Yoon BC , Jung H , Dwivedy A , O'Hare CM , Zivraj KH , Holt CE .

085314/Z/08/Z Wellcome Trust , 085314 Wellcome Trust , WT085314 Wellcome Trust , Wellcome Trust

	Figure 3. LB2 mRNA and Protein Are Expressed in RGC Axons and GCs(A) Axonal lb2 mRNA detected by RT-PCR.(B and C) Lb2 FISH in cultured retinal axons and its quantitation (mean ± SEM; n = no. of GC; ∗p < 0.05; Mann-Whitney).(D–G′) ISH of stage 40–45 embryo sections (RGC: retinal ganglion cell layer; ONH: optic nerve head; and IPL/OPL: inner/outer plexiform layer). The same sections were counterstained for neurofilament and DAPI.(H and I) LB2 immunostaining in cultured retinal axons and its reduction by LB2MO.(J and K′) Immunostaining in tissue sections. The boxed areas are shown in the lower panels.Scale bars, 5 μm in (B) and (H), 25 μm in (D)–(G), (J), and (K), and 10 μm in (J) and (K) lower panels. See also Figure S3.
	Figure S3. Lb2 mRNA and LB2 Protein Localize to Axons, Related to Figure 3(A and B) Lb2 and pax6 ISH in eye sections (RGC: retinal ganglion cell layer; ONH: optic nerve head; ON: optic nerve). The same sections were counterstained for axonal (neurofilament) and nuclear (DAPI) markers. The ONH, where RGC axons collect to exit the eye, is devoid of cell bodies and contains lb2 mRNA but not pax6 mRNA.(C and D) Lb2 and pax6 ISH in brain sections with a counterstained image. The tectal neuropil, where RGC axons terminate, is devoid of cell bodies and contains lb2 mRNA but not pax6 mRNA (e.g., inside the red dashed line).(E) LB2 immunostaining in brain sections shows that LB2 localizes to RGC axons in the optic tectum (Tec) (e.g., inside the red dashed line). Arrowheads indicate axons ascending to the optic tectum.(F and G) Brn3, a nuclear factor abundantly expressed in RGCs, does not localize to the tectal neuropil (e.g., inside the red dashed line) or nearby tegmental neuropil (white arrow), although it is highly expressed by a subset of neurons in the tegmentum (green arrow).(H) LB2 antibody recognizes the nucleus (red) as expected (two left panels) in fibroblast-like cells in eye explant culture. Increasing the exposure reveals localization of LB2 outside the nucleus (upper right panel). LB2MO significantly decreases the nuclear LB2 staining (arrowhead), examined at the same exposure (two middle panels). Increasing the exposure shows that the extranuclear LB2 staining (arrow) is also reduced with LB2MO, indicating that the cytoplasmic LB2 signal is specific (two right panels).(I) Western blot of stage 40 embryo head lysate shows that LB2MO inhibits LB2 translation in a dose-dependent manner.(J) Quantification of the lane intensity of LB2/α-tubulin for the indicated conditions.(K and L) Two additional independent antibodies against LB2 detect a single major band in western blot and extranuclear LB2 in axons and GCs in culture.(M) LB2MO reduces axonal LB2 detected by a different antibody confirming the specificity of this signal (∗∗p < 0.01; unpaired t test).Scale bars: 25 μm in (A)–(E), 5 μm in (F) and (J).
	Figure 1. Visualization of Cue-Induced Protein Synthesis(A) Fluorescent 1D gel image showing AHA incorporation in retinal cultures within 1 hr, which is abolished by 40 μM anisomycin.(B) Coomassie staining of the same gel showing similar protein levels in all lanes.(C) Quantitative analysis (mean ± SEM; ∗∗p < 0.01; 3 replicates; one-way ANOVA and Bonferroni).(D) AHA incorporation in retinal cultures upon 1 hr stimulation of various cues.See also Figure S1.
	Figure 2. En-1 Stimulation Elicits Dynamic Changes in Local Protein Synthesis(A) Axon culture preparation by separating the eye. The absence of DNA in the axonal fraction confirms its purity.(B) DIGE-NCAT detection of newly synthesized axonal polypeptides shows AHA incorporation in spots corresponding to LB2 (red: control; green: En-1; and arrows: LB2 spot).(C and D) Quantitative analysis of putative protein spots between control and En-1 conditions (green: increased; red: decreased; and yellow: unchanged).See also Figure S2 and Table S1.
	Figure 4. LB2 Is Locally Synthesized In Vitro and In Vivo(A–C) LB2 QIF from axon-only culture (mean ± SEM; n = no. of GCs; 3 replicates; ∗∗p < 0.01; one-way ANOVA and Bonferroni).(D) Axon-TRAP experiment. GFP-L10a RNA is expressed by blastomere injections in the CNS of a donor embryo, whose eye is transplanted into an uninjected host. The transplanted eye then extends retinal axons to the contralateral optic tectum of the host brain. The third diagram represents a brain that has been cut at the ventral midline and flattened. The boxed areas were dissected out, from which GFP-L10a-containing ribosomes (green) and associated mRNAs were purified by GFP immunoprecipitation. In the negative control, GFP RNA was used instead of L10a-GFP RNA.(E) RT-PCR from axon-TRAP for β-actin mRNA (TI: total input; IP: immunoprecipitation).(F) RT-PCR for lb2 mRNA.Scale bar, 4 μm. See also Figure S4.
	Figure 5. Inhibiting LB2 Translation Results in Degeneration of RGC Pathway In Vivo(A) MO injection and RGC axon labeling using DiI (green: MO; Tel: telencephalon; Ch: optic chiasm; OT: optic tract; Tec: optic tectum; and red: DiI).(B) DiI-labeled RGC axons in MO-injected embryos with or without LB2-GFP RNA.(C) Characteristic beaded morphology of dying axons in LB2 morphants. The boxed area is shown in the right panel.(D) Quantitative analysis of the LB2MO-induced reduction in RGC axons and its rescue by LB2-GFP (n = no. of brains; 3 replicates; ∗∗∗∗p < 0.0001; Fisher's exact).(E and F) The frequency of active caspase-3-positive cells in retinal sections (mean ± SEM; n = sections analyzed; Mann-Whitney).Scale bars: 65 μm in (B) and (C), 100 μm in (C) right panel, and 25 μm in (E). See also Figure S5.
	Figure 6. LB2MO-Induced Pathway Degeneration Is Mainly Axonal and Results from Inhibition of Local LB2 Synthesis(A) Eye electroporation.(B) DiI-labeled RGC axons in eye-electroporated embryos.(C and D) The mean DiI intensity of the distal 150 μm of the RGC pathway (mean ± SEM; n = no. of brains; 3 replicates; ∗∗∗∗p < 0.0001; Mann-Whitney).(E) Pathway electroporation.(F and G) DiI-labeled RGC axons in pathway-electroporated embryos (mean ± SEM; n = no. of brains; 3 replicates; ∗∗p < 0.01; Fisher's exact).(H) Nuclear LB2 immunofluorescent intensity in the RGC layer normalized to its intensity in the INL (mean ± SEM; for CoMO, total RGC no. = 1648, total INL cell no. = 2813, total no. of sections = 15; for LB2MO, total RGC no. = 1639, total INL cell no. = 3001, total no. of sections = 14; Mann-Whitney).(I) Wild-type LB2 and LB2δNLS localization in HEK293T cells (arrowhead: nuclear; arrow: cytoplasmic).(J) Rescue experiment.(K and L) Ath5:RFP-labeled RGC axons in LB2MO-injected embryos with or without LB2δNLS eye electroporation. Tel: telencephalon; Ch: optic chiasm; OT: optic tract; Tec: optic tectum.Scale bars: 65 μm in (B), (F), and (K) and 10 μm in (I). See also Figure S6.
	Figure 7. LB2 Knockdown Interferes with Mitochondrial Functions in Axons(A and B) LB2 coimmunostaining with CoxIV and VDAC2.(C) A single plane OMX super-resolution image of LB2 and Mito-GFP.(D) TMRM staining of CoMO- or LB2MO-injected axons and GCs.(E) Mitochondrial potential (Fm/Fc) in the distal 30 μm GCs/axons (mean ± SEM; n = no. of mitochondria analyzed; 3 replicates; ∗∗∗p < 0.0001; Mann-Whitney).(F and G) Mitochondrial length (box-and-whisker plot: minimum and maximum) (∗∗∗p < 0.0001; Mann-Whitney).(H) Mitochondrial number per unit length axon (box-and-whisker plot) (n.s.: not significant).(I) Anterograde and retrograde organelle transport measured by lysosome movements (mean ± SEM; 18 CoMO axons and 15 LB2MO axons; ∗∗p < 0.01 and ∗∗∗p < 0.001; Dunn's multiple comparison and Kruskal-Wallis).Scale bars: 1 μm in (C) and 5 μm in the rest. See also Figure S7.
	Figure S1. The Level of LB2 Protein in Retinal Cultures Is Increased by BDNF Stimulation, Related to Figure 1(A) One 2D-DIGE spot (out of ∼1,300 putative spots) that increased in intensity following 24 hr BDNF stimulation of retinal cultures (arrow).(B) Biological variation analysis (BVA) of 2D-DIGE (mean ± SEM; 3 replicates; ∗∗∗p < 0.001; unpaired t test). MS analysis identified the spot as LB2 (Xenopus laevis; Gene name—lmnb2; MASCOT score-66).
	Figure S2. DIGE-NCAT Strategy, Related to Figure 2(A) The distal portions of the axon bundles in culture were severed from the eyes.(B) The axon-only culture was stimulated with a guidance cue and AHA, and followed by RNA/protein coextraction. Total protein lysate concentrations were matched between conditions based on total RNA concentration. We used RNA concentration because the total amount of axonal protein we could recover was too small for protein-based quantitation.(C) AHA-tagged, newly synthesized polypeptides were reacted with TAMRA-alkyne reporter.(D) TAMRA reacted lysate was mixed with CyDye-labeled standard lysates as well as unlabeled lysates. CyDye-labeled standards were used to normalize multiple gels for analysis, and unlabeled lysates were used to increase the amount of proteins per spot.(E) Combined lysates were separated and visualized by 2D-DIGE.(F) Gels from different conditions were standardized using the CyDye-labeled standard lysate, and spots of interest were picked for MS analysis.(G) 3D representation of DIGE-NCAT spots in control versus En-1 condition with spot intensities represented as peaks.(H) A merged image of AHA-labeled, newly synthesized polypeptides with stimulation of 1 hr control (red) or En-1 (green) shows strikingly different 2D-gel patterns between the two conditions. The numbers indicate the spots picked for MS analysis and correspond to the numbers in Table S1.
	Figure S4. Axonal Translation of lb2 mRNA Is Selectively Regulated, Related to Figure 4(A–C) One hour En-1 stimulation does not induce translation of β-actin mRNA, which is localized to RGC axons. ns: not significant; unpaired t test.(D) Lb2 mRNA is expressed at a similar level in the eye to other mRNAs encoding nuclear proteins as revealed by quantitatve RT-PCR.(E) Axon-TRAP analysis shows that other lamin mRNAs are not associated with ribosomes in RGC axons in vivo. Scale bar, 5 μm.
	Figure S5. Experimental Schemes for Axon Degeneration Analysis, Related to Figure 5(A) Injection into two dorsal animal blastomeres at 8-cell stage results in the delivery of MO into the CNS. At stage 45, one eye is labeled by DiI, which anterogradely travels to label RGC axons (optic tract: OT) reaching the contralateral optic tectum (Tec).(B) Electroporation into the eye primordium at stage 28 results in eye-specific MO delivery without affecting the brain.(C) Electroporation in the optic tectum at stage 40 results in the delivery of MO into the RGC axons without affecting their cell bodies in the contralateral eye.(D) Rescue experiment shown in Figure 6J. Ath5:RFP, a plasmid which drives RFP expression in RGCs, and MO are coinjected into one dorsal animal blastomere at 8-cell stage. This results in a unilateral CNS delivery of the MO and mosaic RFP-labeling of RGCs in the same side. The rescue construct is then electroporated into the same eye at stage 28, when RGC axonogenesis occurs. RFP-labeled RGC axons reaching the contralateral optic tectum are imaged at stage 45.
	Figure S6. Axonally Synthesized LB2 Localizes to Mitochondria, Related to Figure 6(A) Detailed schematic representation of pathway electroporation described in Figure 6E. At stage 40, one side of the optic tectum is exposed by removing the eye and skin before MO is electroporated.(B–D) HEK293T cells transfected with Xenopus LB2δNLS plasmid were labeled with MitoTracker, and imaged by a laser-scanning confocal microscope. LB2δNLS does not localize to the nuclear membrane (arrowhead) and instead localize to cytoplasmic structures that include mitochondria (arrow). Cross-sectional views seen from the dashed lines in (D) are shown left and above each image.(E–G) LB2δNLS also localizes to mitochondria in cultured Xenopus RGC axons (arrow).(H) In contrast, wild-type LB2 mainly localizes to the nuclear membrane.Scale bars: (B–D) and (H), 10 μm; (E–G), 5 μm.
	Figure S7. LB2 Localizes to Mitochondria, Related to Figure 7(A) LB2 and Nudel show a relatively weak colocalization.(B) LB2 detected by a second LB2 antibody colocalizes with VDAC2, a mitochondrial protein.(C) Schematic representation of PLA technology. PLA signal from fluorescent oligonucleotides represents proximity of the two proteins of interest (within 40 nm).(D) Representative images of cultured RGC GCs, showing PLA signals obtained with indicated antibody pairs. LB2 interacts with CoxIV, but not with NFPC or neuropilin1. Positive controls (the last two panels) show specific signals within the GC, which is consistent with reported protein-protein interactions.Scale bars, 5 μm.

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