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Experiment details for myod1

Green YS and Vetter ML (2011) Assay

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

Gene Clone Species Stages Anatomy
myod1.S laevis NF stage 14 to NF stage 15 presomitic mesoderm , paraxial mesoderm

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  Fig. 5. EBF2 and EBF3 are sufficient for muscle target gene expression hGR-XEBF2 or hGR-XEBF3 mRNA were injected into one cell of two-cell stage embryos, followed by DEX treatment from the late gastrula stage (stage 11/11.5) to the neurula stage (stage 14/15). hGR mRNA was injected in control embryos. β-gal mRNA was coinjected as a marker of the injected side. In all panels the right side is the injected side, showing the blue color of X-gal staining. The (purple) expression levels of myod (B and C), m-cadherin (E and F), seb-4 (H and I), and myf5 (K and L) are strongly upregulated by EBF2 and EBF3 (brackets), while expression of hGR alone does not change the expression level of the target genes (A, D, G, and J). The expression of actin alpha (N) and tnnc1 (P) is downregulated by EBF3. All panels show dorsal views.

Gene Clone Species Stages Anatomy
myod1.S laevis NF stage 20 to NF stage 21 somite , paraxial mesoderm

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  Fig. 6. EBF2 and EBF3 are necessary for muscle target gene expression (A-L) Two vegetal cells of eight-cell stage embryos were injected with either XEBF2 MO and XEBF3 MO together, or control MO. β-gal mRNA was coinjected as a marker of the injected side. The expression level of target genes was examined at stage 20/21. In all panels the right side is the injected side, showing the blue color of X-gal staining. The (purple) expression levels of myod (B and C), m-cadherin (E and F), seb-4 (H and I), and myf5 (K and L) are downregulated by XEBF2 MO and XEBF3 MO together or by NLS-DN-EBF (brackets), while control MO does not change their expression levels (A, D, G, and J). (M, N) Two vegetal cells of eight-cell stage embryos were coinjected with XEBF2 MO, XEBF3 MO and hGR-XEBF2 mRNA, followed by DEX treatment from the late gastrula stage (stage 11/11.5) to the early tailbud stage (stage 20). The expression of m-cadherin is downregulated without EBF activity (M), but expression was rescued in the presence of EBF activity (N). All panels show dorsal views.

Gene Clone Species Stages Anatomy
myod1.S laevis NF stage 35 and 36 to NF stage 37 and 38 somite , muscle , musculature of face , hypaxial muscle , epaxial muscle , [+]

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  Fig. 4. Expression patterns of muscle target genes In stage 35–37 embryos myod, m-cadherin, actin alpha, seb-4, tnnc1 and myf5 are all expressed in skeletal muscle including somites (black arrows), migrating hypaxial muscle anlagen (black arrowheads) and jaw muscle (yellow arrows). myod, m-cadherin, actin alpha, seb-4, and myf5 (A-D and F) are expressed in the somites, migrating hypaxial muscle anlagen and jaw muscle, and these expression patterns overlap with those of ebf2 and ebf3 (Figure 2). m-cadherin (B) is weakly expressed in a central band in somites, with expression throughout the somite. myf5 (F) expression in somites is weaker than other genes at this stage, and is expressed at the leading edge of migrating hypaxial muscle. tnnc1 (E) is expressed in the somites. actin alpha, seb-4, and tnnc1 are expressed in the heart (yellow arrowheads). All embryos show lateral views.

Gene Clone Species Stages Anatomy
myod1.S laevis NF stage 39 to NF stage 40 somite , muscle , skeletal muscle , musculature of face , hypaxial muscle , [+]

  Fig. 3. Defective skeletal muscle development after knockdown of EBF2 and EBF3 Two vegetal cells of eight-cell stage embryos were injected with control MO or EBF2 MO (2 MO) and EBF3 MO (3 MO), either alone or together. β-gal mRNA was coinjected as a marker of the injected side (light blue). At stage 39/40, myod expression was examined (A-H), and 12/101 antibody was used as a marker of skeletal muscle tissue (I-L). The left column (A, C, E, G, I, and K) shows the uninjected control side of the embryos. The right column (B, D, F, H, J, and L) shows the injected side, and (B and F) in some embryos there is more light blue staining in the pronephros, the functional larval kidney, which largely develops from the two vegetal cells that we targeted (Moody, 1987). All panels show lateral views. After injection of 2 MO or 3 MO, myod expression patterns show that the chevron shape of somites is abnormal (black arrows), the hypaxial muscle anlagen are smaller, and the migration distance is reduced (black arrowheads), compared to the uninjected side. The expression of myod in jaw muscle is also reduced (yellow arrows). When 2 MO and 3 MO were coinjected (H), these defects were more severe than 2 MO or 3 MO alone (D and F). Control MO has no effect (B). 12/101 antibody staining shows that when 2 MO and 3 MO were coinjected, somite segmentation is not complete, and the chevron shape of somites is abnormal (white arrows). Also jaw muscle differentiation is reduced (yellow arrow) and abdominal hypaxial muscle differentiation is strongly reduced (white arrowheads), while control MO shows a mild defect of only hypaxial muscle differentiation (J). To visualize the injected side after immunostaining, β-galactosidase antibody (not shown) was used for coimmunostaining along with 12/101 antibody.