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Fig. 1. The scaffold of axon tracts in the embryonic Xenopus forebrain. (A) The major axon tracts and nuclei of the Xenopus forebrain at 40 h post-fertilization are represented schematically to show the bilateral symmetry of the axon scaffold. The rostral end of the neural tube is curved about the cephalic flexure (c.f.). The dorsal surface of the diencephalon contains the epiphysis (epi). For abbreviations, see below. (B) Double-immunolabeling of control Xenopus forebrains for the NOC-2+ subpopulation of axons (red) and acetylated α-tubulin to show all axons (blue). Axons expressing both NOC-2 and acetylated α-tubulin appear pink. (C) Schematic representation of the trajectory of the NOC-2+ subpopulation of axons in one half of the Xenopus forebrain (arrows). This trajectory is bilaterally symmetrical. The axons in each brain hemisphere arise in the nPT and course within the AC, POC, SOT, ventral TPOC, VLT and VC. Scale bar = 60 μm. AC, anterior commissure; POC, post-optic commissure; nPT, nucleus of the presumptive telencephalon; nTPOC, nucleus of the tract of the post-optic commissure; SOT, supraoptic tract; TPOC, tract of the post-optic commissure; DVDT, dorsoventral diencephalic tract; epi, epiphysis; nTPC, nucleus of the tract of the posterior commissure, PC, posterior commissure; TPC, tract of the posterior commissure; VC, ventral commissure; VLT, ventral longitudinal tract.
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Fig. 2. Neogenin mRNA is expressed in the developing Xenopus forebrain. (A) The predicted structure of Xenopus Neogenin is shown schematically. Xenopus Neogenin contains an open reading frame of 4326 bp and encodes a protein of 1442 amino acids. It has four immunoglobulin-like (Ig) domains, six fibronectin type III (FnIII) domains, a transmembrane region (TM) and a cytoplasmic tail containing three highly conserved domains (P1�P3). The positions of three alternatively spliced exons are indicated (splice 1, bp 1273�1332; splice 2, bp 2534�2581; splice 3, bp 3653�3680). The inclusion of splice 3 (28 bp) imparts a frameshift and premature stop codon, resulting in a truncated protein. Xenopus Neogenin possesses an SQTG amino acid motif in its cytoplasmic tail, instead of the usual conserved C(C/T)TD motif for caspase-3 cleavage, suggesting that Neogenin is unlikely to be involved in caspase-3-mediated cell death in Xenopus. The region used for synthesis of antisense probe is identified. (B) Lateral view of Neogenin mRNA expression on a whole-mount stage 30�31 Xenopus embryo, dorsal to the top and rostral to the left. Neogenin expression is predominant in the head (asterisk) and spinal cord (arrowhead) and is also present in the gut and somites. (C) Lateral view of Neogenin mRNA expression in isolated CNS tissue from a stage 30�31 Xenopus embryo. The forebrain (fb), midbrain (mb), hindbrain (hb), forebrain�midbrain boundary (FMB) and midbrain�hindbrain boundary (MHB) are indicated. Neogenin expression (indicated by the dashed line) is strong in the dorsal forebrain and extends into the ventral midbrain. Expression is also strong in the ventral hindbrain. Neogenin is absent from the hypothalamus (h), epiphysis (e), dorsal midbrain, midbrain�hindbrain boundary and dorsal hindbrain. (D) Dorsorostral view of an isolated stage 30�31 Xenopus brain, dorsal to the top (indicated by the epiphysis (e)), showing strong expression of Neogenin extending from both sides of the midline to the lateral margins of the dorsal forebrain. The estimated position of the nPT relative to these zones of Neogenin expression is indicated (hatched circles), based on double-labeling experiments (see panel F). (E�F) Isolated CNS from stage 30�31 Xenopus embryos, double-labeled for Neogenin mRNA expression (blue) and the NOC-2+ subpopulation of forebrain axons (brown), revealing their spatial relationship. (E) Magnification of a region of the forebrain in lateral view, equivalent to that indicated by a box in panel C, double-labeled for Neogenin (blue) and NOC-2 (brown stain, which appears deep purple in this double label). Neogenin is expressed in the nucleus of the presumptive telencephalon (nPT, indicated by the dashed circle) and in the neuroepithelium underlying the supraoptic tract (SOT) and the longitudinally oriented tract of the post-optic commissure (TPOC). The anterior commissure (AC) is indicated. (F) Dorsorostral view of a double-labeled brain, showing that Neogenin is expressed by the nPT (arrows). The AC is indicated at the rostral margin of the forebrain, and the epiphysis (e) is indicated at the dorsal surface of the brain. (G) Schematic representation of Neogenin mRNA expression in relation to the axon scaffold in the Xenopus forebrain. Scale bar in panel B = 500 μm, panels C�F = 60 μm.
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Fig. 3. Morpholino controls demonstrate efficacy of targeted knock down approach. Bright-field and fluorescent image analysis of stage 30-32 Xenopus embryos co-injected with plasmids encoding EGFP reporter constructs and morpholinos. Rostral is to the left and dorsal to the top in all panels. (A-B) Embryos injected with 5′Neo-EGFP/pCS2+ alone (230 ng) displayed strong mosaic expression of EGFP at stage 32 (40 hpf). (C-D) Embryos co-injected with an equivalent amount of 5′Neo-EGFP/pCS2+ and Neo-ATG MO (11.5 ng) failed to express EGFP. Embryos co-injected with the EGFP reporter plasmid and Neo-UTR MO had the same appearance. (E-F) Embryos co-injected with 5′Neo-EGFP/pCS2+ and a control nonsense MO (Std Control MO, 11.5 ng) showed strong mosaic EGFP expression. (G-H) Co-injection of 5′Neo-EGFP/pCS2+ with a morpholino targeted to an unrelated gene in Xenopus (Semaphorin3; Sema3-ATG MO, 11.5 ng) also produced embryos with strong mosaic expression of EGFP. Scale bar = 1 mm.
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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.
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Fig. 5. The Neogenin ligands RGMa and Netrin-1 are expressed in the developing Xenopus forebrain. In situ hybridization for RGMa (A�E) and Netrin-1 (G�K) was performed on whole-mount stage 30�31 embryos (A, G) and isolated stage 30�31 brains, indicated by dashed lines (B�C, H�I). All staining is bilaterally symmetrical. Combined in situ staining (blue) with immunostaining for the NOC-2+ subset of forebrain axons (brown) was performed to show the relationship between axon tract formation and mRNA expression (D�E, J�K). (A) In lateral view of a whole-mount Xenopus embryo, RGMa expression is detected in the brain, spinal cord somites and branchial arches (ba). (B) In lateral view of an isolated brain, strong RGMa expression is detected in the forebrain (fb), midbrain (mb) and hindbrain (hb). Expression extends rostrally in a band from the dorsal telencephalon, terminating in a ventral �wedge� (asterisk) at the rostral end of the forebrain. RGMa expression is absent from the trajectory of the TPOC (arrowhead) but a patch of expression is detected in the ventral forebrain, dorsal to the hypothalamus (h). RGMa is expressed throughout the ventral half of the midbrain and hindbrain, extending in dorsal peaks at the forebrain�midbrain boundary and midbrain�hindbrain boundary (arrows). (C) In dorsorostral view (epiphysis (e) indicates the dorsal surface of the brain), RGMa expression in the forebrain is detected along the midline and in the neuroepithelium medial to the estimated position of the nPT (hatched circles, compare to double-labeled image in panel E). The rostroventral wedge of RGMa expression in the forebrain (asterisks) extends laterally. Lateral (D) and dorsorostral views (E) of brains double-labeled for NOC-2 shows that RGMa expression is absent from the pathway underlying the nPT, AC and TPOC. The SOT forms along the border of the rostroventral �wedge� of RGMa expression, then grows into a region in devoid of RGMa (unfilled arrowhead in panel D). (G) In lateral view of a whole-mount Xenopus embryo, Netrin-1 is detected in a distinct patch in the brain (arrow) and throughout the ventral CNS (arrowhead). Expression is also detected in the ventral retina and the branchial arches (ba). (H) Netrin-1 expression is strong throughout the ventral forebrain, midbrain and hindbrain. A distinct patch of expression is also present in the dorsal forebrain (arrow). (I) A dorsorostral view shows that Netrin-1 expression in the dorsal forebrain (arrows) is located medially, close to the midline. The estimated positions of the nPT are indicated (hatched circles, compare to double-labeled image in panel K). The epiphysis (e) denotes the dorsal surface of the brain. (I) Double-labeling in lateral (J) and dorsorostral view (K) shows that Netrin-1 expression lies medial to the nPT, and underlies the TPOC (arrowheads). (F, L) Schematic representations of the expression of RGMa and Netrin-1 relative to the axon scaffold is consistent with their potential interaction with Neogenin as ligand-receptor pairs in the guidance of nPT axons to form the SOT (unfilled arrowhead). Scale bar in panel G = 500 μm and also applies to panel A, scale bar in panel J = 60 μm and applies to panels B, D, H, scale bar in panel K = 60 μm and applies to panels C, E, I.
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Fig. 6. Knock down of either RGMa or Netrin-1 phenocopies the SOT defect arising from knock down of Neogenin in vivo. All panels show lateral views of dissected brains from stage 32 embryos, dorsal to the top, rostral to the left, immunolabeled for the NOC-2+ subpopulation of axons unless otherwise indicated. Embryos were co-injected with EGFP mRNA and MOs against RGMa (A�D) or Netrin-1 (E�H). (A, C�D) Three examples of abnormal phenotypes observed after RGMa knock down. The SOT is reduced (A, C) or fails to completely project between the nPT and the TPOC (C�D, arrowhead). The unfilled arrowheads show single axons that have navigated the SOT. Axons from the nPT project aberrantly in the dorsal forebrain (A, arrows). (B) The same brain as in panel A, double-labeled for NOC-2 (red) and acetylated α-tubulin (blue) shows that the axon scaffold is grossly normal. The dashed line indicates the margins of the brain. (E, G�H) Three examples of abnormal phenotypes observed after knock down of Netrin-1. The SOT is reduced (H, unfilled arrowhead) or fails to form (E, G). There are also defects in the formation of the ventral commissure (F, H, arrows). (F) Magnification of the boxed area in panel E showing VC defects. Several axons appear to loop back towards the ipsilateral TPOC (arrows) and fail to cross the midline. The dashed line in panel F indicates the ventral margin of the brain. For control phenotype, see Fig. 4A. Scale bar = 30 μm; scale bar in panel H applies to panels A�E, G�H.
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Fig. 7. Simultaneous partial knock down of Neogenin with either RGMa or Netrin-1 shows genetic interaction in vivo. All panels show lateral views of dissected brains from stage 32 embryos, dorsal to the top, rostral to the left, immunolabeled for the NOC-2+ subpopulation of axons. (A) Animal injected with a reduced dose of Neo-ATG MO (Part-Control), showing normal axon pathways. Embryos injected with a reduced dose of either RGM-ATG MO or Netrin-ATG MO had the same appearance. (B, C) Two examples of abnormal phenotypes seen in animals injected with a combination of MOs to elicit partial knock down of both Neogenin and RGMa. The SOT is reduced (B, unfilled arrowhead) or fails to form (C) and there are abnormal caudoventrally directed nPT axons in the dorsal forebrain (B, arrows). (D, E) Two examples of abnormal phenotypes seen in animals injected with a combination of MOs to elicit simultaneous partial knock downs of Neogenin and Netrin-1. The SOT is reduced or fails to form correctly (D, filled arrowheads in panel E), and there are abnormal caudoventrally directed nPT axons in the dorsal forebrain (arrow in panel E). (F) Animal injected with a combination of MOs to elicit simultaneous partial knock down of Neogenin and an unrelated ligand, Semaphorin3, showing normal axon pathways (compare the phenotype to panel A). Scale bar = 30 μm.
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Fig. 8. Proposed model for axon guidance in the supraoptic tract, mediated by interactions between Neogenin, RGMa and Netrin-1. The Neogenin receptor is expressed by a subset of neurons in the nPT (blue). Axons exiting the nPT are funneled into the SOT via a wedge of the chemorepulsive ligand RGMa (red −), expressed in a stripe of neuroepithelium lying ventral and rostral to the tract. Axons are also simultaneously attracted ventrally by a chemoattractive gradient of Netrin-1 expression underlying the TPOC (green +). When Neogenin, RGMa or Netrin-1 are knocked down, the SOT fails to form correctly and nPT axons follow abnormal trajectories.
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neo1 (neogenin 1)gene expression in Xenopus laevis Borealis embryos, NF stage 32, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
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ntn1 (netrin 1) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
Key: arrow points to telencephalon; arrowhead to expression in the spinal cord floor plate.
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rgma (RGM domain family, member A)gene expression in Xenopus laevis Borealis embryos, NF stage 32, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
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