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. 2012 Apr 26;8(4):e1002665. doi: 10.1371/journal.pgen.1002665

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Demarco et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) A diagram illustrating attractive axon guidance via the UNC-40/DCC receptor. In the absence of UNC-6/Netrin ligand, the extracellular domains of UNC-40 prevent dimerization of the cytoplasmic regions and activation of receptor signal transduction. A myristoylated version of the cytoplasmic domain lacking the transmembrane domain and extracellular domains is believed to constitutively dimerize and is constitutively active, resulting in ectopic protrusion from the AVM neuron. Ig = immunoglobulin domain; FNIII = fibronectin type II domain; TM = transmembrane domain; P1–P3 = conserved proline-rich domains; MYR = covalent myristoyl group. (B) A wild-type AVM neuron visualized with soluble GFP from a mec-4::gfp transgene, with an arrow pointing to the unbranched ventrally-directed axon. The dashed line indicates the ventral nerve cord. (C) An AVM neuron with expression of unc-86p::myr::unc-40, a transgene that drives a myristoylated version of the cytoplasmic domain of UNC-40 in AVM. Fluorescence is soluble GFP expressed from mec-4::gfp to highlight cell shape. A large arrow points to an ectopic lamellipodium, and a smaller arrow points to a misguided and branched AVM axon. In all micrographs, dorsal is up and anterior is to the left. The scale bar represents 5 µm for B and C. (D) A graph charting the percentage of AVM axons with ectopic lamellipodial protrusions (X axis) in different genotypes (Y axis). Unless otherwise noted, all backgrounds are wild type. [myr::unc-40] represents animals harboring a transgene that expresses the myristoylated UNC-40 cytoplasmic domain under the mec-7 promoter (mec-7p::myr::unc-40). M+ indicates wild-type maternal gene function. Alone, none of the single mutants tiam-1(ok772), cdc-42(gk388M+), or unc-73(rh40) displayed ectopic protrusions. (E) A graph plotting the percentage of animals with AVM ventral axon guidance defects in different genotypes. Reported here are defects in AVM guidance that include failure of the axon tom reach the ventral nerve cord and axon wandering. If we just consider failure to reach the ventral nerve cord, similar significant results are found (unc-40 and tiam-1 enhance unc-6(ev400), and tiam-1 does not enhance unc-40). At least 100 AVM neurons were scored for each genotype, and p value significance was determined using Fisher's Exact analysis. Error bars represent 2× standard error of the proportion in both directions. Dashed lines indicate comparisons between genotypes not marked with a p value to those marked with p values. NS = not significantly different.