Role of Zebrafish fhl1A in Satellite Cell and Skeletal Muscle Development

Figures

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  Fig. (1). Expression of fhl1A and its role in skeletal muscle development. (A) At the 3-somite stage, no fhl1A signal was detected in somites. (B, C) Specific expression of fhl1A in somites at the 10- and 20-somite stages. (D, H) Control embryos. (E, I) Knockdown of fhl1A resulted in defective somites. (F, J) fhl1A mRNA successfully recovered the fhl1A morphant. (D’, E’, F’, H’, I’ and J’) Partial amplifications of D, E, F, H, I and J, respectively. (G, K) Statistical analysis of D–F and H–J, respectively. D–F and H–J scale bars: 200 µm; D’–F’ scale bar: 100 µm. The number of embryos analysed is shown in the columns.

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  Fig. (2). Role of fhl1A in skeletal muscle development. (A–I) Embryos stained with myoD probes. (A–D) fhl1A had no effect on myoD at the 3-somite stage. (F–I) At the 12-somite stage, the expression of myoD in somites was upregulated. (H) Overexpression and downregulation of fhl1A. (G) Knockdown of fhl1A (arrowhead). (E, J) Statistical data of (A–D) and (F–I), respectively. Scale bars: 200 µm. (K–N) At the 12-somite stage, anti-MF20 antibody was used for immunostaining, suggesting skeletal muscle was regulated by fhl1A: (K) Control-MO embryos, (L) fhl1A knockdown embryos, (M) embryos injected with fhl1A mRNA, (N) embryos co-injected with fhl1A MO and fhl1A mRNA. (O) Statistical data of (K–N). Scale bar: 100 µm. (P–W) The expression of myoD disappeared from somites in (P) Control-MO and (Q) fhl1A MO and from (S) recovered embryos, but myoD was still expressed in (R) embryos overexpressing fhl1A (arrowhead). (X, Y) Statistical data of (P–S) and (Q–T), respectively. The symbols a–d represent control, fhl1A MO, fhl1A mRNA and Rescue, respectively. The number of embryos analysed is shown in the columns.

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  Fig. (3). Role of fhl1A in satellite cells. (A) At 24 hpf, RT-PCR results demonstrated that satellite cell markers were severely downregulated when fhl1A was knocked down. (B) Statistical data from A; experiments were repeated at least 3 times. (C) Expression of fhl1A was required for formation of satellite cells. At the 10- to 17-somite stage, the number of Pax7+ myogenic precursors in fhl1A knockdown embryos was significantly decreased. Anterior to the left, dorsal up. (D–G) Expression of fhl1A was found to regulate somite development. Somitic expression of mef2ca was (F) upregulated by fhl1A overexpression and (E) downregulated as fhl1A was knocked down (arrowhead). (D) Control embryos. (G) Embryos co-injected with fhl1A MO and fhl1A mRNA. (H) Statistical data of (D–G). The symbols a–d represent control, fhl1A MO, fhl1A mRNA and recovered, respectively. The number of embryos analysed is shown in the column, (** p < 0.01, *** p < 0.001). Error bars indicate ± s.e.m.

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  Fig. (4). Effects of RA signalling on endogenous fhl1A expression. (A–H) Phenotypes obtained by whole-mount in situ hybridization with myoD antisense RNA probe. (B, F) The expression of myoD was upregulated in the presence of exogenous RA and (C, G) downregulated by exogenous DEAB. (D, H) fhl1A MO resulted in downregulated expression of myoD. Black arrows indicate the expression of myoD at somites. (I–K) Endogenous expression of fhl1A was found to be regulated by RA. (L, M) Statistical data from A–D and E–H, respectively. The number of embryos analysed is shown in the columns.