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Fig. 2. Inhibition of p38 blocks the expression of XMyf5 without affecting XMyoD expression. (A) 18 VMZ explants were dissected at stage 10.25 from control embryos and incubated with Noggin and SB (30 μm) [1]. In parallel, embryos at the one-cell stage were injected with 0.5 ng of in vitro transcribed DN MKK6 mRNA [2], or with 7.5 ng of p38α antisense morpholino (p38α MO), or with 7.5 ng of control antisense morpholino (p38α CMO) [3]. At stage 10.25, 18 DLMZ explants were dissected from control and treated embryos. Explants were cultured until sibling embryos reached early neurula stages, and total RNA was isolated. RT PCR analysis was preformed with primers to XMyf5, XMyoD, muscle actin and EF1α serving as a control for quantifying RNA levels of different samples. Control-RT PCR was also performed (not shown). For Western analysis, DLMZ explants from p38α MO, DN MKK6-injected and control embryos were cultured until sibling embryos reached later gastrula stages. Proteins were extracted from explants. Antibodies to HA were used to detect ectopic DN MKK6 protein, and antibodies to p38, to detect endogenous p38α protein. (B, C) Expression patterns of the myogenic transcription factors XMyf5 and XMyoD at different developmental stages. One-cell stage embryos were injected with p38α MO. At stages 13 (B) and 23/24 (C), embryos were fixed and analyzed for the expression of XMyoD and XMyf5 by whole-mount in situ hybridization. (B) 92% of control embryos were expressed XMyoD (n = 51) and 94% of the p38α MO-injected embryos expressed XMyoD (n = 35). 90% of control embryos expressed for XMyf5 (n = 62) and only 20% of the p38α MO-injected embryos expressed XMyf5 (n = 40). Dorsal view, anterior-top. (C) 93% of control embryos expressed XMyoD (n = 30) and 90% of p38α MO-injected embryos expressed XMyoD (n = 27). 97% of control embryos expressed XMyf5 in the somites (n = 35) and 16% of p38α MO-injected embryos were stained for XMyf5 in the somites (n = 40). Anterior-left. |
