Target-selective vertebrate motor axon regeneration depends on interaction with glial cells at a peripheral nerve plexus.

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from 3 nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real-time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

Integration of cooperative and opposing molecular programs drives learning-associated behavioral plasticity.

Habituation is a foundational learning process critical for animals to adapt their behavior to changes in their sensory environment. Although habituation is considered a simple form of learning, the identification of a multitude of molecular pathways including several neurotransmitter systems that regulate this process suggests an unexpected level of complexity. How the vertebrate brain integrates these various pathways to accomplish habituation learning, whether they act independently or intersect with one another, and whether they act via divergent or overlapping neural circuits has remained unclear. To address these questions, we combined pharmacogenetic pathway analysis with unbiased whole-brain activity mapping using the larval zebrafish. Based on our findings, we propose five distinct molecular modules for the regulation of habituation learning and identify a set of molecularly defined brain regions associated with four of the five modules. Moreover, we find that in module 1 the palmitoyltransferase Hip14 cooperates with dopamine and NMDA signaling to drive habituation, while in module 3 the adaptor protein complex subunit Ap2s1 drives habituation by antagonizing dopamine signaling, revealing two distinct and opposing roles for dopaminergic neuromodulation in the regulation of behavioral plasticity. Combined, our results define a core set of distinct modules that we propose act in concert to regulate habituation-associated plasticity, and provide compelling evidence that even seemingly simple learning behaviors in a compact vertebrate brain are regulated by a complex and overlapping set of molecular mechanisms.

Zebrafish behavior as a gateway to nervous system assembly and plasticity.

Nervous system assembly relies on a diversity of cellular processes ranging from dramatic tissue reorganization to local, subcellular changes all driven by precise molecular programs. Combined, these processes culminate in an animal's ability to plan and execute behaviors. Animal behavior can, therefore, serve as a functional readout of nervous system development. Benefitting from an expansive and growing set of molecular and imaging tools paired with an ever-growing number of assays of diverse behaviors, the zebrafish system has emerged as an outstanding platform at the intersection of nervous system assembly, plasticity and behavior. Here, we summarize recent advancements in the field, including how developing neural circuits are refined to shape complex behaviors and plasticity.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

Mitochondrial proteins encoded by the 22q11.2 neurodevelopmental locus regulate neural stem and progenitor cell proliferation.

Microdeletion of a 3Mb region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes individuals to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha, encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Single mutant analysis shows that both mrpl40 and prodha mutants display aberrant neural stem and progenitor cell proliferation, with each gene regulating distinct cell populations. Finally, double mutants for both mrpl40 and prodha display aggravated behavioral phenotypes and neural stem and progenitor cell analysis reveals a previously unrecognized partially redundant role for mrpl40 and prodha in regulating radial glia-like cell proliferation. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1.

The acoustic startle response is an evolutionarily conserved avoidance behavior. Disruptions in startle behavior, particularly startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. We uncovered mutations in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 and Dolk play critical roles in the spinal cord to regulate movement magnitude during the startle response and spontaneous swim movements. Moreover, we show that Kv1.1 protein is mislocalized in dolk mutants, suggesting they act in a common genetic pathway. Combined, our results identify a diverse set of eight genes, all associated with human disorders, that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway.

Diverging roles for Lrp4 and Wnt signaling in neuromuscular synapse development during evolution.

Motor axons approach muscles that are prepatterned in the prospective synaptic region. In mice, prepatterning of acetylcholine receptors requires Lrp4, a LDLR family member, and MuSK, a receptor tyrosine kinase. Lrp4 can bind and stimulate MuSK, strongly suggesting that association between Lrp4 and MuSK, independent of additional ligands, initiates prepatterning in mice. In zebrafish, Wnts, which bind the Frizzled (Fz)-like domain in MuSK, are required for prepatterning, suggesting that Wnts may contribute to prepatterning and neuromuscular development in mammals. We show that prepatterning in mice requires Lrp4 but not the MuSK Fz-like domain. In contrast, prepatterning in zebrafish requires the MuSK Fz-like domain but not Lrp4. Despite these differences, neuromuscular synapse formation in zebrafish and mice share similar mechanisms, requiring Lrp4, MuSK, and neuronal Agrin but not the MuSK Fz-like domain or Wnt production from muscle. Our findings demonstrate that evolutionary divergent mechanisms establish muscle prepatterning in zebrafish and mice.

Robo2 Drives Target-Selective Peripheral Nerve Regeneration in Response to Glia-Derived Signals.

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration.SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets.

Dynein promotes sustained axonal growth and Schwann cell remodeling early during peripheral nerve regeneration.

Following injury, axons of the peripheral nervous system have retained the capacity for regeneration. While it is well established that injury signals require molecular motors for their transport from the injury site to the nucleus, whether kinesin and dynein motors play additional roles in peripheral nerve regeneration is not well understood. Here we use genetic mutants of motor proteins in a zebrafish peripheral nerve regeneration model to visualize and define in vivo roles for kinesin and dynein. We find that both kinesin-1 and dynein are required for zebrafish peripheral nerve regeneration. While loss of kinesin-1 reduced the overall robustness of axonal regrowth, loss of dynein dramatically impaired axonal regeneration and also reduced injury-induced Schwann cell remodeling. Chimeras between wild type and dynein mutant embryos demonstrate that dynein function in neurons is sufficient to promote axonal regrowth. Finally, by simultaneously monitoring actin and microtubule dynamics in regenerating axons we find that dynein appears dispensable to initiate axonal regrowth, but is critical to stabilize microtubules, thereby sustaining axonal regeneration. These results reveal two previously unappreciated roles for dynein during peripheral nerve regeneration, initiating injury induced Schwann cell remodeling and stabilizing axonal microtubules to sustain axonal regrowth.

Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

Zebrafish foxc1a drives appendage-specific neural circuit development.

Neural connectivity between the spinal cord and paired appendages is key to the superior locomotion of tetrapods and aquatic vertebrates. In contrast to nerves that innervate axial muscles, those innervating appendages converge at a specialized structure, the plexus, where they topographically reorganize before navigating towards their muscle targets. Despite its importance for providing appendage mobility, the genetic program that drives nerve convergence at the plexus, as well as the functional role of this convergence, are not well understood. Here, we show that in zebrafish the transcription factor foxc1a is dispensable for trunk motor nerve guidance but is required to guide spinal nerves innervating the pectoral fins, equivalent to the tetrapod forelimbs. In foxc1a null mutants, instead of converging with other nerves at the plexus, pectoral fin nerves frequently bypass the plexus. We demonstrate that foxc1a expression in muscle cells delineating the nerve path between the spinal cord and the plexus region restores convergence at the plexus. By labeling individual fin nerves, we show that mutant nerves bypassing the plexus enter the fin at ectopic positions, yet innervate their designated target areas, suggesting that motor axons can select their appropriate fin target area independently of their migration through the plexus. Although foxc1a mutants display topographically correct fin innervation, mutant fin muscles exhibit a reduction in the levels of pre- and postsynaptic structures, concomitant with reduced pectoral fin function. Combined, our results reveal foxc1a as a key player in the development of connectivity between the spinal cord and paired appendages, which is crucial for appendage mobility.

SNPfisher: tools for probing genetic variation in laboratory-reared zebrafish.

Single nucleotide polymorphisms (SNPs) are the benchmark molecular markers for modern genomics. Until recently, relatively few SNPs were known in the zebrafish genome. The use of next-generation sequencing for the positional cloning of zebrafish mutations has increased the number of known SNP positions dramatically. Still, the identified SNP variants remain under-utilized, owing to scant annotation of strain specificity and allele frequency. To address these limitations, we surveyed SNP variation in three common laboratory zebrafish strains using whole-genome sequencing. This survey identified an average of 5.04 million SNPs per strain compared with the Zv9 reference genome sequence. By comparing the three strains, 2.7 million variants were found to be strain specific, whereas the remaining variants were shared among all (2.3 million) or some of the strains. We also demonstrate the broad usefulness of our identified variants by validating most in independent populations of the same laboratory strains. We have made all of the identified SNPs accessible through 'SNPfisher', a searchable online database (snpfisher.nichd.nih.gov). The SNPfisher website includes the SNPfisher Variant Reporter tool, which provides the genomic position, alternate allele read frequency, strain specificity, restriction enzyme recognition site changes and flanking primers for all SNPs and Indels in a user-defined gene or region of the zebrafish genome. The SNPfisher site also contains links to display our SNP data in the UCSC genome browser. The SNPfisher tools will facilitate the use of SNP variation in zebrafish research as well as vertebrate genome evolution.

Schwann cells and deleted in colorectal carcinoma direct regenerating motor axons towards their original path.

After complete nerve transection, a major challenge for regenerating peripheral axons is to traverse the injury site and navigate toward their original trajectory. Denervated Schwann cells distal to the lesion site secrete factors promoting axonal growth and serve as an axonal substrate, yet whether Schwann cells also actively direct axons toward their original trajectory is unclear. Using live-cell imaging in zebrafish, we visualize for the first time how in response to nerve transection distal Schwann cells change morphology as axons fragment, and how Schwann cell morphology reverses once regenerating growth cones have crossed the injury site and have grown along distal Schwann cells outlining the original nerve path. In mutants lacking Schwann cells, regenerating growth cones extend at rates comparable with wild type yet frequently fail to cross the injury site and instead stray along aberrant trajectories. Providing growth-permissive yet Schwann cell-less scaffolds across the injury site was insufficient to direct regenerating growth cones toward the original path, providing compelling evidence that denervated Schwann cells actively direct regenerating axons across the injury site toward their original trajectory. To identify signals that guide regenerating axons in vivo, we examined mutants lacking the deleted in colorectal carcinoma (DCC) guidance receptor. In these dcc mutants, a significant fraction of regenerating motor axons extended along aberrant trajectories, similar to what we observe in mutants lacking Schwann cells. Thus, Schwann cell and dcc-mediated guidance are critical early during regeneration to direct growth cones across the transection gap and onto their original axonal trajectory.

Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

Temporal requirement for SMN in motoneuron development.

Proper function of the motor unit is dependent upon the correct development of dendrites and axons. The infant/childhood onset motoneuron disease spinal muscular atrophy (SMA), caused by low levels of the survival motor neuron (SMN) protein, is characterized by muscle denervation and paralysis. Although different SMA models have shown neuromuscular junction defects and/or motor axon defects, a comprehensive analysis of motoneuron development in vivo under conditions of low SMN will give insight into why the motor unit becomes dysfunctional. We have generated genetic mutants in zebrafish expressing low levels of SMN from the earliest stages of development. Analysis of motoneurons in these mutants revealed motor axons were often shorter and had fewer branches. We also found that motoneurons had significantly fewer dendritic branches and those present were shorter. Analysis of motor axon filopodial dynamics in live embryos revealed that mutants had fewer filopodia and their average half-life was shorter. To determine when SMN was needed to rescue motoneuron development, SMN was conditionally induced in smn mutants during embryonic stages. Only when SMN was added back soon after motoneurons were born, could later motor axon development be rescued. Importantly, analysis of motor behavior revealed that animals with motor axon defects had significant deficits in motor output. We also show that SMN is required earlier for motoneuron development than for survival. These data support that SMN is needed early in development of motoneuron dendrites and axons to develop normally and that this is essential for proper connectivity and movement.

Initiation of synapse formation by Wnt-induced MuSK endocytosis.

In zebrafish, the MuSK receptor initiates neuromuscular synapse formation by restricting presynaptic growth cones and postsynaptic acetylcholine receptors (AChRs) to the center of skeletal muscle cells. Increasing evidence suggests a role for Wnts in this process, yet how muscle cells respond to Wnt signals is unclear. Here, we show that in vivo, wnt11r and wnt4a initiate MuSK translocation from muscle membranes to recycling endosomes and that this transition is crucial for AChR accumulation at future synaptic sites. Moreover, we demonstrate that components of the planar cell polarity pathway colocalize to recycling endosomes and that this localization is MuSK dependent. Knockdown of several core components disrupts MuSK translocation to endosomes, AChR localization and axonal guidance. We propose that Wnt-induced trafficking of the MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis.

The tumor suppressor gene retinoblastoma-1 is required for retinotectal development and visual function in zebrafish.

Mutations in the retinoblastoma tumor suppressor gene (rb1) cause both sporadic and familial forms of childhood retinoblastoma. Despite its clinical relevance, the roles of rb1 during normal retinotectal development and function are not well understood. We have identified mutations in the zebrafish space cadet locus that lead to a premature truncation of the rb1 gene, identical to known mutations in sporadic and familial forms of retinoblastoma. In wild-type embryos, axons of early born retinal ganglion cells (RGC) pioneer the retinotectal tract to guide later born RGC axons. In rb1 deficient embryos, these early born RGCs show a delay in cell cycle exit, causing a transient deficit of differentiated RGCs. As a result, later born mutant RGC axons initially fail to exit the retina, resulting in optic nerve hypoplasia. A significant fraction of mutant RGC axons eventually exit the retina, but then frequently project to the incorrect optic tectum. Although rb1 mutants eventually establish basic retinotectal connectivity, behavioral analysis reveals that mutants exhibit deficits in distinct, visually guided behaviors. Thus, our analysis of zebrafish rb1 mutants reveals a previously unknown yet critical role for rb1 during retinotectal tract development and visual function.

A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration.

Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.

Chemical modulation of memory formation in larval zebrafish.

Whole organism-based small-molecule screens have proven powerful in identifying novel therapeutic chemicals, yet this approach has not been exploited to identify new cognitive enhancers. Here we present an automated high-throughput system for measuring nonassociative learning behaviors in larval zebrafish. Using this system, we report that spaced training blocks of repetitive visual stimuli elicit protein synthesis-dependent long-term habituation in larval zebrafish, lasting up to 24 h. Moreover, repetitive acoustic stimulation induces robust short-term habituation that can be modulated by stimulation frequency and instantaneously dishabituated through cross-modal stimulation. To characterize the neurochemical pathways underlying short-term habituation, we screened 1,760 bioactive compounds with known targets. Although we found extensive functional conservation of short-term learning between larval zebrafish and mammalian models, we also discovered several compounds with previously unknown roles in learning. These compounds included a myristic acid analog known to interact with Src family kinases and an inhibitor of cyclin dependent kinase 2, demonstrating that high-throughput chemical screens combined with high-resolution behavioral assays provide a powerful approach for the discovery of novel cognitive modulators.