Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart
(A–C) Average transvalvular flow direction as a function of time for wild-type hearts as seen in the AV canal (light magenta box) or atrium (light blue box) between 36, 48, and 58 hpf in the area highlighted in the heart drawings (ventral view, anterior to the top). Anterograde flow from the atrium to ventricle is shown in black, retrograde flow from the ventricle to the atrium in red, and no flow between the chambers is shown in white. The sequence of time segments with retrograde, anterograde, and no-flow fractional periods are depicted in red, black, and white, respectively. The retrograde flow fraction (RFF) is the fraction of the cardiac cycle that is red. (D–F) cmlc2 expression reveals changes in heart morphology. cmlc2 is expressed in the heart tube in the anterior region at 36 hpf (D), and is expressed strongly in the ventricle and weakly in the atrium at 48 and 54 hpf (E and F). (G–O) Expression of notch1b, klf2a, and bmp4 progressively becomes localized to the AV canal during valve specification. mRNA distribution of notch1b (G–I), klf2a (J–L), bmp4 (M–O) at 36 hpf (D, G, J, and M), 48 hpf (E, H, K, and N), and 56 hpf (F, I, L, and O). notch1b is found in the anterior part of the heart tube at 36 hpf (G), and becomes stronger in the AV canal and in the ventricle at 48 hpf (H). At 54 hpf, notch1b expression becomes restricted in the AV canal and the outflow tract (I). klf2a expression is found throughout the heart tube at 36 hpf (J) and becomes stronger in the AV canal and in the atrium at 48 hpf (K). At 56 hpf, klf2a is exclusively expressed in the AV canal and the outflow tract, displaying an expression pattern very similar to notch1b (L). bmp4 expression is found in the anterior part of the heart tube at 36 hpf (M) and becomes progressively concentrated at the level of the AV canal from 48 to 54 hpf (N and O). Anterior to the top, white arrows point to the AV canal, black arrows to the outflow tract. Scale bar indicates 50 µm.