April 1, 2012;
14-3-3 proteins regulate retinal axon growth by modulating ADF/cofilin activity.
Precise navigation of axons to their targets is critical for establishing proper neuronal networks during development. Axon
elongation, whereby axons extend far beyond the site of initiation to reach their target cells, is an essential step in this process, but the precise molecular pathways that regulate axon
growth remain uncharacterized. Here we show that 14-3-3/14-3-3ς proteins-adaptor proteins that modulate diverse cellular processes including cytoskeletal dynamics-play a critical role in Xenopus retinal ganglion cell
elongation in vivo and in vitro. We have identified the expression of 14-3-3/14-3-3ς transcripts and proteins in retinal growth cones, with higher levels of expression occurring during the phase of rapid pathway extension. Competitive inhibition of 14-3-3/14-3-3ς by expression of a genetically encoded peptide, R18, in RGCs resulted in a marked decrease in the length of the initial retinotectal projection
in vivo and a corresponding decrease in axon
elongation rate in vitro (30-40%). Furthermore, 14-3-3/14-3-3ς (R1
) co-localized with Xenopus actin depolymerizing factor (ADF
)/cofilin (XAC) in RGC
growth cones. Inhibition of 14-3-3/14-3-3ς function with either R18 or morpholinos reduced the level of inactive pXAC and increased the sensitivity to collapse by the repulsive cue, Slit2
. Collectively, these results demonstrate that14-3-3/14-3-3ς participates in the regulation of retinal axon
elongation, in part by modulating XAC activity.
Xla Wt + Difopein
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References [+] :
Figure 1 Localization of 14-3-3ς mRNA in retinal axons and growth cones. (A) RT-PCR of mRNAs purified from heads and eyes of various stages. RT only control was performed to rule out genomic contaminations. (B) Representative images of FISH with either anti-sense or sense probes (scale bar = 5 μm) (R1). (C) Quantification of FISH mean intensity per unit area (n = no. of growth cones; **** = p<0.0001; Mann–Whitney). (D) In situ hybridization of stage 40 embryo sections (ONH = optic nerve head, LS = lens, RGC = retinal ganglion cells, IPL = inner plexiform layer, OPL = outer plexiform layer, OT = optic tectum; scale bars = 25 μm for left panel and 100 μm for the middle panel).
Figure 2 14-3-3/14-3-3ς proteins are expressed in retinal growth cones. (A) Western blot of head or eye lysates using 14-3-3 or 14-3-3ς antibodies. The 14-3-3ς antibody recognizes both the unphosphorylated and the phosphorylated form of 14-3-3ς at a predicted molecular weight of * 29 kD (R1), which is more apparently visualized at low exposures (inset). (B) Immunofluorescence staining of retinal growth cones using 14-3-3 or 14-3-3ς antibodies. The area marked by square shows localization of 14-3-3ς within filopodia. A magnified view of the squared area is shown in the inset (scale bar = 5 μm).
Figure 3 The level of 14-3-3ς in growth cones decreases with age. (A) Representative 14-3-3ς IF staining in growth cones cultured for 8, 24, and 48 h (scale bars = 5 μm). (B) Quantitation of mean 14-3-3ς IF intensity/unit area in growth cones cultured 8, 24, 48 h (n = no. of growth cones; three replicates; ** = p<0.01; one-way ANOVA with Bonferroni post-test).
Figure 4 Inhibition of 14-3-3/14-3-3ς shortens retinal axon projection in vivo. (A) Schematics of R18 and R18K constructs tagged with YFP. (B) Schematics of eye electroporation. FITC-tagged morpholinos are electroporated into the eye at stage 26. (C) Dissected brains from stage 40 embryos that have been electroporated with either R18-YFP or R18K-YFP. The arrowhead indicates markedly shorter axons found in R18-YFP transfected brains (dashed line = anterior tectal border; scale bar = 50 μm; CH = optic chiasm; TEL = telencephalon; OT = optic tectum). (D) The length of the pathway was measured in two ways: (1) following the curvature of the pathway (a) and (2) measuring the shortest distance from the optic chiasm to the longest axon (LA; b) divided by the shortest distance from the chiasm to the posterior tectum (PT; c). (E) Measurement of the pathway length (equivalent to \a" in Fig. 4D). (F) Measurement of CH to LA length/CH to PT length (equivalent to \b/c" in Fig 4.4). (G) Measurement of CH to PT length to rule out significant differences in brain size. (Note: in E, F, and G, n = number of brains analyzed; three replicates; **p<0.01; ***p<0.001; unpaired t-test).
Figure 5 Shortened retinal projection due to retarded axon elongation rate. (A) Brains of stage 43 embryos that have been eye-electroporated with R18-YFP or R18K-YFP (R1). Axons reach the tectum even in those that were electroporated with R18-YFP. The rightmost panel represents the magnified view of the R18 pathway with an axon taking an unusually tortuous path (arrowhead; dashed line = anterior tectal border; scale bar = 50 μm). (B) Measurement of the pathway length. (C) Distance from CH-LA/CH-PT. (D) Distance from CH-PT (For B, C, D, n = no. of brains analyzed; unpaired t-test). (E) Whole eye culture of eyes that have been electroporated with R18-YFP or R18K-YFP (scale bar = 50 μm). (F) The elongation rate of axons transfected with R18-YFP or R18K-YFP (n = no. of growth cones; *p<0.05; Mann–Whitney).
Figure 6 Colocalization of 14-3-3ς with pXAC and XAC. (A) Growth cone staining with Alexa 488-conjugated pXAC antibody and Alexa 594-conjugated 14-3-3ς antibody. The squared area shows colocalization in a filopodium. The inset shows more magnified view of the squared area (scale bar = 5 μm; image intensity adjusted). (B) Growth cone staining with Alexa 488-conjugated XAC antibody and Alexa 594-conjugated 14-3-3ς antibody. (C) Growth cone staining with Alexa 488 and Alexa 594 fluorophores without antibody conjugation. (D) Measurement of Pearson coefficient for determination of colocalization (n = no. of growth cones;***p<0.001; statistical analysis by Kruskal–Wallis test with Dunn post-test). (R1)
Figure 7 14-3-3/14-3-3ς knockdown decreases phospho-XAC in retinal growth cones. (A, B) QIF analysis of pXAC (A) and XAC (B) in growth cones transfected with either R18K- or R18-YFP (n = no. of growth cones; four replicates; *p<0.05; Mann–Whitney). (C) Western blot of eye lysates showing 14-3-3ς knockdown with the antisense morpholino. a-Tubulin was used as a loading control. (D) QIF analysis of 14-3-3ς in growth cones injected with either control or 14-3-3ς morpholino (*** p<0.001; n = no. of growth cones; three replicates; unpaired t-test). (E, F) QIF analysis of pXAC (E) and XAC (F) in growth cones injected with control or 14-3-3ς morpholino (n = no of growth cones; *** p<0.001; three replicates; unpaired t-test).
Figure 8 14-3-3/14-3-3ς inhibition sensitizes growth cones to Slit1-induced collapse. (A) R18K transfected growth cones stimulated with either control or Slit2 CM at sub-threshold concentration (scale bar = 5 μm). (B) R18-YFP transfected growth cones stimulated with either control or Slit2 CM at sub-threshold concentration. (C) Collapse rate of R18K- or difopein-transfected growth cones stimulated with either control or Slit2 CM at sub-threshold concentration (n = number of growth cones; three replicates; * = p<0.05; one-way ANOVA with Bonferroni post-test).
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