Abstract:Floor-plate-derived extracellular signaling molecules, including canonical axon guidance cues of the Netrin family, control neuronal circuit organization. Despite the importance of the floor plate as an essential signaling center in the developing vertebrate central nervous system, no systematic approach to identify binding partners for floor-plate-expressed cell-surface and secreted proteins has been carried out. Here, we used a high-throughput assay to discover extracellular protein-protein interactions, whi… Show more
“…Using AVEXIS [ 6 ] we had previously determined that Vasna is an Islr2 ligand [ 47 ]. By screening an expanded library totaling 194 zebrafish receptor ectodomains [ 17 ], we confirmed the Islr2-Vasna interaction and additionally detected specific binding with its paralog, Vasnb (Fig. 5a ).…”
Section: Resultsmentioning
confidence: 62%
“…Both Islr2-Vasna and Islr2-Vasnb interactions were detected in both prey/bait orientations, demonstrating the specificity of the observed binding events (Fig. 5a ; [ 17 ]). Fig.…”
BackgroundIn the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline.ResultsHere, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the “glial knot” in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract.ConclusionsAlthough stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse.
“…Using AVEXIS [ 6 ] we had previously determined that Vasna is an Islr2 ligand [ 47 ]. By screening an expanded library totaling 194 zebrafish receptor ectodomains [ 17 ], we confirmed the Islr2-Vasna interaction and additionally detected specific binding with its paralog, Vasnb (Fig. 5a ).…”
Section: Resultsmentioning
confidence: 62%
“…Both Islr2-Vasna and Islr2-Vasnb interactions were detected in both prey/bait orientations, demonstrating the specificity of the observed binding events (Fig. 5a ; [ 17 ]). Fig.…”
BackgroundIn the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline.ResultsHere, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the “glial knot” in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract.ConclusionsAlthough stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse.
“…A more robust mechanism that has been proposed involves a contact-dependent switch: the contact between a growth cone and its intermediate target could induce a transcriptional change in the neuron and thus result in the insertion of guidance receptors in a precisely timed manner. This has been Gao et al, 2015;Ahmed et al, 2011 Slit (Robo) Plexin A1 identified as alternative Slit receptor Delloye-Bourgeois et al, 2015 Cis binding with RPTP69D turns DsCAM into Slit receptor Alavi et al, 2016…”
During nervous system development, neurons extend axons to reach their targets and form functional circuits. The faulty assembly or disintegration of such circuits results in disorders of the nervous system. Thus, understanding the molecular mechanisms that guide axons and lead to neural circuit formation is of interest not only to developmental neuroscientists but also for a better comprehension of neural disorders. Recent studies have demonstrated how crosstalk between different families of guidance receptors can regulate axonal navigation at choice points, and how changes in growth cone behaviour at intermediate targets require changes in the surface expression of receptors. These changes can be achieved by a variety of mechanisms, including transcription, translation, protein-protein interactions, and the specific trafficking of proteins and mRNAs. Here, I review these axon guidance mechanisms, highlighting the most recent advances in the field that challenge the textbook model of axon guidance.
“…A linker with 19 amino acid residues seems minimally required for separating the functional FN4 and FN5 domains in the context of cell-binding. To verify the difference in netrin-1 adhesion in vitro between the wild-type versions of the human long and short isoforms of DCC, an AVEXIS assay (Bushell et al, 2008 ; Gao et al, 2015 ) was then performed. A fragment of the long isoform of human DCC FN456 was inserted into the AVEXIS prey vector.…”
mentioning
confidence: 99%
“…Scale bar: 20 μm. (B) The in vitro binding of human DCC ectodomains and netrin-1s was examined using an Avexis assay (Bushell et al, 2008 ; Gao et al, 2015 ). Briefly, netrin was biotinylated and attached to a streptavidin plate.…”
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