Leung LC , Harris WA , Holt CE , Piper M .

100329 Wellcome Trust , 085314/Z/08/Z Wellcome Trust , Medical Research Council , WT100329 Wellcome Trust , Wellcome Trust

	Fig 1. Electroporation of NFPC morpholinos disrupts RGC axon outgrowth in vivo. (A-D) A method for investigating intraretinal guidance. (A) RGCs were lipofected with either GAP-GFP or NFδE constructs at stage 19, or electroporated with control (Con-MO) or NFPC morpholinos (NFPC-MO) at stage 24. Embryos were then allowed to develop until stage 40, at which time eyes were dissected from the embryo. After removal of the lens, immunostaining for acetylated α-tubulin was performed to mark retinal axons. (B) Brightfield image of an uninjected eye with lens removed. (C) Fluorescence labelling of the same eye as in B, revealing expression of acetylated α-tubulin in retinal axons. (D) Magnified view of the boxed region in C, showing immunolabelled retinal axons coursing towards the optic disc. (E, F) Acetylated α-tubulin staining in a retina loaded with the Con-MO (E) and a retina loaded with the NFPC-MO (F). In all cases analyzed regions of interest are delineated by dashed lines. Immunostaining reveals that in all cases RGC axon bundles (arrows in E and F) form and are oriented towards the optic disc (double arrowheads in E and F). However, quantification of the number of RGC axon bundles per unit area of the retina (G) reveals significantly reduced number of axon bundles in NFPC-MO-loaded retinae in comparison to Con-MO-loaded eyes. Values were normalized against the Con-MO group and the number of retinae analysed are presented within the bars. * p <0.05, Kruskal-Wallis test. Scale bar in F: 75 μm (B, C), 30 μm (D), 40 μm (E, F). https://doi.org/10.1371/journal.pone.0141290.g001
	Fig 2. Cultured retinal growth cones loaded with NFPC morpholino do not exhibit chemoattraction to Netrin-1. (A-C) Phase contrast images of retinal neurites cultured from uninjected (A), control morpholino-loaded (Con-MO; B) and NFPC morpholino-loaded (NFPC-MO; C) retinae. Neurites from uninjected (A) and Con-MO-loaded (B) retinae exhibit robust turning towards the point source of Netrin-1. Neurites loaded with the NFPC-MO, however, are not attracted towards Netrin-1 (C). (D) Cumulative distributions of turning angles of each sample group. (E) Mean turning angles of the experimental groups reveals that, whereas uninjected and Con-MO-loaded neurites exhibit attraction to Netrin-1, NFPC-MO-loaded neurites do not. * p < 0.05, Kolmogorov-Smirnov test. Panel F shows a summary of the trajectory plots from the uninjected and NFPC-MO experimental groups exposed to Netrin-1. Each line represents a single growth cone trajectory; the origin represents the centre of the growth cone at 0 min, and positive (+ve) and negative (-ve) turning angles are indicated. Scale bar in A: 10 μm. https://doi.org/10.1371/journal.pone.0141290.g002
	Fig 3. Netrin-1 dynamically regulates NFPC in retinal growth cones. (A-D) Cultured stage 24 retinal neurites were stimulated with Netrin-1 or a vector-only control for 0 (A), 10 (B), 30 (C) or 60 (D) min, and then assayed for NFPC expression via immunofluorescence labelling. Panels E-H reveal the quantification of immunofluorescence as an indicator of total NFPC levels within the growth cone (open bars–vector only control; black bars–Netrin-1 treatment). In each case, data were normalized to the 0 min control treatment. (E) Netrin-1 induced a significant decrease in the levels of NFPC within the growth cone after 10 min. By 30 min, however, NFPC localized to the growth cone had returned to levels comparable to that in the control. (F) The decrease in NFPC immunoreactivity localized to the growth cone after 10 min was abolished when explants were pre-treated with the proteasomal inhibitors lactacystin (Lacta) or LLnL. (G) The decrease in NFPC localized to the growth cone after 10 min was also abolished when the explants were treated with the endocytosis inhibitors phenylarsine oxide (PAO) or monodansylcadaverine (MDC). (H) Blocking protein translation with anisomycin (Aniso) in isolated retinal neurites suppressed the recovery in growth cone NFPC levels seen after 30 min of Netrin-1 exposure. However, inhibition of transcription with α-amanatin (α-aman) did not prevent the recovery of growth cone NFPC levels. *** p < 0.001, Kruskal Wallis test. Numbers within the bars indicate the number of growth cones assayed. Scale bar in A: 5 μm. https://doi.org/10.1371/journal.pone.0141290.g003
	Fig 4. Perturbation of NFPC binding leads to tectum entry defects. Open brain embryos were incubated with Con-Fc (A) or NFPC-Fc (B) from stage 35. At stage 40, retinal axons were labelled with DiI. Brains were then dissected and mounted in the contralateral view to enable visualization of the optic tract. Inverse greyscale images show that the axon bundle trajectories of Con-Fc-treated brains appeared normal (A, higher magnification shown in A’), with RGC axons entering the tectum normally (the tectum is delineated with a dashed line in A’). Brains treated with the NFPC-Fc ectodomain construct (B), however, exhibit various phenotypes including axons avoiding the tectum and growing along the anterior tectal boundary (B’). (C) Graph showing the proportion of brains incubated with the NFPC-Fc peptide that display axons avoiding either the anterior or posterior boundary of the tectum. Statistical significance was calculated against the Con-Fc proportions. * p<0.05, ** p<0.01, 1p = 0.0934, Fisher’s exact test (6 independent experiments). Brains electroporated with the Con-MO within the optic tectum (D) do not exhibit defects in axon pathfinding, as RGC axons grew through the electroporated region into the tectum (D’ is an inverse greyscale image showing the trajectory of DiI-filled RGC axons). Panel E shows a representative image of a brain electroporated with the NFPC-MO within the tectum. Perturbation of NFPC binding culminated in phenotypes including looping and projection along the posterior tectal boundary (E’ is an inverse greyscale image showing the trajectory of DiI-filled RGC axons). Panel F reveals the proportion of brains loaded with the NFPC-MO that exhibit abnormal projections into the tectum. Statistical significance was calculated against Con-MO proportions. *** p<0.001, Fisher’s exact test (7 independent experiments). Scale bar in A: 300 μm (A, B, D, E), 75 μm (A’, B’), 50 μm (D’, E’). https://doi.org/10.1371/journal.pone.0141290.g004
	(A) An example of an uninjected eye. Immunostaining with acetylated α-tubulin (red) revealed a normal pattern of axon outgrowth (arrow) that converged on the optic disc (double arrowhead) to exit the eye. (B) Lipofection with a GAP-GFP construct did not affect the number or the directionality of axon bundles (arrow) converging on the optic disc (double arrowhead). Exposed retinae are marked by dashed white lines. Scale bar in B: 30 μm.
	S2 Fig. Dominant negative disruption of NFPC activity disrupts RGC axon outgrowth in vivo. (A) An example of a contralateral, uninjected eye. Immunostaining with acetylated α-tubulin (red) revealed a normal pattern of axon outgrowth (arrow) converging on the optic disc (double arrowhead) to exit the eye. (B) Lipofection with the NFδE construct culminated in disrupted retinal axon outgrowth, with these retinae exhibiting a significantly reduced number of axon bundles per unit area of retina in comparison with the GAP-GFP-expressing controls (C; * p < 0.05, Kruskal-Wallis test. Values were normalized against the GAP-GFP-injected group and the number of retinae analysed are presented within the bars.). However, the remaining axon bundles (arrows in B) converged on the optic disc (double arrowhead). Scale bar in B: 30 μm. https://doi.org/10.1371/journal.pone.0141290.s002 (TIF)
	S3 Fig. Cultured retinal neurites exhibit chemoattraction to Netrin-1. (A-C) Phase contrast images of retinal neurites from uninjected (A, B) and mock electroporated (C) retinae exposed to either a culture medium control (CM; A) or netrin-1 (B, C). (D) Cumulative distributions of turning angles of each sample group. (E) Mean turning angles of the experimental groups reveals that, whereas uninjected neurites exposed to CM did not exhibit any turning bias, neurites exposed to Netrin-1 were attracted to this guidance cue. * p < 0.05, Kolmogorov-Smirnov test. Scale bar in A: 10 μm. https://doi.org/10.1371/journal.pone.0141290.s003 (TIF)
	S4 Fig. Protocol for preparing morpholino-loaded RGC growth cones. (A) Embryonic retinal primordia were electroporated with either a control, FITC-tagged morpholino (Con-MO) or a FITC-tagged anti-NFPC morpholino (NFPC-MO) using a specifically designed electroporation chamber. Eyes were then removed and cultured in vitro for 24 h. Examples of Con-MO-loaded (B, red) and NFPC-MO-loaded (C, green) neurites are shown. https://doi.org/10.1371/journal.pone.0141290.s004 (TIF)

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