Wnt signaling, but not Hh signaling, is reduced in tmem67 morphants. (a) Zebrafish embryos were injected with either control or tmem67 MOs [e2i2], subjected to WISH at 2.5 dpf with ptch1, ptch2 and gli1 riboprobes, and imaged under a light microscope. NS: not significant. Scale bar = 200 μm. (b) Total RNAs were extracted from WT or tmem67 morphant embryos at 2.5 dpf and subjected to quantitative real-time PCR with ptch1, ptch2, gli1 and axin2 primers. Expression levels of indicated mRNA were normalized to those of actin primers. NS: not significant. ** P < 0.01. (c) One-cell stage zebrafish embryos were injected with either control or tmem67 MOs [e2i2], probed with axin2 riboprobes at 2.5 dpf and imaged. For a rescue experiment, one-cell stage zebrafish embryos were sequentially injected with tmem67 MO [e2i2] and RNA encoding the indicated TMEM67 mutant, probed with axin2 riboprobes at 2.5 dpf and imaged. p.Tyr920* represents p.Tyr920ThrfsX40. Arrows indicate reduction of signals compared to those in control embryos. Scale bar = 200 μm. (d) Tg(Tcf/Lef-miniP:dGFP) embryos were injected with either control or tmem67 MOs [e3i3] and imaged at 1 dpf under a fluorescent microscope. Scale bar = 200 μm.
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