XB-ART-1167Dev Dyn 2006 Feb 01;2352:524-9. doi: 10.1002/dvdy.20628.
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Spatio-temporal expression of MRF4 transcripts and protein during Xenopus laevis embryogenesis.
Whereas there have been extensive studies of the expression of XMyf5 and XMyoD during Xenopus embryogenesis, nothing is known about the spatio-temporal accumulation of XMRF4 transcripts and protein. In this report, we describe the cloning and characterization of two full-length MRF4 cDNAs and of their proximal promoters in Xenopus laevis. The comparison of the relative transcript levels of the XMRF4-a and -b genes in developing and adult muscles is highly suggestive of specific functions for the corresponding XMRF4 proteins. Whole-mount embryo in situ hybridization revealed the first XMRF4 transcripts in the more differentiated anterior myocytes of the embryo when the myosin heavy chain E3 mRNA begins to be detectable. XMRF4 mRNA accumulation later extended posteriorly but was never detected in the posterior unsegmented mesoderm, in contrast to XMyoD and XMyf-5. Whole-mount embryo immunohistochemistry revealed that XMRF4 protein accumulated in somite nuclei slightly after XMRF4 transcripts.
PubMed ID: 16258964
Article link: Dev Dyn
Species referenced: Xenopus laevis
Genes referenced: myf5 myf6 myh4 myh6 myod1
Antibodies: Myf6 Ab1
Article Images: [+] show captions
|Figure 2. Whole-mount in situ hybridization at stage 18-19 (A) 21-22 (B), and 27-28 (C) with labeled antisense probes against XMRF4, XMyf5, XMyoD, and embryonic MHC E3 mRNAs. The same results were obtained using the three MRF4 probes. The anterior side of the embryos is on the left. Lateral view (C, right in B), dorsal view (A, left in B). Arrowhead: posterior unsegmented mesoderm|
|Figure 3. A: Control of specificity of the anti-XMRF4 antibody: One nanogram of synthetic Flag-tagged XMRF4-a (1) or -b (2) mRNA was bilaterally injected into the animal pole of two-cell stage embryos. Ten micrograms of egg homogenates was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. The affinity-purified anti-XMRF4 antibody recognizes the two XMRF4 proteins in the same way as the anti-Flag antibody. No band was obtained in uninjected embryos (data not shown). B: Whole mount immunostaining using an affinity-purified polyclonal antibody against Xenopus MRF4 at stage 24 (up), 26 (middle), 28 (down). The anterior side of the embryos is on the left. Arrowhead, posterior unsegmented mesoderm. C: Indirect immunofluorescent detection of Anti-XMRF4 (green) and 4 prime ,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining (blue) on longitudinal cryosection of somitic muscles at stage 28. No labels were detected with secondary antibody alone (data not shown).|