Figure 5

Role of lefty2 downregulation in Nodal/Pitx2 signaling and heart L-R asymmetry. (A–D, F–H) In situ hybridization showing lft1 (A–D) and lft2 (F–H) expression in surface fish (A, B, F) and cavefish (C, D, G, H) at 50% epiboly. (A, C) Dorsal views. (B, D, F–H) Lateral views. Arrows in (A, C) indicate the shield region and in (F–H) indicate the germ ring. The numbers in the frames indicate embryos with the indicated expression pattern compared to the total number of in situ hybridized embryos. Scale bar in B: 150 µm; magnification is the same in all frames. (E) Bar graphs showing relative fold changes in lft1, lft2, and ndr1 mRNA in cavefish compared to surface fish gastrulae determined by qPCR at the 50% epiboly stage. Black bars: surface fish. Yellow bars: cavefish. Error bars: SEM. Number of replicates shown at the bottom of the columns. Asterisk: p < 0.01. (I) Effects of the Nodal inhibitors SB-431542 and SB-505124 on heart L-R asymmetry in cavefish. Bar graphs showing the proportion of D-looped, non-looped, and L-looped hearts in cavefish controls and 1 µM and 5 µM SB431542 or SB-505124 treated larvae stained with MF-20 antibody at 3 days post-fertilization (dpf). The numbers of assayed fish are shown at the right of each bar. Asterisk: Chi² statistic = 62.2813; p < 0.00001. (J, K) Effects of lft2 CRISPR-Cas9 on heart looping in surface fish at 3 dpf. (J) Examples of larvae (L) with axial defects (L1–4, top) and mutated lft2 sequences (bottom, underlined in green) in 4 injected surface fish compared to a wild type surface fish control (WT) from the same clutch. Red arrows: sgRNA target. Scale bar is 100 µm; magnification is the same in all frames. (K) Bar graphs showing proportion of heart looping asymmetry in genotyped injected surface fish with (top) or without (middle) mild axial phenotypes compared to controls from the same clutch (bottom). The numbers of assayed fish are shown at the right of each bar. Asterisk: Chi² statistic = 6.1667; p = .187043.