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Fig. 3. Rfx2 is required for Shh signaling. (A–C) In situ hybridization of a Shh downstream gene, Nkx2.2.Dorsal view, anterior left. Stage 24. Expression of Nkx2.2 in the ventral neural tube (A) is dramatically reduced in the spinal cord of Rfx2 morphants (21/29) (B). The reduced expression of Nkx2.2 can be rescued by co-injection of 300 pg GFP-Rfx2 mRNA (C). (D–F) In situ hybridization of a Shh downstream gene, Vax1. Lateral view, anterior left. Stage 35.Expression of Vax1 in the ventral forebrain (D) is robustly reduced in Rfx2 morphants (16/24) (E) and can be rescued (F). |
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Fig. 7. TTC25 is required for proper neural tube cilia formation and Shh signaling pathway. (A–B) Nkx2.2 expression at stage 24, dorsal view, anterior left. Expression of Nkx2.2 in the ventral neural tube (A) is reduced in the spinal cord of TTC25 morphants (B). (C–D) Vax1b expression at stage 35, lateral view, anterior left. Expression of Vax1b in the ventral forebrain (C) is reduced, but still present, in TTC25 morphants (D). (E–F) Confocal Z-projections of stage 24 neural tube sections stained for cilia markers acetylated α-tubulin (green)and Arl13b (red). (E) In control embryos, most cilia project into the lumen of the neural tube; ventral neural cilia are longer than dorsal ones and express less Arl13b. (F) In TTC25 morphants, the neural tube fails to close and cilia are not found at the luminal boundary (top). Cilia are fewer, shorter, and stain more intensely with Arl13b. (G) Ventral cilia were measured in 3 sections (as in E, F) from 5 embryos each. The 20 longest ventral cilia from each embryo are plotted. Horizontal lines indicate the mean, vertical lines SEM, pb0.0001 Mann–Whitney test. |
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Suppl. Fig. 4. Rfx2 is required for proper neural tube cilia formation and Shh signaling. (A) Transverse section view of neural tube lumen of stage 26 control embryos. Cilia are stained with acetylated α-tubulin. (B) Transverse section view of neural tube lumen of Rfx2 morphants. Fewer and shorter cilia are observed. (C) Ventral cilia were measured in 12 μm projection confocal images. The average length of control cilia is 5.95 0.21 μm (mean SEM, n = 34). However, the average length significantly reduces to 2.6 0.07 μm in Rfx2 morphants (mean SEM, n = 61). Horizontal lines indicate the mean, vertical lines SEM, *** p < 0.0001 Mannhitney test. (D) In situ hybridization of a Shh downstream gene, Nkx2.2.Dorsal view, anterior top. Stage 24. Expression of Nkx2.2 in the ventral neural tube (D) is dramatically reduced in the spinal cord of Rfx2 morphants (72%, 21/29) (E). The reduced expression of Nkx2.2 is partially rescued by co-injection of GFP-Rfx2 mRNA. (F) In embryos co-injected with 100 pg GFP-Rfx2 mRNA, 55% (12/22) show robustly reduced Nkx2.2 expression. (G) 33% (6/20) of embryos show dramatically reduced Nkx2.2 expression in embryos co-injected with 200 pg GFP-Rfx2. (H) In embryos co-injected with 300 pg GFP-Rfx2 mRNA, 22% (4/18) of embryos show dramatically reduced Nkx2.2 expression. |
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nkx2.2 (NK2 homeobox 2 ) gene expression in Xenopus laevis embryos, NF stage 24, as assayed by in situ hybridization, dorsal view, anterior left. |
