Inhibition of MAPK activity causes defects in muscle and nervous system development.

<p>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. <b>A–A″′, C–C″′, E–E″′.</b> 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>B, D</b> and <b>F</b> represent <i>Pdu-Mhc</i> detections (blue) after <i>in situ</i> hybridization. <b>A–A″′.</b> Normally developed muscles and nerves in a 66 hpf larvae of the 0.5% DMSO control group were classified as phenotype 0 (P0). <b>B.... </b><i>Pdu-Mhc</i> expression in a P0 larva at 66 hpf. <b>C–C’’’.</b> 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. <b>D.... </b><i>Pdu-Mhc</i> expression in a P1 larva. <b>E–E’’’.</b> 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. <b>F.... </b><i>Pdu-Mhc</i> expression in a P2 larva. <b>G.</b> 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. <b>H–I.... </b><i>Pdu-twist</i> expression in control (H) and U0126 treated larva (I) at 24 hpf. Improper positioning of <i>Pdu-twist</i> 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.</p

MAPK activation during early development in <i>Platynereis dumerilii</i>.

<p>Antibody staining against di-phosphorylated, activated MAPK/ERK (dpERK) in red or white, DNA-staining with Hoechst appears in blue, actin was marked by FITC-coupled Phalloidin (in green). <b>A, B</b> Embryos at the 38- and the 46-cell stage exhibit no dpERK staining in the mesentoblast (4d) and its descendants ML and MR. <b>C.</b> Initial dpERK staining was detected within the nephroblasts (n) in the animal hemisphere of a 7.5 hpf early blastula. <b>D.</b> MAPK activation is still visible during further head kidney development in the mid-blastula (10.5 hpf). <b>E–G″.</b> dpERK staining is visible during gastrulation in nuclei of small cells (arrowheads) and macromeres (M) in the region of the blastopore. <b>F–F′.</b> Micromeres with MAPK activity show an accumulation of filamentous actin at 15 hpf. <b>G′–G″.</b> MAPK positive cells in the region of the blastopore at 15 hpf. <b>G″′.</b> dpERK positive macromere nuclei in the same embryo as in G but different focal plane. <b>H, H′. </b><i>Pdu-twist in situ</i> hybridization in combination with dpERK staining in a 15 hpf embryo. Activated MAPK and <i>Pdu-twist</i> positive cells are in close proximity at the region of the blastopore (asterisk). Arrows point towards two dpERK-positive nuclei that are in the same focal plane as the nuclei (arrowheads) of two <i>Pdu-twist</i> (black) expressing cells. Scale bars are 50 µm and 10 µm in whole embryo views and close-ups, respectively.</p