Additional Supporting Information may be found in the online version of this article.

DVDY_21929_sm_SupplFigS1.tif3521KSupp. Fig. S1. vmhc gene expression profile. In situ hybridization results of embryos at various stages. The MyoD riboprobe was included in A–C to ensure proper staging. The vmhc transcript could be detected in the anterior lateral plate mesoderm as early as the 10-somite stage (A). Expression persists in the heart progenitor region, which migrates toward the midline (B), fuses to form a heart tube (C), and differentiates into two distinct chambers (D–F). vmhc expression can be detected in the extraocular muscles and pharyngeal muscles after 3 dpf (F).
DVDY_21929_sm_SupplFigS2.tif195KSupp. Fig. S2. Synteny of the vmhc gene between the zebrafish and Fugu genomes. Approximately 150 kb of the zebrafish genomic sequence (chromosome 2, 19.73–19.88 Mb) was retrieved from the annotated Ensembl database; 130 kb of the Fugu genomic sequence (scaffold 109, 790–920 kb) was retrieved from the Ensembl database and annotated by GENSCAN and Augustus software. Arrows represent predicted genes and the direction of transcription; the names listed on top are Entrez gene IDs. Homologous genes between the zebrafish and Fugu are connected by lines, and the percentage of peptide identity/similarity between the two species is listed.
DVDY_21929_sm_SupplFigS3.tif4733KSupp. Fig. S3. Transgenic fish also express GFP in different muscles. A,B: Tg(V-0.7k:egfp) embryos at 72 hpf exhibited an early onset and strong GFP expression in the extraocular and pharyngeal muscles (A), while Tg(V-0.1k:egfp) embryos did not (B). C–E: Day-6 larval fish from different transgenic lines express GFP in multiple muscles. Similar to Tg(V-0.7k:egfp) fish expressing GFP in the extraocular muscles, pharyngeal muscles, and fin musculature (D), Tg(V-1.9k:egfp) fish also express GFP in myocytes in the body midline (C). In contrast, Tg(V-0.1k:egfp) fish display very weak GFP expression in the cephalic musculature, but strong expression in trunk muscles (E). F–H: Adult 6-month-old fish from different transgenic lines also exhibit varied GFP expression patterns. Most lines have strong (F, H) or weak (G) GFP expression around the eye, jaw, and on the operculum. Muscles near the pectoral and tail fins frequently show GFP signals, as do the dorsal, ventral, and anal fins (H).
DVDY_21929_sm_SupplFigS4.tif154KSupp. Fig. S4. Predicted NKE sites located upstream of the Fugu vmhc gene. Inter-species sequence comparison of upstream sequences between the zebrafish and Fugu vmhc genes. VISTA software was used with the Fugu sequence as the base genome. We defined the start codon as 0, as the transcription start site of Fugu vmhc is unknown. Yellow and orange rectangles correspond to the distal and proximal elements in the zebrafish vmhc promoter, respectively. Diamonds, nkx2.5-binding sites (NKE sites), as predicted by the TESS software.

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