Analysis of zebrafish sidetracked mutants reveals a novel role for Plexin A3 in intraspinal motor axon guidance

One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis we show that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, we show that sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, we show that sidetracked (plexin A3) plays an additional role in motor axonal morphogenesis. Together, our data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. We propose that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance.     

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Celsr3 and Fzd3 in axon guidance

The assembly of functional neuronal circuits depends on the correct wiring of axons and dendrites. To reach their targets, axons are guided by a variety of extracellular guidance cues, including Netrins, Ephrins, Semaphorins and Slits. Corresponding receptors in the growth cone, the dynamic structure at the tip of the growing axon, sense and integrate these positional signals, and activate downstream effectors to regulate cytoskeletal organization. In addition to the four canonical families of axon guidance cues mentioned above, some proteins that regulate planar cell polarity were recently found to be critical for axon guidance. The seven-transmembrane domain receptors Celsr3 and Fzd3, in particular, control the development of most longitudinal tracts in the central nervous system, and axon navigation in the peripheral, sympathetic and enteric nervous systems. Despite their unequivocally important role, however, underlying molecular mechanisms remain elusive. We do not know which extracellular ligands they recognize, whether they have co-receptors in the growth cone, and what their downstream effectors are. Here, we review some recent advances and discuss future trends in this emerging field.     

Severe developmental defects in Dictyostelium null mutants for actin-binding proteins

The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, cross-linking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, reflect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of alpha-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the efficiency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin null mutants.     

ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.     

Mouse Dach2 mutants do not exhibit gross defects in eye development or brain function

Drosophila dachshund is a critical regulator of eye, brain, and limb formation. Vertebrate homologs, Dach1 and Dach2, are expressed in the developing retina, brain, and limbs, suggesting functional conservation of the dachshund/Dach gene family. Dach1 mutants die postnatally, but exhibit grossly normal development. Here we report the generation of Dach2 mutant mice. Although deletion of Dach2 exon 1 results in abrogation of RNA expression, Dach2 mutants are viable and fertile. Histochemical analysis reveals grossly normal Dach2 mutant eye development. In addition, a battery of neurological assays failed to yield significant differences in behavior between Dach2 mutants and controls. We discuss these findings in the light of published observations of DACH2 mutations in the human population. Finally, to test the functional conservation hypothesis, we generated Dach2; Dach1 double mutant mice. Dach double mutants die after birth, similar to Dach1 homozygotes. However, unlike Drosophila dachshund mutants that lack eyes and exhibit leg truncations, the eyes and limbs of Dach double mutants are present, suggesting differences between Dach and dachshund gene function during embryonic eye and limb formation.     

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila

At specific choice points in the periphery, subsets of motor axons defasciculate from other axons in the motor nerves and steer into their muscle target regions. Using a large-scale genetic screen in Drosophila, we identified the sidestep (side) gene as essential for motor axons to leave the motor nerves and enter their muscle targets. side encodes a target-derived transmembrane protein (Side) that is a novel member of the immunoglobulin superfamily (IgSF). Side is expressed on embryonic muscles during the period when motor axons leave their nerves and extend onto these muscles. In side mutant embryos, motor axons fail to extend onto muscles and instead continue to extend along their motor nerves. Ectopic expression of Side results in extensive and prolonged motor axon contact with inappropriate tissues expressing Side.     

Ashkenazi Jewish centenarians do not demonstrate enrichment in mitochondrial haplogroup J

Background

Association of mitochondrial haplogroup J with longevity has been reported in several population subgroups. While studies from northern Italy and Finland, have described a higher frequency of haplogroup J among centenarians in comparison to non-centenarian, several other studies could not replicate these results and suggested various explanations for the discrepancy.

Methodology/Principal Findings

We have evaluated haplogroup frequencies among Ashkenazi Jewish centenarians using two different sets of matched controls. No difference was observed in the haplogroup J frequencies between the centenarians or either matched control group, despite adequate statistical power to detect such a difference. Furthermore, the lack of association was robust to population substructure in the Ashkenazi Jewish population. Given this discrepancy with the previous reported associations in the northern Italian and the Finnish populations, we conducted re-analysis of these previously published data, which supported one of several possible explanations: i) inadequate matching of cases and controls; ii) inadequate adjustment for multiple comparison testing; iii) cryptic population stratification.

Conclusions/Significance

There does not exist a universal association of mitochondrial haplogroup J with longevity across all population groups. Reported associations in specialized populations may reflect genetic or other interactions specific to those populations or else cryptic confounding influences, such as inadequate matching attributable to population substructure, which are of general relevance to all studies of the possible association of mitochondrial DNA haplogroups with common complex phenotypes.     

Loss of phosphatase activity in Ptp69D alleles supporting axon guidance defects

PTP69D is a receptor protein tyrosine phosphatase that was identified as a key regulator of neuromuscular axon guidance in Drosophila, and has subsequently been shown to play a similar role in the central nervous system and retina. Three Ptp69D alleles with mutations involving catalytically important residues exhibit a high degree of phenotypic variation with viability of mutant adult flies ranging from 0 to 96%, and ISNb motor nerve defects ranging from 11 to 57% [Desai and Purdy, 2003]. To determine whether mutations in Ptp69D affecting axon guidance and viability demonstrate losses of phosphatase activity and whether differences in catalytic potential underlie phenotypic variability, we expressed full-length wild-type and mutant PTP69D protein in Schneider 2 cells, and assessed phosphatase activity using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferone phosphate (DiFMUP). Detailed biochemical characterization of wild-type PTP69D, including an examination of sensitivity to various inhibitors, in vitro catalytic efficiency, and the pH-k(cat) profile of the enzyme, suggests a common tyrosine phosphatase reaction mechanism despite lack of sequence conservation in the WPD loop. Analysis of mutant proteins revealed that every mutant had less than 1% activity relative to the wild-type enzyme, and these rates did not differ significantly from one another. These results indicate that mutations in Ptp69D resulting in axon guidance defects and lethality significantly compromise catalytic activity, yet the range of biological activity exhibited by Ptp69D mutants cannot be explained by differences in catalytic activity, as gauged by their ability to hydrolyze the substrate DiFMUP.     

SNARE proteins play a role in motor axon guidance in vertebrates and invertebrates

Axonal growth and guidance rely on correct growth cone responses to guidance cues, both in the central nervous system (CNS) and in the periphery. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the cross‐talk mechanisms between guidance and membrane dynamics and turnover in the axon. Our studies have shown that Netrin‐1/deleted in colorectal cancer signaling triggers exocytosis through the SNARE Syntaxin‐1 (STX‐1) during the formation of commissural pathways. However, limited in vivo evidence is available about the role of SNARE proteins in motor axonal guidance. Here we show that loss‐of‐function of SNARE complex members results in motor axon guidance defects in fly and chick embryos. Knock‐down of Syntaxin‐1, VAMP‐2, and SNAP‐25 leads to abnormalities in the motor axon routes out of the CNS. Our data point to an evolutionarily conserved role of the SNARE complex proteins in motor axon guidance, thereby pinpointing an important function of SNARE proteins in axonal navigation in vivo . © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 963–974, 2017     

Ephrin-mediated cis-attenuation of Eph receptor signaling is essential for spinal motor axon guidance

Axon guidance receptors guide neuronal growth cones by binding in trans to axon guidance ligands in the developing nervous system. Some ligands are coexpressed in cis with their receptors, raising the question of the relative contribution of cis and trans interactions to axon guidance. Spinal motor axons use Eph receptors to select a limb trajectory in response to trans ephrins, while expressing ephrins in cis. We show that changes in motor neuron ephrin expression result in trajectory selection defects mirrored by changes in growth cone sensitivity to ephrins in vitro, arguing for ephrin cis-attenuation of Eph function. Furthermore, the relative contribution of trans-signaling and cis-attenuation is influenced by the subcellular distribution of ephrins to membrane patches containing Eph receptors. Thus, growth cone ephrins are essential for axon guidance in vivo and the balance between cis and trans modes of axon guidance ligand-receptor interaction contributes to the diversity of axon guidance signaling responses.     

A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila

Background: The neuromuscular system of the Drosophila larva contains a small number of identified motor neurons that make genetically defined synaptic connections with muscle fibers. We drove high-level expression of genes in these motor neurons by crossing 2293 GAL4-driven EP element lines with known insertion site sequences to lines containing a pan-neuronal GAL4 source and UAS-green fluorescent protein elements. This allowed visualization of every synapse in the neuromuscular system in live larvae.Results: We identified 114 EPs that generate axon guidance and/or synaptogenesis phenotypes in F1 EP x driver larvae. Analysis of genomic regions adjacent to these EPs defined 76 genes that exhibit neuromuscular gain-of-function phenotypes. Forty-one of these (known genes) have published mutant alleles; the other 35 (new genes) have not yet been characterized genetically. To assess the roles of the known genes, we surveyed published data on their phenotypes and expression patterns. We also examined loss-of-function mutants ourselves, identifying new guidance and synaptogenesis phenotypes for eight genes. At least three quarters of the known genes are important for nervous system development and/or function in wild-type flies.Conclusions: Known genes, new genes, and a set of previously analyzed genes with phenotypes in the Adh region display similar patterns of homology to sequences in other species and have equivalent EST representations. We infer from these results that most new genes will also have nervous system loss-of-function phenotypes. The proteins encoded by the 76 identified genes include GTPase regulators, vesicle trafficking proteins, kinases, and RNA binding proteins.     

Bulge defects do not destabilize negatively supercoiled DNA

The conformational properties of DNA lesions such as extrahelical bulges are presumed to be essential for recognition of defects in DNA structure by a cell's genomic repair machinery. Efficient recognition and repair of lesions by DNA-repair systems occurs despite the wide range of normal heterogeneities in DNA structure, including features such as sequence-dependent bends. The effects of global negative supercoiling on the structure of DNA lesions have been largely unexplored. We have investigated the behavior of several plasmid DNAs containing bulge defects with up to five extrahelical adenine residues. Using two-dimensional agarose-gel electrophoresis, we show that there is no spontaneous cooperative unwinding of these bulge loci up to native levels of negative supercoiling (sigma = -0.055) under our conditions.     

A chemoattractant role for NT-3 in proprioceptive axon guidance

Neurotrophin-3 (NT-3) is required for proprioceptive neuron survival. Deletion of the proapoptotic gene Bax in NT-3 knockout mice rescues these neurons and allows for examination of their axon growth in the absence of NT-3 signaling. TrkC-positive peripheral and central axons from dorsal root ganglia follow proper trajectories and arrive in close proximity to their targets but fail to innervate them. Peripherally, muscle spindles are absent and TrkC-positive axons do not enter their target muscles. Centrally, proprioceptive axons branch in ectopic regions of the spinal cord, even crossing the midline. In vitro assays reveal chemoattractant effects of NT-3 on dorsal root ganglion axons. Our results show that survival factor NT-3 acts as a short-distance axon guidance molecule for muscle sensory afferents as they approach their proper targets.