Tbx16 functions cell-autonomously in DFCs to control KV morphogenesis and LR development. (A–D) Live embryos at 24 hpf. (B) tbx16 morphants show defects seen in tbx16 mutants including a ‘spade’ tail (arrow in panel B) and trunk defects (arrowhead in panel B). In contrast, DFCtbx16 MO (C) and yolktbx16 MO (D) embryos appeared similar to uninjected controls (A). (E–H) Fluorescent images corresponding to (A–D). Fluorescent tbx16 MO was found in all cells in tbx16 morphants (F), but remained only in the yolk cell (arrow in panel G) and a small group of cells in the tail of DFCtbx16 MO embryos (arrowhead in panel G). tbx16 MO was restricted to the yolk cell (arrow in panel H) in yolktbx16 MO embryos (H). (I–L) sonic hedgehog (shh) marks midline structures such as the floor plate (fp) and notochord (n). Kinks in the midline often seen in tbx16 morphants (arrow in panel J) were not observed in DFCtbx16 MO (K), yolktbx16 MO (L) or uninjected (I) embryos. (M–P) aPKC/aTubulin co-immunostaining of KV cells between 6 and 8 SS. KV morphology defects were observed in DFCtbx16 MO embryos (K) similar to defects in tbx16 morphant (J) and mutant (Figs. 2G–L) embryos. Control yolktbx16 MO embryos (L) formed a normal KV similar to uninjected embryos (I). (Q–X) Left–right asymmetry was analyzed (see Table 2) by RNA in situ hybridization of spaw in LPM at 17–18 SS (Q–T) and lft1 and lft2 in precursors of the brain and heart respectively at 22–24 SS (U–X). sonic hedgehog (shh) marks the midline. Views are dorsal and left (L) and right (R) sides are indicated in panel Q. In wild-type embryos, spaw (Q) and lft1 and lft2 (U) were asymmetrically expressed on the left. Expression of these markers was altered in tbx16 morphant (R, V) and DFCtbx16 MO embryos (S, W). LR markers were unaffected in yolktbx16 MO embryos (T, X).
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