous mechanisms in axon guidance, and has generated a resource of mouse lines with striking patterns of axonal labelling, which facilitates analysis of the normal wiring diagram of the brain. Studies of two of these mouse lines have identified an in vivo guidance function for a vertebrate transmembrane semaphorin, Sema6A, and have helped re-evaluate that of the Eph receptor EphA4.

We labeled axonal projections using carbocyanine dyes in the developing rat brain to study cellular interactions that might underlie the establishment of thalamocortical connectivity. By embryonic day 14 (E14), groups of neurons in the ventral diencephalon and the primitive internal capsule have established projections to the dorsal thalamus, and thalamic fibers pass in topographic order among them. Simultaneously, axons from the early-born cells in both subplate and marginal zone (i.e., the original cortical preplate) establish an ordered array that fills the intermediate zone. Thalamic axons and preplate fibers meet in the lateral part of the internal capsule (at E15 for occipital cortex and dorsolateral thalamus). Subsequently, selective labeling of corresponding thalamic and early corticofugal projections reveals thalamic fibers growing in association with early corticofugal axons, right up to the cortical subplate. A small carbocyanine crystal implanted at any point in the cortex shortly after the arrival of thalamic axons (E16 for the occipital cortex) labels a single, tight bundle containing both descending and ascending fibers, rather than two separate tracts, providing further evidence for intimate topographic association of the two axon systems. Crystals placed in a row, parasagittally or coronally along the hemisphere, reveal separate, topographically distributed, discrete fiber bundles throughout the pathway, leading to spatially ordered groups of back-labeled thalamic cells. These results indicate that the topography of thalamic axons is maintained throughout the pathway and that they reach the cortex by associating with the projections of a number of preexisting cells, including the preplate scaffold.

An intermediate target for axons leaving the cerebral cortex in embryonic mammals is the ganglionic eminence (GE), the embryonic precursor of the basal ganglia. The cues that direct these axons over the initial portion of their trajectory are not well understood, but could include both short-range and long-range attractants and repellents. In the present study, we provide evidence that corticofugal axons might be guided at least partly by a diffusible factor or factors originating in the lateral GE and the sulcus between the lateral and medial ridges of the GE (ISS), as well as evidence implicating the axonal chemoattractant netrin-1 in mediating these effects. Explants of lateral GE and ISS obtained from E12.5 and E13.5 mouse forebrain have a strong effect on both the outgrowth and orientation of corticofugal axons when cultured at a distance with explants of embryonic cortex in collagen gels. Netrin-1 mRNA is detected in these target tissues by in situ hybridization, and both netrin-1 protein and heterologous cells secreting netrin-1 can mimic the outgrowth-promoting effect of these target tissues in vitro. Furthermore, the growth of corticofugal axons is oriented toward an ectopic source of netrin-1 in vitro, and a function blocking anti-netrin-1 antiserum specifically abolishes the cortical axon outgrowth elicited by explants of lateral GE and the ISS in collagen gel cocultures. Taken together, these results suggest a role for netrin-1 in the attraction at a distance of early cortical axons by the GE. Thus in mammals – as is also observed in nematodes – the development of non-commissural projections in anterior regions of the embryo might be directed by mechanisms similar to those involved in directing the development of commissural projections in more posterior regions of the central nervous system.

Pioneer axons in insect legs are experimentally accessible model systems for the molecular identification and cellular localization of guidance cues regulating the path of axon growth. A detailed study of the Fe2 pioneer axons in the legs of the cockroach was performed to examine the diversity of guidance mechanisms. A detailed microscopic analysis of the axons at various points in their trajectory indicates that the Fe2 axons grow on a mesodermal substratum which contains the cues guiding their growth along a stereotyped path. An identified pair of muscle pioneer cells (MPC) are likely to play an important role in enabling the Fe2 growth cones to respond to mesodermal guidance cues. The addition of heparan sulfate, heparitinase, and phosphatidylinositol-specific phospholipase C to the medium perturbs thein situpath of growth of the Fe2 axons and the location of the MPC in cultured embryos. This indicates a role for heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored proteins in axon guidance. When these results are compared to those of similar experiments performed on the well-characterized Ti1 axons, they indicate significant differences in the mechanisms that are used for axon guidance. The Fe2 neurons are a good model for elucidating the mechanisms used to guide axon growth on nonmuscle mesodermal substrates often encountered in the periphery of vertebrate embryos.

During nervous system formation nerve cells extend axons in order to form precise patterns of neuronal connectivity. These connections are often established after the neuronal growth cones have pioneered or navigated through complex pathways to their target area both within the CNS and to and from the periphery. Recent studies have provided evidence that the process of specific pathway formation may rely on a number of molecular guidance mechanisms and cues such as selective adhesion, growth cone avoidance, surface gradients, guidepost cells, and chemotropism. Analysis of the molecular basis for these guidance mechanisms show that the molecules involved often belong to distinct multigene families and that they can provide both short- and long-range attractive as well as repulsive cues. Many of these molecules have a modular structure that is made up of different tandemly arranged domains that allow for multiple functional interactions with a range of other molecules. This allows the same molecule to be multifunctional, for example, by attracting certain neurons while repelling others. This review is an overview of the molecular structure, as it relates to function and mechanisms of action of some of the major gene families thought to be mediating specific axonal guidance and pathway formation.

During nervous system development, spinal commissural axons project toward floor plate cells and trochlear motor axons extend away from these cells. Netrin-1, a diffusible protein made by floor plate cells, can attract spinal commissural axons and repel trochlear axons in vitro, but its role in vivo is unknown. Netrin-1 deficient mice exhibit defects in spinal commissural axon projections that are consistent with netrin-1 guiding these axons. Defects in several forebrain commissures are also observed, suggesting additional guidance roles for netrin-1. Trochlear axon projections are largely normal, predicting the existence of additional cues for these axons, and evidence is provided for a distinct trochlear axon chemorepellent produced by floor plate cells. These results establish netrin-1 as a guidance cue that likely collaborates with other diffusible cues to guide axons in vivo.

In the nervous system of many species, growing axons associate transiently with cellular groupings along their path. Whether this mechanism applies to the development of corticothalamic and thalamocortical projections is unknown. Using carbocyanine dyes, we studied the early growth of both corticofugal and thalamocortical fibers in hamster embryos. At embryonic day 11.5 (E11.5), corticofugal fibers invade the lateral ganglionic eminence (LGE), and thalamocortical fibers invade the medial ganglionic eminence (MGE). At this age, both sets of fibers are not yet in contact with each other. At the same time, neurons in each subdivision of the GE grow toward the cortex and thalamus. During the next 24 hr, corticofugal and thalamocortical fibers remain within the confines of the GE, where they course at different radial levels and bear large and complex growth cones. In the LGE, corticofugal fibers are often found in close association with cells that are likely to be neuronal. Starting on E13.5, both early projections from the GE decrease, and corticothalamic and thalamocortical fibers invade their definitive target regions. To test whether the GE specifically orients the growth and trajectories of cortical fibers even in the absence of the reciprocal thalamic projection, we cocultured explants of cortex and GE from either hamster or mouse embryos. These experiments showed that the GE, but not other tested brain regions, is able specifically to orient the growth of cortical axons. We therefore suggest that the GE may be an intermediate target in the pathfinding of axons between the cortex and the thalamus.

The guidance of axons to their targets in the developing nervous system is believed to involve diffusible chemotropic factors secreted by target cells. Floor plate cells at the ventral midline of the spinal cord secrete a diffusible factor or factors that promotes the outgrowth of spinal commissural axons and attracts these axons in vitro. Two membrane-associated proteins isolated from brain, netrin-1 and netrin-2, possess commissural axon outgrowth-promoting activity. We show here that netrin-1 RNA is expressed by floor plate cells, whereas netrin-2 RNA is detected at lower levels in the ventral two-thirds of the spinal cord, but not the floor plate. Heterologous cells expressing recombinant netrin-1 or netrin-2 secrete diffusible forms of the proteins and can attract commissural axons at a distance. These results show that netrin-1 is a chemotropic factor expressed by floor plate cells and suggest that the two netrin proteins guide commissural axons in the developing spinal cord.

In vertebrates, commissural axons pioneer a circumferential pathway to the floor plate at the ventral midline of the embryonic spinal cord. Floor plate cells secrete a diffusible factor that promotes the outgrowth of commissural axons in vitro. We have purified from embryonic chick brain two proteins, netrin-1 and netrin-2, that each possess commissural axon outgrowth-promoting activity, and we have also identified a distinct activity that potentiates their effects. Cloning of cDNAs encoding the two netrins shows that they are homologous to UNC-6, a laminin-related protein required for the circumferential migration of cells and axons in C. elegans. This homology suggests that growth cones in the vertebrate spinal cord and the nematode are responsive to similar molecular cues.

Guidepost cells are essential structures for the establishment of major axonal tracts. How these structures are specified and acquire their axon guidance properties is still poorly understood. Here, we show that in mouse embryos appropriate levels of Bone Morphogenetic Protein 7 (Bmp7), a member of the TGF-β superfamily of secreted proteins, are required for the correct development of the glial wedge, the indusium griseum, and the subcallosal sling, three groups of cells that act as guidepost cells for growing callosal axons. Bmp7 is expressed in the region occupied by these structures and its genetic inactivation in mouse embryos caused a marked reduction and disorganization of these cell populations. On the contrary, infusion of recombinant Bmp7 in the developing forebrain induced their premature differentiation. In both cases, changes were associated with the disruption of callosal axon growth and, in most animals fibers did not cross the midline forming typical Probst bundles. Addition of Bmp7 to cortical explants did not modify the extent of their outgrowth nor their directionality, when explants were exposed to a focalized source of the protein. Together, these results indicate that Bmp7 is indirectly required for corpus callosum formation by controlling the timely differentiation of its guidepost cells. © 2010 Wiley Periodicals, Inc.