miR-92 loss of function. Loss-of-function experiments were performed by injection of antisense MOs against miR-92 into single-cell zebrafish embryos followed by localization of markers, as indicated. (A) Percentages of left (L), right (R) and midline (M) localized foxa3 in NIC (n=188), miR-92 MO1-injected (n=55), miR-92 MO2-injected (n=15), MO1+2-injected (n=126) and control MO-injected (n=56) embryos. (B) Percentages of left, right and midline localized cmcl2 in NIC (n=103), MO1-injected (n=102), MO2-injected (n=97), MO1+2-injected (n=62) and control MO-injected (n=53) embryos. (C-F) Confocal z-stacks of Kupffer′s vesicle (KV) in NICs, MO1-injected and MO2-injected embryos and in miR-92 morphants co-injected with miR-92 RNA into the dorsal forerunner cells (DFCs) (Rescue) at the10-somite stage using a sox17:gfp transgenic line that labels KV cells green. Motile cilia were identified by immunohistochemistry with antibodies against acetylated tubulin (red). Scale bars: 5 μm. (G) GFP-positive KV cells were counted and cilia length measured in the indicated embryos. Error bars represent s.e.m. *, P<0.01 for KV cell number between miR-92 morphants and NICs, P<0.05 for cilia length and P<0.05 for KV cell number between miR-92 morphants and rescue embryos; Student′s t-test. MO refers to miR-92 MO. (H-K) Localization of sox17-expressing cells in wild-type embryos (NIC) and miR-92 morphants (MO) at 90% epiboly. (H,J) Dorsal views with anterior to the top. (I,K) Lateral views with dorsal to the right. (L) Numbers of sox17-expressing cells in NICs (n=16) and miR-92 morphants (MO; n=13). Error bars represent s.e.m. *, P<0.01; Student′s t-test.
|
