Teneurin trans-axonal signaling prunes topographically missorted axons.

Building precise neural circuits necessitates the elimination of axonal projections that have inaccurately formed during development. Although axonal pruning is a selective process, how it is initiated and controlled in vivo remains unclear. Here, we show that trans-axonal signaling mediated by the cell surface molecules Glypican-3, Teneurin-3, and Latrophilin-3 prunes misrouted retinal axons in the visual system. Retinotopic neuron transplantations revealed that pioneer ventral axons that elongate first along the optic tract instruct the pruning of dorsal axons that missort in that region. Glypican-3 and Teneurin-3 are both selectively expressed by ventral retinal ganglion cells and cooperate for correcting missorted dorsal axons. The adhesion G-protein-coupled receptor Latrophilin-3 signals along dorsal axons to initiate the elimination of topographic sorting errors. Altogether, our findings show an essential function for Glypican-3, Teneurin-3, and Latrophilin-3 in topographic tract organization and demonstrate that axonal pruning can be initiated by signaling among axons themselves.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics.

Axonal regeneration in the central nervous system is an energy-intensive process. In contrast to mammals, adult zebrafish can functionally recover from neuronal injury. This raises the question of how zebrafish can cope with this high energy demand. We previously showed that in adult zebrafish, subjected to an optic nerve crush, an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair. We postulate a 'dendrites for regeneration' paradigm that might be linked to intraneuronal mitochondrial reshuffling, as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously. Here, we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments (dendrites, somas, and axons) during the regenerative process. Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction, whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth. Upon dendritic regrowth in the retina, mitochondrial density inside the retinal dendrites returned to baseline levels. Moreover, a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage. Taken together, these findings suggest that during optic nerve injury-induced regeneration, mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

To Stick or Not to Stick: The Multiple Roles of Cell Adhesion Molecules in Neural Circuit Assembly.

Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly.

Analyzing the Role of Heparan Sulfate Proteoglycans in Axon Guidance In Vivo in Zebrafish.

One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly wired to form functional circuits. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, less is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins.

Live imaging of retinotectal mapping reveals topographic map dynamics and a previously undescribed role for Contactin 2 in map sharpening.

Organization of neuronal connections into topographic maps is essential for processing information. Yet, our understanding of topographic mapping has remained limited by our inability to observe maps forming and refining directly in vivo. Here, we used Cre-mediated recombination of a new colorswitch reporter in zebrafish to generate the first transgenic model allowing the dynamic analysis of retinotectal mapping in vivo. We found that the antero-posterior retinotopic map forms early but remains dynamic, with nasal and temporal retinal axons expanding their projection domains over time. Nasal projections initially arborize in the anterior tectum but progressively refine their projection domain to the posterior tectum, leading to the sharpening of the retinotopic map along the antero-posterior axis. Finally, using a CRISPR-mediated mutagenesis approach, we demonstrate that the refinement of nasal retinal projections requires the adhesion molecule Contactin 2. Altogether, our study provides the first analysis of a topographic map maturing in real time in a live animal and opens new strategies for dissecting the molecular mechanisms underlying precise topographic mapping in vertebrates.

From whole organism to ultrastructure: progress in axonal imaging for decoding circuit development.

Since the pioneering work of Ramón y Cajal, scientists have sought to unravel the complexities of axon development underlying neural circuit formation. Micrometer-scale axonal growth cones navigate to targets that are often centimeters away. To reach their targets, growth cones react to dynamic environmental cues that change in the order of seconds to days. Proper axon growth and guidance are essential to circuit formation, and progress in imaging has been integral to studying these processes. In particular, advances in high- and super-resolution microscopy provide the spatial and temporal resolution required for studying developing axons. In this Review, we describe how improved microscopy has revolutionized our understanding of axonal development. We discuss how novel technologies, specifically light-sheet and super-resolution microscopy, led to new discoveries at the cellular scale by imaging axon outgrowth and circuit wiring with extreme precision. We next examine how advanced microscopy broadened our understanding of the subcellular dynamics driving axon growth and guidance. We finally assess the current challenges that the field of axonal biology still faces for imaging axons, and examine how future technology could meet these needs.

Trans-Axonal Signaling in Neural Circuit Wiring.

The development of neural circuits is a complex process that relies on the proper navigation of axons through their environment to their appropriate targets. While axon-environment and axon-target interactions have long been known as essential for circuit formation, communication between axons themselves has only more recently emerged as another crucial mechanism. Trans-axonal signaling governs many axonal behaviors, including fasciculation for proper guidance to targets, defasciculation for pathfinding at important choice points, repulsion along and within tracts for pre-target sorting and target selection, repulsion at the target for precise synaptic connectivity, and potentially selective degeneration for circuit refinement. This review outlines the recent advances in identifying the molecular mechanisms of trans-axonal signaling and discusses the role of axon-axon interactions during the different steps of neural circuit formation.

Syntaphilin-Mediated Docking of Mitochondria at the Growth Cone Is Dispensable for Axon Elongation In Vivo.

Mitochondria are abundantly detected at the growth cone, the dynamic distal tip of developing axons that directs growth and guidance. It is, however, poorly understood how mitochondrial dynamics relate to growth cone behavior in vivo, and which mechanisms are responsible for anchoring mitochondria at the growth cone during axon pathfinding. Here, we show that in retinal axons elongating along the optic tract in zebrafish, mitochondria accumulate in the central area of the growth cone and are occasionally observed in filopodia extending from the growth cone periphery. Mitochondrial behavior at the growth cone in vivo is dynamic, with mitochondrial positioning and anterograde transport strongly correlating with growth cone behavior and axon outgrowth. Using novel zebrafish mutant lines that lack the mitochondrial anchoring proteins Syntaphilin a and b, we further show that Syntaphilins contribute to mitochondrial immobilization at the growth cone. Syntaphilins are, however, not required for proper growth cone morphology and axon growth in vivo, indicating that Syntaphilin-mediated anchoring of mitochondria at the growth cone plays only a minor role in elongating axons.

Characterization of the caspase family in zebrafish.

First discovered for their role in mediating programmed cell death and inflammatory responses, caspases have now emerged as crucial regulators of other cellular and physiological processes including cell proliferation, differentiation, migration, and survival. In the developing nervous system, for instance, the non-apoptotic functions of caspases have been shown to play critical roles in the formation of neuronal circuits by regulating axon outgrowth, guidance and pruning. How caspase activity is spatially and temporally maintained at sub-lethal levels within cells remains however poorly understood, especially in vivo. Thanks to its transparency and accessibility, the zebrafish offers the unique ability to directly visualize caspase activation in vivo. Yet, detailed information about the caspase family in zebrafish is lacking. Here, we report the identification and characterization of 19 different caspase genes in zebrafish, and show that caspases have diverse expression profiles from cleavage to larval stages, suggesting highly specialized and/or redundant functions during embryonic development.

EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.

Heparan sulfate proteoglycans: a sugar code for vertebrate development?

Heparan sulfate proteoglycans (HSPGs) have long been implicated in a wide range of cell-cell signaling and cell-matrix interactions, both in vitro and in vivo in invertebrate models. Although many of the genes that encode HSPG core proteins and the biosynthetic enzymes that generate and modify HSPG sugar chains have not yet been analyzed by genetics in vertebrates, recent studies have shown that HSPGs do indeed mediate a wide range of functions in early vertebrate development, for example during left-right patterning and in cardiovascular and neural development. Here, we provide a comprehensive overview of the various roles of HSPGs in these systems and explore the concept of an instructive heparan sulfate sugar code for modulating vertebrate development.

Wnt/ß-catenin signaling is required for radial glial neurogenesis following spinal cord injury.

Spinal cord injury results in permanent sensorimotor loss in mammals, in part due to a lack of injury-induced neurogenesis. The regeneration of neurons depends upon resident neural progenitors, which in zebrafish persist throughout the central nervous system as radial glia. However the molecular mechanisms regulating spinal cord progenitors remain uncharacterized. Wnt/ß-catenin signaling is necessary for the regenerative response of multiple tissues in zebrafish as well as other vertebrates, but it is not known whether the pathway has a role in spinal cord regeneration. Here we show that spinal radial glia exhibit Wnt/ß-catenin activity as they undergo neurogenesis following transection. We then use Cre-mediated lineage tracing to label the progeny of radial glia and show that Wnt/ß-catenin signaling is required for progenitors to differentiate into neurons. Finally, we show that axonal regrowth after injury also requires Wnt/ß-catenin signaling, suggesting coordinated roles for the pathway in functional recovery. Our data thus establish Wnt/ß-catenin pathway activation as a necessary step in spinal cord regeneration.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

Analyzing the role of heparan sulfate proteoglycans in axon guidance in vivo in zebrafish.

One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly formed. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell-surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, relatively little is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins, including morpholino, DNA, or RNA injections.

Proteoglycan-mediated axon degeneration corrects pretarget topographic sorting errors.

Proper arrangement of axonal projections into topographic maps is crucial for brain function, especially in sensory systems. An important mechanism for map formation is pretarget axon sorting, in which topographic ordering of axons appears in tracts before axons reach their target, but this process remains poorly understood. Here, we show that selective axon degeneration is used as a correction mechanism to eliminate missorted axons in the optic tract during retinotectal development in zebrafish. Retinal axons are not precisely ordered during initial pathfinding but become corrected later, with missorted axons selectively fragmenting and degenerating. We further show that heparan sulfate is required non-cell-autonomously to correct missorted axons and that restoring its synthesis at late stages in a deficient mutant is sufficient to restore topographic sorting. These findings uncover a function for developmental axon degeneration in ordering axonal projections and identify heparan sulfate as a key regulator of that process.

Specific serine-proline phosphorylation and glycogen synthase kinase 3ß-directed subcellular targeting of stathmin 3/Sclip in neurons.

During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3ß both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3ß target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3ß induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.

Analyzing retinal axon guidance in zebrafish.

How neuronal connections are established during development is one of the most fascinating questions in the field of neurobiology. The zebrafish retinotectal system offers distinct advantages for studying axon guidance in an in vivo context. Its accessibility and the larva's transparency not only allow its direct visualization, but also facilitate experimental manipulations to address the mechanisms of its development. Here we describe methods for labeling and visualizing retinal axons in vivo, including transient expression of DNA constructs, injection of lipophilic dyes, and time-lapse imaging. We describe in detail the available transgenic lines for marking retinal ganglion cells (RGCs); a protocol for very precise lipophilic dye labeling; and a protocol for single cell electroporation of RGCs. We then describe several approaches for perturbing the retinotectal system, including morpholino or DNA injection; localized heat shock to induce misexpression of genes; a comprehensive list of known retinotectal mutants; and a detailed protocol for RGC transplants to test cell autonomy. These methods not only provide new ways for examining how retinal axons are guided by their environment, but also can be used to study other axonal tracts in the living embryo.

The microtubule network and neuronal morphogenesis: Dynamic and coordinated orchestration through multiple players.

Nervous system function and plasticity rely on the complex architecture of neuronal networks elaborated during development, when neurons acquire their specific and complex shape. During neuronal morphogenesis, the formation and outgrowth of functionally and structurally distinct axons and dendrites require a coordinated and dynamic reorganization of the microtubule cytoskeleton involving numerous regulators. While most of these factors act directly on microtubules to stabilize them or promote their assembly, depolymerization or fragmentation, others are now emerging as essential regulators of neuronal differentiation by controlling tubulin availability and modulating microtubule dynamics. In this review, we recapitulate how the microtubule network is actively regulated during the successive phases of neuronal morphogenesis, and what are the specific roles of the various microtubule-regulating proteins in that process. We then describe the specific signaling pathways and inter-regulations that coordinate the different activities of these proteins to sustain neuronal development in response to environmental cues.

SCLIP is crucial for the formation and development of the Purkinje cell dendritic arbor.

Cerebellar Purkinje cells elaborate one of the most complex dendritic arbors among neurons to integrate the numerous signals they receive from the cerebellum circuitry. Their dendritic differentiation undergoes successive, tightly regulated phases of development involving both regressive and growth events. Although many players regulating the late phases of Purkinje cell dendritogenesis have been identified, intracellular factors controlling earlier phases of dendritic development remain mostly unknown. In this study, we explored the biological properties and functions of SCLIP, a protein of the stathmin family, in Purkinje cell dendritic differentiation and cerebellum development. Unlike the other stathmins, SCLIP is strongly expressed in Purkinje cells during cerebellar development and accumulates in their dendritic processes at a critical period of their formation and outgrowth. To reveal SCLIP functions, we developed a lentiviral-mediated approach on cerebellar organotypic cultures to inhibit or increase its expression in Purkinje cells in their tissue environment. Depletion of SCLIP promoted retraction of the Purkinje cell primitive process and then prevented the formation of new dendrites at early stages of postnatal development. It also prevented their elongation and branching at later phases of differentiation. Conversely, SCLIP overexpression promoted dendritic branching and development. Together, our results demonstrate for the first time that SCLIP is crucial for both the formation and proper development of Purkinje cell dendritic arbors. SCLIP appears thus as a novel and specific factor that controls the early phases of Purkinje cell dendritic differentiation during cerebellum development.

Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting.

BACKGROUND INFORMATION: Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule-regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3'/RB3'' are localized to the Golgi and vesicle-like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. RESULTS: We show that their conserved N-terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle-like localization. Using palmitoylation-deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co-operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin-like domain) extension. CONCLUSIONS: Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.