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Manojlovic Z, Earwood R, Kato A, Stefanovic B, Kato Y.
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Regulatory Factor X (RFX) transcription factors are important for development and are likely involved in the pathogenesis of serious human diseases including ciliopathies. While seven RFX genes have been identified in vertebrates and several RFX transcription factors have been reported to be regulators of ciliogenesis, the role of RFX7 in development including ciliogenesis is not known. Here we show that RFX7 in Xenopus laevis is expressed in the neural tube, eye, otic vesicles, and somites. Knockdown of RFX7 in Xenopus embryos resulted in a defect of ciliogenesis in the neural tube and failure of neural tube closure. RFX7 controlled the formation of cilia by regulating the expression of RFX4 gene, which has been reported to be required for ciliogenesis in the neural tube. Moreover, ectopic expression of Foxj1, which is a master regulator of motile cilia formation, suppressed the expression of RFX4 but not RFX7. Taken together, RFX7 plays an important role in the process of neural tube closure at the top of the molecular cascade which controls ciliogenesis in the neural tube.
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Fig. 1. RFX7 and RFX4 are expressed in the nervous system. (A) Spatial expression profiles of RFX7 and RFX4 genes. (B) Temporal expression profiles of RFX7 and RFX4 genes. (C) RFX7 and RFX4 are not expressed in the GRP at the stage 14. The GRP is indicated by red arrows. d: dorsal, v: ventral, a: anterior, p: posterior, b: brain, op: optic vesicles, ot: otic vesicles, sc: spinal cord, s: somites.
Fig. 2. Isolation of X. laevis RFX7 5′ and X. laevis RFX4 5′ clones. (A) Sequences of X. laevis RFX7 5′ (GenBank accession number: KF543241) and X. laevis RFX4 5′ (GenBank accession number: KF543240). The predicted initiation codon is indicated by red characters. (B) RFX7MO and RFX4MO blocked protein translation of RFX7 5′ and RFX4 5′, respectively. MOs and RNAs were co-injected into 2 cell stage embryos and embryos were collected at stage 10. Protein extracts from these embryos were used for immunoblotting.
Fig. 3. RFX7 and RFX4 are required for neural tube closure. (A) Dorsal view of bilaterally injected embryos at stage 18. Thirty-five nanograms of RFX7MO and 60 ng of RFX4MO were injected into two dorsal blastomeres of 4-cell stage embryos. One nanogram of Flag-xtRFX7 and Flag-RFX4 RNAs were used for rescue experiments. Brackets show the distance between the neural folds in RFX7 or RFX4 morphants. a: anterior, p: posterior. (B) The quantitative assessment of the injections in (A). At least three independent experiments were performed. “n†indicates the number of injected embryos.
Fig. 4. RFX7 and RFX4 are necessary for ciliogenesis in the neural tube. (A) Transverse section view of the neural tube at stage 23. Cilia are visualized by staining with acetylated α-tubulin (green) and ARL13B (red) antibodies. ARL13B is also expressed in cells without cilia at the neural tube. In RFX7 and RFX4 morphants, cilia were not detected. All sections from whole embryos were examined to detect cilia. White dashed lines outline the lumen of the neural tube. The number of embryos examined is indicated. (B) MOs and RNA injected embryos were collected at stage 23 and RNAs isolated from injected embryos were used to examine the gene expression. ODC was used as an internal control.
Fig. 5. The expression of RFX4 is regulated by RFX7 and Foxj1. (A) RFX4 rescued NTD in RFX7 morphants. The bar-graphs show the quantitative assessment of the injections. At least three independent experiments were performed. “n†indicates the number of injected embryos. (B) Five hundred picograms of Foxj1 RNA was injected into a dorsal blastomere of 4-cell stage embryos and the gene expression was examined at stage 14 by whole mount in situ hybridization. β-gal staining (red) indicates the injected side. The number of embryos examined is indicated. (C) Foxj1 RNA was injected into two dorsal blastomeres of 4-cell stage embryos and the gene expression was examined at stage 14 by RT-PCR.
rfx4 (regulatory factor X4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
rfx4 (regulatory factor X4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
rfx7 (regulatory factor X7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
rfx7 (regulatory factor X7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
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