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Fig. 4. Knock down of Neogenin or expression of dominant-negative Neogenin causes axon pathfinding errors. Embryos were co-injected with the Std Control MO and EGFP mRNA (A�C, J), Neogenin MOs and EGFP mRNA (D�I), (K�L) or dominant-negative Neogenin mRNA and EGFP mRNA (N�O). All are lateral views of dissected brains from stage 32 embryos, immunolabeled for the NOC-2+ subpopulation of axons unless otherwise indicated. Dorsal is to the top and rostral is to the left in all panels. (A�C) Injection of the Std Control MO does not affect the trajectory of the NOC-2+ subpopulation of axons. (A) The SOT appears normal (unfilled arrowhead), coursing ventrocaudally from the nPT in the dorsal forebrain to the ventral TPOC. The boxed region is magnified in panel J. (B) Double-labeling for NOC-2 (red) and acetylated α-tubulin (blue) for all axons reveals normal forebrain morphology (compare to Fig. 1B). The dashed line in panels A and B indicates the margins of the dissected brain. (C) Injected cells were traced by EGFP fluorescence (green). (D�I) Four examples of abnormal phenotypes observed after Neogenin knock down using Neo-ATG or Neo-UTR MOs, as indicated. The SOT is reduced in size or fails to form properly (arrowheads in panels D�I, boxed regions in panels H�I are magnified in panels K�L, respectively) and nPT axons are observed to follow aberrant trajectories in the dorsal forebrain (arrows in panels D�F, H�I, K�L). (E�F) The same brain as in panel D, double- or triple-labeled as in panels B and C, to show that the axon scaffold in Neogenin MO-injected animals is grossly normal. (J�L) Magnification of the boxed areas in panels A, D and I, respectively, to show the SOT phenotype in control (J) and Neogenin knock down animals (K�L). Loss of Neogenin either prevented some nPT axons from growing ventrally within the SOT (arrowheads in panels K�L, unfilled arrowhead in panel K shows a reduced SOT comprised of a single axon) or caused them to grow along abnormal longitudinal trajectories in the dorsal forebrain (arrows in panels K�L). (M) Schematic representation of the Neogenin truncation construct (DN-Neo) which lacked the cytoplasmic signaling domain. Embryos were injected with mRNA transcripts encoding DN-Neo and EGFP (N�O). Overexpression of DN-Neo caused defects in SOT formation, similar to that described for the Neogenin MO knock down animals. The SOT often failed to develop (arrowhead in panels N�O, compare to panel G). Co-labeling for acetylated α-tubulin (blue in panel O) showed that axon scaffold development was grossly normal in these animals. Scale bar = 30 μm. Scale bar in panel O applies in panels A�I, N; scale bar in panel L applies in panels J�K. |
