TMEM67 mutants show decrease in protein function. (a) HEK293T cells were transfected with plasmids individually encoding WT, p.Gly132Ala or p.Tyr920ThrfsX40 TMEM67, all fused C-terminally to a FLAG epitope, and processed for WB with anti-FLAG antibody. Anti-actin antibody was used for a loading control. Shown is a representative image of three independent experiments. Full-length blots are presented in Supplementary Fig. S1. (b) The transfected cells in (a) were processed for quantitative real-time PCR with TMEM67-specific primers. Expression levels of TMEM67 were normalized to those of ACTIN. ***P < 0.001 by the two-tailed Student’s t-test (n = 3). (c) The HEK293T cells transfected as described in (a) were treated with DMSO (dimethyl sulfoxide; vehicle control) or MG132 (25 μM) for 4 hr before harvest and processed for WB as described in (a). Cropped blots are presented. (d) A schema showing the binding site of tmem67 splice-blocking morpholino (MO [e2i2]). Dashed lines indicate splicing events. Not drawn to scale. E: exon; FP: forward primer; RP: reverse primer. (e) One-cell stage zebrafish embryos were injected with tmem67 MOs and processed for RT-PCR. β-actin was used as a loading control. Arrow indicates a PCR product with deletion of a part of exon2. (f) An electropherogram of an exon junction area of the PCR product indicated by arrow in (e). (g) One-cell stage zebrafish embryos were injected with either control MOs or tmem67 MOs [e2i2] and imaged at 2.5 days post-fertilization (dpf). For a rescue experiment, one-cell stage zebrafish embryos were sequentially injected with tmem67 MO [e2i2] and RNA encoding the indicated TMEM67 mutant and imaged at 2.5 dpf. Dashed lines mark the hindbrain ventricles. Arrows indicate enlarged ventricles (hydrocephalus). p.Tyr920* represents p.Tyr920ThrfsX40. Scale bar = 100 μm. (h) Embryos with hydrocephalus were counted in each group in (g). The hindbrain ventricles larger than 5,000 μm² were considered to be hydrocephalic. *P < 0.05; ***P < 0.001 by the two-tailed Student’s t-test; NS: not significant. Number of larvae used in the analysis of each group is over 40.

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.

TMEM67 mutants show decrease in protein function. (a) A schema showing the binding site of tmem67 splice-blocking morpholino (MO [e3i3]). Dashed lines indicate splicing events. Not drawn to scale. E: exon; FP: forward primer; RP: reverse primer. (b) One-cell stage zebrafish embryos were injected with tmem67 MOs and processed for RT-PCR. Αctin was used as a loading control. Arrow indicates a PCR product with deletion of exon3 and retention of a part of intron3. (c) An electropherogram of an exon junction area of the PCR product indicated by arrow in (b). (d) One-cell stage zebrafish embryos were injected with either control MOs or tmem67 MOs [e3ie] and imaged at 2.5 days post-fertilization (dpf). For a rescue experiment, one-cell stage zebrafish embryos were sequentially injected with tmem67 MO [e3i3] and RNA encoding the indicated TMEM67 mutant and imaged at 2.5 dpf. Dashed lines mark the hindbrain ventricles. Arrows indicate enlarged ventricles (hydrocephalus). p.Tyr920* represents p.Tyr920ThrfsX40. Scale bar = 100 μm. (e) Embryos with hydrocephalus were counted in each group in (d). The hindbrain ventricles larger than 5,000 μm2 were considered to be hydrocephalic. *: P < 0.05; ***: P < 0.001 by the two-tailed Student’s t-test; NS: not significant. Number of larvae used in the analysis of each group is over 40.