The YSL regulates myocardial migration. Embryos injected with a 4 bp mismatch mtx1 control MO (A,C,G), mtx1 MO(A) (B,D-F,H,N-O,Q), mtx1 MO(B) (I) or mtx1 MO(C) (J,K), into the YSL at the 1000-cell stage (3 hpf), embryos injected with mtx1 MO(C) into the YSL at the 1000-cell stage and mtx1 mRNA into the YSL at dome stage (L), and uninjected control embryos (M,P). (A-F) Live embryos at 34 hpf. (A,B) Lateral views, anterior to the left. mtx1 MO(A)-injected embryos exhibit cardiac edema (arrowhead) and a flattened hindbrain ventricle (arrow) (B). (C-F) Frontal views reveal the cardiac phenotype of control and mtx1 MO(A)-injected embryos. Approximately 30% of mtx1 MO(A)-injected embryos showed a clear cardia bifida phenotype at 34 hpf (D). Rhodamine Dextran was co-injected with mtx1 MO(A) to visualize the distribution of the injected solution, and this method revealed that it was mostly restricted to the YSL. Bright field (D) and fluorescent views (E) are merged in F. White arrowheads point to the cardiac tissues. Expression of the myocardial-specific markers, cmlc2 (G-J,P,Q) and Tg(cmlc2:GFP) (K-O), in embryos injected with mtx1 MOs into the YSL. (G-O) Dorsal views, anterior to the top. (P,Q) Frontal views, arrowheads point to the myocardial clusters. At the 21-somite stage (19.5 hpf), myocardial cells have fused at the midline and form a shallow cardiac cone in control MO-injected embryos (G), whereas they remained bilateral in mtx1 MO-injected embryos (H-K). (H-K) Three independent mtx1 MOs, MO(A) (H), MO(B) (I), and MO(C) (J,K), caused the same phenotype in a fully penetrant manner when examined at the 21-somite stage (19.5 hpf). (L) mtx1 mRNA injection into the YSL at dome stage rescued the myocardial migration defect of mtx1 MO(C)-injected embryos (21-somite stage, 19.5 hpf, shown). At 24 hpf, myocardial cells have fused at the midline and begun to form a tube that extends toward the left anterior part of control uninjected embryos (M), whereas myocardial cells have just fused (N) or not yet fused (O) in mtx1 MO(A)-injected embryos. At 38 hpf, myocardial cells form a single tubular functioning heart in uninjected control embryos (P), but these cells formed two clearly distinct structures in approximately 30% of mtx1 MO(A)-injected embryos (Q).
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