Inhibition of MAPK activity causes defects in muscle and nervous system development.
Embryos were treated with 10 µM, 25 µM or 50 µM of the MEK inhibitor U0126. Control groups were incubated in 0.5% DMSO/NSW or pasteurized NSW from 13.5 to 16.5 hpf. All treated embryos from a clutch were collected and fixed 66 hpf for further analysis. A–A″′, C–C″′, E–E″′. Musculature of larvae was labeled with FITC-Phalloidin (actin in green or white), the nervous system was stained with an antibody against acetylated tubulin (aat in red or white), Hoechst labeling of DNA appears blue. B, D and F represent Pdu-Mhc detections (blue) after in situ hybridization. A–A″′. Normally developed muscles and nerves in a 66 hpf larvae of the 0.5% DMSO control group were classified as phenotype 0 (P0). B.... Pdu-Mhc expression in a P0 larva at 66 hpf. C–C’’’. Larvae with a shortened body axis and reduced parapodial development were classified as phenotype 1 (P1). Muscle pattern defects occur due to abnormal positioned and orientated muscles. The nervous system is formed, but nerve fibers are messily arranged. D.... Pdu-Mhc expression in a P1 larva. E–E’’’. Larvae classified as phenotype 2 (P2) lack a secondary body axis, fail to elongate and appear rounded in shape. Muscle accumulation is clearly observed and fibers of the ventral nervous system are missing. F.... Pdu-Mhc expression in a P2 larva. G. Quantification of the proportions of non-developed eggs (n.d.), affected (phenotypes 1&2) and unaffected (phenotype 0) larvae in treatment and control groups. Means and standard errors of means are shown. Significance levels revealed by the Tukey HSD post hoc test are indicated for selected groups (* = p<0.05, ** = p<0.01, n.s. = not significant/p>0.05). Data were obtained from three experimental replicates. Total Numbers (n) of counted larvae: NSW: n = 913, 0.5% DMSO: n = 1545, 10 µM: n = 1387, 25 µM: n = 1351 and 50 µM: n = 1503. H–I.... Pdu-twist expression in control (H) and U0126 treated larva (I) at 24 hpf. Improper positioning of Pdu-twist expressing cells was observed in 45 of 310 larvae after 0.5% DMSO vehicle treatment and 161 of 270 larvae after treatment with 10 µM U0126. Scale bars are 50 µm and 10 µm for whole embryos and close ups, respectively.