Figure 4: Developmental regulation of YAP during postnatal growth

(a) The internodal lengths of mSCs infected with Crb3 (black square) or control (white circle) shRNA viruses injected at 3–5 dPN were measured at 15, 30 and 60dPN. Cells infected with Crb3 shRNA virus are significantly longer than cells infected with control ShRNA virus at all time points. Number of cell measured between 63 and 83, and three animals for each condition. Statistical analysis compares Crb3 sh with Cont sh at each time point. (b) Representative immunostaining of YAP immunostaining (red) in the mSC nucleus (DAPI, blue) during development at 15, 30 and 60 dPN (P). (c) Left axis: Quantification of nuclear YAP immunostaining (black circle) in mSCs at 15, 30 and 60 dPN. n between 62 and 164 cells from three animals at each age. Right axis: mean internodal extension rate at median time point 22.5 and 45 (white circle). Mean internodal extension (μm) was calculated between 15 and 30 dPN (median time point 22.5 dPN) and between 30 and 60 dPN (median time point 45 dPN) using the mean internodal lengths at 15, 30 and 60 dPN of control shRNA cells shown in a. The mean internodal extension rate is the ratio of mean internodal extension in μm over 15 (15–30 dPN) or 30 (30–60 dPN) days. (d) Representative western blots for YAP and P-YAP in mouse sciatic nerve at 15, 30 and 60 dPN (P). Each lane represents independent sciatic nerves. (e) Quantification of western blots for YAP and P-YAP at 15, 30 and 60 dPN (n=3, 4 and 4 independent experiments, respectively). Levels were first normalized for protein loading with actin, then the fold change in expression of individual replicates over time was expressed relative to the average level seen at 15 dPN. Statistical analysis compares YAP/Actin or P-YAP/Actin at 30 or 60 d to 15 dPN. Statistical two-tailed Student’s t-tests: *P<0.05; **P<0.01; ***P<0.001; NS, P>0.05 not significant (NS). Scale bars, 10 μm (b). Error bars show s.e.m (a) and (c) and s.d. in e.