Fig 1: RGC Axon-Axon Interactions Require CYFIP2 Function(A) Representative western blots and quantification of CYFIP1 and CYFIP2 levels in Xenopus eye lysates at stages 34 and 41 (n = 3, normalized to a-Tubulin).(B) Representative western blots and quantification of CYFIP2 (n = 6, normalized to a-Tubulin) and CYFIP1 (n = 3, normalized to a-Tubulin) levels in CYFIP2MO- compared to CoMO-injected embryos at stage 34.(C) Examples of stalling growth cones (GCs) during homotypic and heterotypic responses after CYFIP2 depletion.(D) Quantification of the homotypic interaction responses after CYFIP2 knockdown.(E and F) Quantification of the number (E) and duration (F) of filopodia contacts during fasciculation and stalling events in CYFIP2MO (n = 7 GC, n = 27 filopodia) compared to CoMO (n = 6 GC, n = 20 filopodia) conditions for homotypic interactions.(G) Quantification of the heterotypic interaction responses after CYFIP2 knockdown.(H and I) Quantification of the number (H) and duration (I) of filopodia contacts during tracking and stalling events in CYFIP2MO (n = 8 GC, n = 26 filopodia) compared to CoMO (n = 5 GC, n = 29 filopodia) conditions for heterotypic interactions.(D and G) Numbers of events analyzed are indicated on the graph (n = 12 independent experiments).(E–I) Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., non-significant (Mann-Whitney test for A, B, E, F, H and I) and (Fisher’s exact test for D and G). Time stamps are in the format of min:s. Scale bars: 5 µm (C).
Fig 2: Differential Function of CYFIP1 and CYFIP2 during RGC Axonal Development(A) Sequence analysis of the whole zebrafish embryo injected with cas9 mRNA + cyfip1 or cyfip2 gRNAs. The target site is indicated on the sequence (green), followed by 4 examples of corresponding mutated regions.(B) DiI (red) and DiO (green) fluorescent dyes were injected in the zebrafish embryo retina at 5 dpf. The dashed line denotes the confocal imaging area of the optic tract (OT).(C1–D3′′′) Dorsal (D) (C1′–C3′, D1′–D3′) and Ventral (V) (C1′′–C3′′, D1′′–D3′′) RGC projections were analyzed in control embryos (cas9 mRNA + gRNA control) (C1, D1), cyfip1 CRISPR-injected embryos (cas9 mRNA + gRNA cyfip1) (C2, D2), and cyfip2 CRISPR-injected embryos (cas9 mRNA + gRNA cyfip2) (C3, D3) at 48 hpf (C) and 5 dpf (D). Arrows in (D3′) show missorted dorsal axons in the OT. Yellow lines in (D1)–(D3) indicate the reference line used for quantification of the missorting index (MI). Examples of DiI (dorsal) and DiO signals plotted along the reference line corresponding to sorted (D1′′′, D2′′′) or misprojected (D3′′′) RGC axons (int., intensity; A.U, Arbitrary Units).(E) Quantifications of D and V axonal projection area in the OT at 48 hpf.(F) The missorting index (MI) was quantified as the ratio of the intensity signal of the missorted D (Dm) axons to all the D axons (Dm+Ds). For gRNA cyfip1-injected embryos, only the embryos showing an axon growth phenotype were quantified (n = 18 embryos).Error bars represent SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, n.s., non significant (Mann-Whitney test for E and F). The number of zebrafish analyzed is indicated on the bars. Scale bars: 50 μm (C1–D3). See also Figure S1.
Supplier Page from Abcam for Anti-CYFIP1 antibody