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The p38MAPK signaling pathway is essential for skeletal muscle differentiation in tissue culture models. We demonstrate a novel role for p38MAPK in myogenesis during early Xenopus laevis development. Interfering with p38MAPK causes distinct defects in myogenesis. The initial expression of Myf5 is selectively blocked, while expression of MyoD is unaffected. Expression of a subset of muscle structural genes is reduced. Convergent extension movements are prevented and segmentation of the paraxial mesoderm is delayed, probably due to the failure of cells to withdraw from the cell cycle. Myotubes are properly formed; however, at later stages, they begin to degenerate, and the boundaries between somites disappear. Significant apoptotic cell death occurs in most parts of the somites. The ventralbody wall muscle derived from migratory progenitor cells of the ventralsomite region is poorly formed. Our data indicate that the developmental defects caused by p38alpha-knockdown were mediated by the loss of XMyf5 expression. Thus, this study identifies a specific intracellular pathway in which p38MAPK and Myf5 proteins regulate a distinct myogenic program.
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.
Fig. 5. Somite boundaries and myotube structure are disrupted in p38α-knockdown embryos. (A) Embryos (n = 15) were injected with p38α MO and cultured until they reached stage 25. Embryos were fixed and taken for whole-mount in situ hybridization for detecting muscle actin (upper panel). Somite patterning was observed on longitudinal sections immunostained with the 12/101 antibody and analyzed by confocal microscopy (lower panel) (n = 7). (B) Longitudinal sections of MO-injected and uninjected embryos (stage 25) were immunostained with the 12/101 antibody and analyzed under light microscopy (n = 6). Double arrows show myotube diameter. (C) 18 DLMZ explants were removed from each uninjected and p38α MO-injected embryos and cultured until sibling embryos reached stage 18 and RNA was isolated. RT-PCR analysis was preformed with primers to MHC, desmin, tropomyosin, muscle actin and EF1α serving as a control for quantifying RNA levels in the different samples. Control-RT PCR was performed (not shown). All injected embryos in panels A and B displayed similar phenotypes.
Fig. 6. The ventral portions of the somite undergo apoptotic cell death in tailbudp38-knockdown embryos. (A) Two-cell embryos were injected unilaterally into one blastomere with p38α MO (7.5 ng). Embryos were cultured to stage 25. Four embryos were transversely-sectioned and TUNEL analysis was performed followed by TRO-PRO3 staining. One sectioned embryo is shown. Similar results were obtained with all other embryos (n = 4). Cells undergoing apoptosis are stained in light blue. Approximate somite borders are schematically presented. Som. is somite and N.T is neural tube. (B) Two cell embryos (n = 5) were injected unilaterally with p38α MO (7.5 ng). Embryos were cultured to stage 31. Injected embryos were longitudinally sectioned at different dorso-ventral levels of the somites and immunostained with the 12/101 antibody (red staining) followed by TRO-PRO3 staining (blue staining). One sectioned embryo is shown. Similar results were obtained with all other embryos (n = 5). (C) Embryos were injected with p38α MO. Embryos were fixed and taken for whole-mount in situ hybridization to pax3 and muscle actin. Arrows point at migrating muscle progenitor cells. Embryos were injected with p38α MO and cultured to stage 46. Whole embryos were fixed, immunostained with the 12/101 antibody and analyzed by light microscopy. Arrows point at muscle cells (lower panel).
Fig. 7. Exogenous expression of XMyf5 rescues the phenotype of p38-knockdown embryos. (A) Embryos were injected with XMyf5 MO. DLMZ explants were dissected at stage 10.25 and cultured until sibling embryos reached early neurula stages. (B) Embryos were injected with XMyf5 MO and cultured to tadpole stage. Embryos were fixed and taken for whole-mount in situ hybridization using a probe of muscle actin. 95% of control embryos displayed segmental phenotype (n = 38). 92% of MO-injected embryos lost the segmented phenotype (n = 41). (C) Embryos were injected with the XMyf5 MO. 18 DLMZ explants were dissected at stages 10.25 and cultured until sibling embryos reached early neurula stages (stage 18). RNA was isolated and RT PCR analysis was performed with primers to MHC, desmin, tropomyosin, muscle actin and EF1α serving as a control for quantifying RNA levels in each sample. Control-RT PCR reactions were performed (not shown). (D) Embryos were injected with XMyf5-encoding RNA (0.8 ng), and p38α MO and cultured until control embryos reached stage 29/30. Embryos were fixed and analyzed by whole-mount in situ hybridization using the muscle actin probe (upper panel). Statistical analysis relates to the percentage of embryos displaying normal segmented phenotypes. Embryos were injected as described above. 18 DLMZ explants were dissected at stages 10.25 and cultured until sibling embryos reached early neurula stages (stage 18). RNA was isolated and RT PCR analysis was performed with primers to desmin, tropomyosin, muscle actin and EF1α (lower panel).
Fig. 1. p38MAPK and its substrate CREB are phosphorylated in DLMZ but not in VMZ explants. (A) VMZ and DLMZ explants were dissected at stage 10.25, and cultured until sibling embryos reached later gastrula and neurula stages as indicated. Proteins were extracted from 18 explants and CREB, phospho CREB (pCREB), α tubulin, p38 and phospho p38 (pp38) were detected by Western analysis. The asterisk represents an unidentified cross-reacting protein. (B) VMZ explants were isolated at stage 10+, incubated with Noggin or SB203580 (30 μM) and cultured until sibling embryos reached stage 16. Proteins were extracted from 18 explants. CREB and phospho CREB were detected by Western blotting.
Fig. 3. Inhibition of p38 reduces convergent extension of presomitic mesoderm. (A) Embryos were injected with p38α MO. DLMZ and DMZ explants were dissected at stage 10.25 and cultured until sibling embryos reached early neurula stages. (B) Longitudinal sections of embryos (n = 5) at stages 16 and 22, injected with p38α MO in one blastomere at the two-cell stage, were immunostained, using the 12/101 antibody and viewed by confocal microscopy. Red staining represents presomitic and somitic mesoderm (12/101) and blue represents nuclei staining (TO-PRO3 iodide). Results shown are from one embryo. Similar results were obtained with all five embryos.
Fig. 4. Inhibition of p38 prevents cell cycle arrest in the presomitic mesoderm. Two-cell embryos were injected unilaterally into one blastomere with p38α MO (7.5 ng). Embryos were cultured until late gastrula-early neurula stages, then fixed and longitudinally-sectioned. Proliferation cell nuclear antigen (PCNA) antibody was used to detect nuclei of proliferating cells (red staining). Nuclear counterstaining was done with TO-PRO3 iodide (blue staining). Section of one embryo is shown. Similar results were obtained with all other embryos (n = 4). Statistical analysis from the four embryos is presented.
