Green YS and Vetter ML (2011), EBF proteins participate in transcriptional reg...

XB-ART-43792

Dev Biol 2011 Oct 01;3581:240-50. doi: 10.1016/j.ydbio.2011.07.034.

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EBF proteins participate in transcriptional regulation of Xenopus muscle development.

Green YS , Vetter ML .

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EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells.

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Species referenced: Xenopus
Genes referenced: acta1 actl6a cdh15 ebf2 ebf3 gal.2 myf5 myod1 nog rbm24 tbxt tnnc1
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	Fig. 1. Confirmation of candidate EBF targets by RT-PCR hGR-Xebf3 mRNA and Noggin mRNA were injected into embryos at the single-cell stage. At the blastula stage, the animal caps were dissected then divided into two groups and either treated with DEX or left as untreated controls. Following a 4.5 hour incubation, total RNA was isolated and RT-PCR performed. The column labeled –RT is a negative control in which reverse transcriptase was omitted at the cDNA synthesis step. Total embryo (TE) cDNA from stage 12 (for myf5, brachyury and histone h4) or stage 27 (for the remaining genes) was used as a positive RT-PCR control. brachyury was analyzed to ensure there was no mesoderm contamination in animal caps. histone h4 was used as a loading control. All tested genes (except the loading and contamination controls) were upregulated in the presence of DEX.
	Fig. 2. Expression patterns of ebf2 and ebf3 in Xenopus muscle ebf2 (A-C) and ebf3 (D-F) are expressed in multiple developing neural and muscle tissues. At stage 22, ebf2 and ebf3 are expressed in pre-somitic mesoderm (yellow arrows). At all three stages, ebf2 and ebf3 are expressed in the developing somites (black arrows). At stage 37, they are also expressed in the migrating hypaxial muscle anlagen (arrowheads).
	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.
	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.
	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.
	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.
	Figure 7. The identification of direct and indirect candidate targets of EBF3 hGR-XEBF3 mRNA was injected into one-cell stage embryos, and animal caps were collected at the blastula stage (stage 9). The animal caps were divided into four groups, based on CHX and DEX treatment: −C−D, −C+D, +C−D, and +C+D. After a 3.5-hour incubation with CHX and/or a 3-hour incubation with DEX, total RNA was isolated from the animal caps and RT-QPCR was conducted with the isolated RNA. The expression level was normalized with the expression level of histone h4 and then normalized to the expression level of −C+D, for each gene, at 100 arbitrary units. The expression level of myf5 in the condition of +C+D is comparable to the condition of −C+D (A) and the expression level of myod in the condition of +C+D is only partially reduced compared to the condition of −C+D (B). The expression level of m-cadherin in the condition of +C+D is similar to the levels of the two control conditions (C). The expression level of seb-4 in +C+D is similar to the expression level in −C+D but also similar to the expression level in one control condition, +C−D so it is not conclusively a direct or indirect target of EBF activity. Error bars represent standard error of the mean. N = 5 replicates, 20 to 30 animal caps per condition.
	Fig. 8. MYOD drives expression of ebf2 and ebf3 (A-D) MYOD-hGR mRNA or control, hGR mRNA was 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). β-gal mRNA was coinjected as a marker of the injected side. In all panels the right side is the injected side, showing the light blue color of X-gal staining. The expression of ebf2 (B) and ebf3 (D) is strongly upregulated by activated MYOD-hGR (brackets), while control hGR injection does not change the expression level of ebf2 (A) or ebf3 (C). All panels show dorsal views. (E, F) Two vegetal cells of eight-cell stage embryos were co-injected with XEBF2 MO, XEBF3 MO and MYOD-hGR mRNA, followed by DEX treatment from the late gastrula stage (stage 11/11.5) to the early tailbud stage (stage 20). The expression level of m-cadherin is downregulated without MYOD activity (E), but the expression level was rescued in the presence of MYOD activity (F).
	acta1 (actin, alpha 1, skeletal muscle) gene expression in Xenopus laevis embryos, NF stage 35-37, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	tnnc1 (troponin C type 1 (slow)) gene expression in Xenopus laevis embryos, NF stage 35-37, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.

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Anyanful, The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans. 2004, Pubmed