Figure 1.
Physical map and gene organization of the Japanese lamprey fast skeletal myosin heavy chain genes (MYHs).
(A) The position, orientation, size and gene organization of fast skeletal MYHs that compose the cluster. The vertical and horizontal lines indicate exons and introns, respectively. (B) The detailed gene organization of MYHs. Note that only the positions of coding exons 3-41 are presented. The boxes and lines indicate exons and introns, respectively.
Figure 2.
Phylogenetic analysis of myosin heavy chains (MYHs) from Japanese lamprey, torafugu and humans.
The neighbor-joining (A) and maximum-likelihood (B) trees based on the full-length amino acid sequences of fast skeletal MYHs from Japanese lamprey, torafugu and humans. Cardiac and slow skeletal MYHs of torafugu and humans and sarcomeric MYH of bay scallops were used as outgroups. Lc MYHs from the Japanese lamprey Lethenteron camtschaticum were compared with Hs MYH1 (adult fast IId/x), Hs MYH2 (adult fast IIa), Hs MYH8 (perinatal fast), Hs MYH4 (adult fast IIb), Hs MYH3 (embryonic fast), Hs MYH13 (extraocular fast), Hs MYH6 (alpha cardiac), Hs MYH7 (beta cardiac) and Hs MYH14/7B (slow A) from human, Homo sapiens, and Ai MYH (striated muscle) from the bay scallop, Argopecten irradians. MYH sequences of the torafugu, Takifugu rubripes (Tr), were cited from our previous data [16-19].
Figure 3.
Transient expression of the Japanese lamprey MYH1-3kb-hrGFP and MYH2-3kb-DsRed transgenes in zebrafish embryos.
A, B: Lateral view showing the expression of the MYH1-3kb-hrGFP in the trunk myotome of zebrafish embryos at 1 (A) and 3 (B) days post-fertilization (dpf). C: Lateral view showing the expression of the MYH2-3kb-DsRed in the trunk myotome of an embryo at 3 dpf. D: Lateral view showing the expression of the MYH1-3kb-hrGFP and MYH2-3kb-DsRed in the trunk myotome of an embryo at 3 dpf. Two muscle fibers clearly co-expressed the 2 transgenes in the same cells (merged, arrowheads). E-G: Lateral (E) and right (F) and left (G) ventrolateral side views showing the expression of the MYH1-3kb-hrGFP in cranial muscles of an embryo at 5 dpf. H-K: Transverse sections at the middle trunk of an embryo at 5 dpf. Cells were labeled with an antibody against GFP (H) and F59 antibody raised against chicken slow skeletal MYH (I). Differential interference contrast (DIC) microscopy image (J) and merged image from panels H, I and J (K). Asterisks indicate crosstalk signals due to strong GFP expression in the red channel. Note that superficial slow skeletal muscle fibers were labeled with F59, and the MYH1-3kb-hrGFP transgene was found to be expressed in fast muscle fibers. Scale bar = 20 μm.
Figure 4.
Activity of Japanese lamprey, zebrafish and mouse myosin heavy chain gene (MYH) promoter in C2C12 cells.
A: C2C12 myoblasts were transfected with a promoter-less control pGL3 vector (control), -3 kb Japanese lamprey MYH1 (MYH1) or MYH2 (MYH2) and -2.8 kb mouse MYH1 (IId/x) (mouse MYH1) promoter luciferase constructs along with pRL-TK as a transfection control. B: C2C12 myoblasts were transfected with a promoter-less control pGL3 vector (control), -3 kb zebrafish myhz2 (myhz2) or myhc4 (myhc4) and -2.8 kb mouse MYH1 (IId/x) (mouse MYH1) promoter luciferase constructs along with pRL-TK as a transfection control. Cells were grown for 24 h in a growth medium, and myoblasts were harvested for luciferase assays. Other cells were cultured for an additional 4 days in differentiation medium, and myotubes were harvested for luciferase assays. The results are expressed as firefly luciferase activity per Renilla luciferase activity from each sample. The data represent the average of triplicate data, and all bars are the mean ± SD. Data were analyzed using Student's t test (*, P < 0.05; **, P < 0.01).