Figure 1

Analysis of differentially expressed mRNAs and lncRNAs in three stages of zebrafish embryonic development. (a) Differentially expressed lncRNAs. (b) Differentially expressed mRNAs.
Figure 2

GO enrichment analysis of DEGs at different stages of zebrafish embryonic development. BP: biological process. CC: cellular component. MF: molecular function. Down means downregulated genes, Up means upregulated genes. Underline indicates the terms related with muscle development.
Figure 3

Spatiotemporal expression of lncRNA gas5 in zebrafish. (a) Spatial expression at different developmental stages of zebrafish embryos using in situ hybridization. Microscope’s magnification: 45 ×. (b) Expression at different developmental stages, analyzed using qPCR. The expression level of 0 hpf was set as 1, and the relative expression levels of other stages were compared with that of 0 hpf. (c) Expression profiles in various tissues of adult zebrafish, detected using qPCR. The expression level of the eyes was set as 1, and the relative expression levels of other tissues were compared with that of the eyes. Data of qPCR derived from three independent experiments are shown as mean ± SEM. * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001.
Figure 4

The effects of lncRNA gas5 knockout. (a) The expression level of lncRNA gas5 knockout embryos, detected by qPCR. Data derived from three independent experiments are shown as mean ± SEM. **** indicates p < 0.0001. (b) The expression level of lncRNA gas5 knockout embryos, detected by in situ hybridization. NI represents no injection, control represents only injection of Cas9 protein, Cas9 represents co-injection of sgRNA and Cas9 protein. Microscope’s magnification: 45 × (c–h) The effects of knockout on embryonic development. Microscope’s magnification: 45 ×. #: enlargement of yolk extension. *: small or absent eyes, arrows indicate nonseparation of two otoliths.
Figure 5

Effect of lncRNA gas5 knockout on expression levels of related genes. (a) Expression of genes related to muscle development in zebrafish embryos 24 hpf. (b) Expression of genes adjacent to lncRNA gas5 in zebrafish embryos 24 hpf. Control represents only injection of Cas9 protein. Data from qPCR derived from three independent experiments are shown as mean ±SEM. * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001.
Figure 6

Whole-mount in situ hybridization of skeletal myogenesis marker myod in control and knockout zebrafish embryos at 10, 24, and 36 hpf. (a) Whole-mount in situ hybridization of skeletal myogenesis marker myod in non-injected (NI) embryos at 10, 24, and 36 hpf. (b) Whole-mount in situ hybridization of skeletal myogenesis marker myod in only Cas9-injected embryos at 10, 24, and 36 hpf. (c) Whole-mount in situ hybridization of skeletal myogenesis marker myod in knockout embryos (KO) at 10, 24, and 36 hpf. Arrows indicate the abnormal expression of myod in myotomes. NI indicates no injection. KO indicates knockout. Microscope’s magnification: 45 ×
Acknowledgments
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