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Fig. 4. Rfx2 is required for GRP cilia assembly and the left–right asymmetry pattern. (A) GRP tissue of a membrane RFP-injected control embryo. Acetylated bα-tubulin labels cilia (green) and RFP labeled cell boundary (red). (B) GRP tissue of a Rfx2 morphant. Membrane-RFP is co-injected as a tracer. (C) GRP tissue of an embryo injected with Rfx2 morpholino and GFP-Rfx2 mRNA. (A′), (B′), (C′) are zoom in view of (A), (B), (C), respectively. (D) Average length of GRP cilia. While the average length of control GRP cilia is 8.59± 0.18 μm (mean±SEM, n=69), it's significantly reduced to 2.34±0.07 μm in Rfx2 morphants (mean±SEM, n=113). The shorter cilia phenotype is partially rescued to 4.74± 0.14 μm(mean±SEM, n=63) by co-injecting with GFP-Rfx2 mRNA. Horizontal lines indicate the mean, vertical lines SEM, *** pb0.0001 Mann–Whitney test. (E–F, I) Pitx2c expression at stage 26, lateral view. In control embryos, Pitx2c is expressed in the left LPM (black arrow) but not the right LPM (blue arrow). (G–H) In Rfx2 morphants, bilateral LPM of Pitx2c expression is observed. (blue arrow). (I) Quantification of Pitx2c expression patterns in control embryos and Rfx2 morphants. |
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Fig. 8. Rfx2 is required for GRP cilia assembly and the left–right asymmetry. (A–B) Pitx2c expression at stage 26, lateral view. In wildtype embryos, Pitx2c is expressed in the left LPM (A, A′, C). In TTC25 morphants, only 33% of embryos show normal left LPM expression (C), and Pitx2c is often expressed in the right LPM (B′, C). (D–E) Confocal images of the posterior GRP (node) stained with acetylated α-tubulin to visualize cilia and injected with membrane-RFP to outline cells. (F) GRP cilia were measured in confocal images as in D, E for 5 embryos each. Horizontal lines indicate the mean, vertical lines SEM, pb0.0001 Mann–Whitney test. |
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pitx2 ( paired-like homeodomain 2 ) gene expression in Xenopus laevis embryos, NF stage 26, as assayed by in situ hybridization. Lateral view: anterior left in "E", right in "F", dorsal up. |
