Axon tracts of the ventral nerve cord in <i>ptc</i> mutant embryos progressively narrow with age in an anterior-posterior direction.

<p>(A-D): Wild-type and <i>ptc</i> mutant embryos aged 10 and 15 hours of development were stained with Fas II (A, B) and BP102 (C, D) and analyzed with diamino benzidine (DAB) staining. Fas II stains medial (M), Intermediate (I) and Lateral (L) tracts, which become clearly defined by 15 hours of development. BP102 stains anterior commissure (AC), posterior commissure (PC) and longitudinal connective (LC). LC contains both the longitudinal axons and the portion of commissural axons that crosses the midline. Note the well-defined position of tracts along the nerve cord, parallel to the midline and to each other in wild-type. In older mutant embryos, the tracts progressively shift towards the midline. Scale bar: Panels A and C: 22 μm, panels B and D: 8 μm. (E-F): Wild-type and <i>ptc</i> mutant embryos were double stained with Fas II and Even-skipped (Eve) antibodies and analyzed with confocal microscopy. Eve was used as a position-marker. In panel E, 10 hpf and 14 hpf wild-type and <i>ptc</i> embryos are shown. While the newly formed individual tracts are well-defined in 10 hpf embryos, the tracts are defasciculated in 14 hpf <i>ptc</i> mutant embryos (arrow head). In pane F, 15 hpf embryos are shown. By 15 hours of development, the tracts in <i>ptc</i> show the funneling phenotype. In <i>ptc</i> mutant embryos axons inappropriately cross the midline, but this defect is related to the misspecification of neuronal identity [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007094#pgen.1007094.ref020" target="_blank">20</a>]. Scale bar: Panel E: 20 μm (upper images) and 8 μm (lower images), and Panel F: 8 μm. (G): Defasciculation in the anterior segments is less severe compared to the posterior segments in <i>ptc</i> embryos. Wild-type and <i>ptc</i> mutant embryos were double-stained for Fas II and Eve. In the anterior segments in <i>ptc</i> mutant, the tracts are much better defined compared to the posterior segments. Scale bar: 8 μm. All the images shown are representative from at least N = 3 independent observations unless otherwise stated.