The Stripe Assay: Studying Growth Preference and Axon Guidance on Binary Choice Substrates In Vitro

AbstractThe Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well as in vitro structural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. Here, we define the minimal ectodomain structure required for Slit binding and midline repulsive signaling in vivo by Drosophila Robo1. We find that the majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. We show that a significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and that the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, we find that deleting four out of five Robo1 Ig domains (ΔIg2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats (ΔFn1-3 and ΔIg2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1’s Fn repeats. Our results provide an in vivo perspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observed in vitro for domains other than Ig1 may have limited significance for in vivo signaling in the context of midline repulsion.

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Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

eLife ◽

10.7554/elife.06585 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 36

Author(s):

Scott D Hansen ◽

R Dyche Mullins

Keyword(s):

Axon Guidance ◽

Actin Filaments ◽

Network Formation ◽

Focal Adhesions ◽

Leading Edge ◽

Polymerase Activity ◽

Actin Assembly ◽

Filament Assembly

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

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Nell2 regulates the contralateral-versus-ipsilateral visual projection as a layer-specific positional cue

10.1101/392597 ◽

2018 ◽

Author(s):

Chizu Nakamoto ◽

Elaine Durward ◽

Masato Horie ◽

Masaru Nakamoto

Keyword(s):

Axon Guidance ◽

Dorsal Lateral Geniculate Nucleus ◽

Retinal Ganglion ◽

Guidance Cue ◽

Retinal Axons ◽

Visual Projection ◽

Summary Statement ◽

Retinogeniculate Projection ◽

Dorsal Lateral Geniculate

SUMMARY STATEMENTNell2 is an ipsilateral layer-specific axon guidance cue in the visual thalamus and contributes to establishment of the eye-specific retinogeniculate projection by specifically inhibiting contralateral retinal axons.ABSTRACTIn mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific layers in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN). Although layer-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specificretinogeniculate projection, the molecular nature of such cues has remained elusive. Here we show that the extracellular glycoprotein Nell2 (also known as Nel) is expressed in the dorsomedial region of the dLGN, which corresponds to the layer receiving ipsilateral RGC axons. In Nell2 mutant mice, contralateral RGC axons invaded the ipsilateral layer of the dLGN, and ipsilateral axons terminated in partially fragmented patches, forming a mosaic pattern of contralateral and ipsilateral axon termination zones. In vitro, Nell2 exerted inhibitory effects on contralateral, but not ipsilateral, RGC axons. These results provide evidence that Nell2 acts as a layer-specific positional label in the dLGN that discriminates contralateral and ipsilateral RGC axons, and that it plays essential roles in establishment of the eye-specific projection patterns in the retinogeniculate system.

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RNA-based mechanisms underlying axon guidance

The Journal of Cell Biology ◽

10.1083/jcb.201305139 ◽

2013 ◽

Vol 202 (7) ◽

pp. 991-999 ◽

Cited By ~ 25

Author(s):

Toshiaki Shigeoka ◽

Bo Lu ◽

Christine E. Holt

Keyword(s):

Axon Guidance ◽

Translational Control ◽

Growth Cones ◽

Receptor Activation ◽

Neural Pathways ◽

Neuronal Circuitry ◽

Guidance Cues ◽

Spatiotemporal Control

Axon guidance plays a key role in establishing neuronal circuitry. The motile tips of growing axons, the growth cones, navigate by responding directionally to guidance cues that pattern the embryonic neural pathways via receptor-mediated signaling. Evidence in vitro in the last decade supports the notion that RNA-based mechanisms contribute to cue-directed steering during axon guidance. Different cues trigger translation of distinct subsets of mRNAs and localized translation provides precise spatiotemporal control over the growth cone proteome in response to localized receptor activation. Recent evidence has now demonstrated a role for localized translational control in axon guidance decisions in vivo.

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Topographically Specific Effects of ELF-1 on Retinal Axon Guidance In Vitro and Retinal Axon Mapping In Vivo

Cell ◽

10.1016/s0092-8674(00)80150-6 ◽

1996 ◽

Vol 86 (5) ◽

pp. 755-766 ◽

Cited By ~ 289

Author(s):

Masaru Nakamoto ◽

Hwai-Jong Cheng ◽

Glenn C Friedman ◽

Todd McLaughlin ◽

Michael J Hansen ◽

...

Keyword(s):

Axon Guidance ◽

Specific Effects ◽

Retinal Axon

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SEMAPHORIN 3A - A NEW INTRATHYMIC SUPPRESSOR FACTOR

Medical academic journal ◽

10.17816/maj191s1203-205 ◽

2019 ◽

Vol 19 (1S) ◽

pp. 203-205

Author(s):

K V Rutto ◽

E P Kisseleva

Keyword(s):

Immune Responses ◽

Axon Guidance ◽

Cell Activity ◽

Thymic Epithelial Cells ◽

Neuronal System ◽

Semaphorin 3A ◽

Therapeutic Tool ◽

Suppressor Factor ◽

Immunosuppressive Factor

Semaphorins were originally identified as axon guidance factors involved in the development of the neuronal system. However, accumulating evidence indicates that several semaphorins, so-called ‘immune semaphorins’, are also involved in various phases of immune responses. One of such factors is semaphorin 3A - a member of class 3 semaphorins, which are secretory molecules in vertebrates. There are multiple mechanisms involved in the process of semaphorin 3A-mediated regulation. One of them is down-regulation of peripheral T-cell activity in consequence of which semaphorin 3A is considered as an immunosuppressive factor. But semaphorin 3A is also expressed in the thymus while its function there remains obscure. Here are discussed new data on immunosuppressive function of this factor towards thymocytes and thymic epithelial cells, obtained in vitro. Because it is involved both in physiological immunoregulation and in the pathogenesis of many autoimmune, atopic, and malignant diseases, semaphorin 3A turns to be a promising therapeutic tool to be studied and applied in these diseases.

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RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons

Development ◽

10.1242/dev.128.20.3963 ◽

2001 ◽

Vol 128 (20) ◽

pp. 3963-3974 ◽

Cited By ~ 4

Author(s):

Hideki Enomoto ◽

Peter A. Crawford ◽

Alexander Gorodinsky ◽

Robert O. Heuckeroth ◽

Eugene M. Johnson ◽

...

Keyword(s):

Axon Guidance ◽

Blood Vessels ◽

Sympathetic Neurons ◽

Sympathetic Innervation ◽

Axonal Growth ◽

Initial Contact ◽

Neuronal Precursors ◽

Sympathetic Nervous ◽

Deficient Mice

Sympathetic axons use blood vessels as an intermediate path to reach their final target tissues. The initial contact between differentiating sympathetic neurons and blood vessels occurs following the primary sympathetic chain formation, where precursors of sympathetic neurons migrate and project axons along or toward blood vessels. We demonstrate that, in Ret-deficient mice, neuronal precursors throughout the entire sympathetic nervous system fail to migrate and project axons properly. These primary deficits lead to mis-routing of sympathetic nerve trunks and accelerated cell death of sympathetic neurons later in development. Artemin is expressed in blood vessels during periods of early sympathetic differentiation, and can promote and attract axonal growth of the sympathetic ganglion in vitro. This analysis identifies RET and artemin as central regulators of early sympathetic innervation.

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