Figure 7.
SFK inhibition by Csk attenuates EphB2- and EphA4-induced LMC motor axon redirection. A, D, G, J, M, P, Neurofilament detection in the limb nerves of chick HH st. 28/29 EphB2::GFP (A), Csk and EphB2::GFP (D), EphA4::GFP (G), Csk and EphA4::GFP (J), EphA4 and GFP (M), or EphA4Y602E and GFP (P) expressing embryos. B, E, H, K, N, Q, GFP detection in the limb nerves of HH st. 28/29 EphB2::GFP (B), Csk and EphB2::GFP (E), EphA4::GFP (H), Csk and EphA4::GFP (K), EphA4 and GFP (N), or EphA4Y602E and GFP (Q) expressing embryos. C, F, I, L, O, R, Quantification of GFP+ axons within dorsal and ventral limb nerves expressed as the percentage of total GFP+ axons [GFP Fluo (%)]. In EphB2::GFP electroporated embryos, 35 ± 4% of GFP+ axons were detected in the dorsal limb nerves, and 65 ± 4% of GFP+ axons were detected in the ventral limb nerves (v/d ratio of 1.9; C). In Csk and EphB2::GFP coelectroporated embryos, 52 ± 6% of GFP+ axons were detected in the dorsal limb nerves, and 48 ± 6% of GFP+ axons were detected in the ventral limb nerves (v/d ratio of 0.9; F). In EphA4::GFP electroporated embryos, 91 ± 4% of GFP+ axons were detected in the dorsal limb nerves, and 9 ± 4% of GFP+ axons were detected in the ventral limb nerves (v/d ratio of 0.1; I). In Csk and EphA4::GFP coelectroporated embryos, 76 ± 4% of GFP+ axons were detected in the dorsal limb nerves, and 24 ± 4% of GFP+ axons were detected in the ventral limb nerves (v/d ratio of 0.3; L). In EphA4 and GFP coelectroporated embryos, 85 ± 6% of GFP+ axons were detected in the dorsal limb nerves, and 15 ± 6% of GFP+ axons were detected in the ventral limb nerves (v/d ratio of 0.2; O). In EphA4Y602E and GFP coelectroporated embryos, 59 ± 6% of GFP+ axons were detected in the dorsal limb nerves, and 41 ± 6% of GFP+ axons were detected in the ventral limb nerves (v/d ratio of 0.7; R). Number of embryos quantified: C, n = 8; F, n = 9; I, n = 8; L, n = 7; O, n = 5; R, n = 4. Proportions of GFP+ axons in Csk and EphB2::GFP expressing embryos and those in EphB2::GFP expressing embryos are significantly different (p < 0.001) using Student's unpaired t test. Proportions of GFP+ axons in Csk and EphA4::GFP expressing embryos and those in EphA4::GFP expressing embryos are significantly different (p < 0.001) using Student's unpaired t test. Proportions of GFP+ axons in EphA4Y602E and GFP expressing embryos and those in EphA4 and GFP expressing embryos are significantly different (p < 0.001) using Student's unpaired t test. v/d ratio = (average ventral GFP Fluo %)/(average dorsal GFP Fluo %); NF, neurofilament; d, dorsal; v, ventral; error bars = SD. ***p < 0.001; all values expressed as mean ± SD. Scale bar, 100 μm in all images.