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Biochem Biophys Res Commun
2016 Aug 26;4773:419-25. doi: 10.1016/j.bbrc.2016.06.083.
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IFT46 plays crucial roles in craniofacial and cilia development.
Park I
,
Lee HK
,
Kim C
,
Ismail T
,
Kim YK
,
Park JW
,
Kwon OS
,
Kang BS
,
Lee DS
,
Park TJ
,
Park MJ
,
Choi SC
,
Lee HS
.
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The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development.
Fig. 1.
Spatio-temporal expression patterns of IFT46 during embryogenesis. (A) Xenopus embryos were harvested at various stages and RT-PCR was performed using standard methods. The numbers indicate embryonic stages and ornithine decarboxylase (ODC) was a loading control. IFT46 is expressed as a maternal gene. (B) Xenopus embryos were harvested at stage 32 and processed via whole mount in situ hybridization using Dig-labeled antisense probes of IFT46. IFT46 was expressed in the central nervous system and multiciliated cells. The right figure is higher magnification view. (C) Both blastomeres of 2-cell stage embryos were injected with IFT46 mRNA containing a Flag tag and harvested at stage 27. Embryos were immunostained with acetylated tubulin-monoclonal antibody (green) and Flag-polyclonal antibodies (red). IFT46 was localized along the axoneme of motile cilia of epithelial cells. White arrows indicate IFT46 proteins. Right figure is higher magnification view (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 2.
Loss of IFT46 prevents motile cilia formation in Xenopus epithelial cells. (A) Both blastomeres of 2-cell staged embryos were injected with IFT46 MO and mutant mRNA, IFT46 (5MT), not recognized by MOs. Embryos were harvested at stage 32 and immunofluorescence staining of motile cilia with acetylated tubulin-monoclonal antibody (red) was carried out. (B) Relative quantification of green fluorescence per motile cilia of control MO- or IFT46 MO-injected embryos with IFT46 (5MT) mRNA. IFT46 MO and IFT46 (5MT) mRNA co-injected embryos showed partially rescued ciliogenesis. Data are shown as mean ± SD, and the number of embryos per sample is 25. ***p < 0.001 compared with control MO-injected. (C) Western blot analysis of exogenous IFT46-Flag mRNA expression (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 3.
Loss of IFT46 hinders primary cilia formation. (A) Both blastomeres of 2-cell staged embryos were injected with IFT46 MO and fixed embryos at stage 32. Primary cilia in the neural tube lumen were detected by immunofluorescence staining using acetylated tubulin-antibody (gray) and confocal microscopy. (B) Treatment of IFT46 siRNA for 24 h and serum starvation was performed to induce primary cilia for 48 h. We performed immunofluorescence staining of the nucleus and primary cilia using DAPI (blue) and acetylated tubulin-monoclonal antibody (red) respectively. Compared with the control, IFT46 siRNA-treated cells show short or no primary cilia. Data are shown as mean ± SD. ***p < 0.001 compared with control siRNA-treated. (C) RT-PCR was performed to evaluate the knockdown of IFT46. β-actin was used as a loading control (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 4.
Loss of IFT46 induces severe reduction in neural development and cartilage formation. (A) 10 ng of IFT46 MO was injected into the both blastomeres of 2-cell staged embryos with 2 ng of dominant-negative BMP-4 receptor (DN-BR) mRNA. Animal caps were dissected from injected embryos at stage 8.5 and collected at stage 18. The animal cap was processed for RT-PCR using standard methods. ODC was used as a loading control. No-RT indicates the control in the absence of reverse transcriptase. Quantification of the relative expression levels of BF-1, XAG, N-CAM, AP2α, and ZIC3 normalized to ODC expression. (B) Both blastomeres of 2-cell staged embryos were injected with 5 ng of IFT46 MO. Tadpoles were fixed at stage 50 and subjected to Alcian blue staining for cartilage formation. IFT46 morphants showed severe defects on cartilage formation (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. S1.
Loss of IFT46 causes ventralized phenotype (A) Embryos were injected with 10 ng of IFT46 MO into the both blastomeres of 2-cell staged embryos. IFT46 morphants showed defects in head and eye formation and convergent extension. (B) Relative quantification of the number of control and IFT46 morphants. Most embryos injected with IFT46 MO showed similar phenotypes. Data are shown as mean ± SD, and the number of embryos per sample is 25. ***p < 0.001 compared with control MO-injected.
Fig. S2.
Loss of IFT46 affects neural development (A) MO-injected embryos at stage 17 of neural plate were visualized by whole mount in situ hybridization for SOX3. The neural plate was wider in IFT46 MO-injected embryos. (B) We measured the length of half of the neural plate for SOX3. Data are shown as mean ± SD, and the number of embryos per sample is 25. ***p < 0.001 compared with control MO-injected. (C) Using the neural crest cell marker PAX3, neural tube closure was abnormal in IFT46 MO-injected compared with control MO-injected embryos. (D) We calculated the length of the gap between the neural fold for PAX3. Data are shown as mean ± SD, and the number of embryos per sample is 25. ***p < 0.001 compared with control MO-injected.2
