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First published online 3 August 2005
doi: 10.1242/dev.01959

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An essential role of Xenopus Foxi1a for ventral specification of the cephalic ectoderm during gastrulation

Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan

View larger version (61K): [in a new window]   Fig. 1. Temporal and spatial expression of Xfoxi1a/b. Whole-mount in situ hybridization using albino embryos was performed with Xfoxi1a (A-C,E,F,I) or Xfoxi1b (J-L) probes. Double in situ hybridization was performed with (D) Xfoxi1a (turquoise; BCIP) and Sox2 (indigo; BM purple) probes, (G) Xfoxi1a (indigo; BM purple) and Sox2 (turquoise; BCIP) probes, and (H) Xfoxi1a (purple; magenta-phosphate) and Xsix1 (turquoise; BCIP) probes. (A) Animal view; (B) lateral view; (C,E,I,J,L) lateral view (anterior towards the left); (D,F,G,H,K) anterior view (dorsal towards the top). The embryo stage is shown in each panel. A, anterior; An, animal; D, dorsal; P, posterior; V, ventral; Vg, vegetal. (M) Amino acid sequence alignment of Xfoxi1a and Xfoxi1b. Identical and similar amino acid residues are marked with asterisks and double dots, respectively. Gaps are indicated by dashes.

View larger version (74K): [in a new window]   Fig. 2. Regulation of Xfoxi1a expression by Bmp and Wnt signals. (A-J) Effects of Bmp or Wnt signals on Xfoxi1a expression in the gastrula and neurula were analyzed by injecting pCS2-BMP4 (2.5 pg DNA/cell) (B,G), Chd (50 pg RNA /cell) (C,H), pCS2-Wnt1 (2.5 pg DNA/cell) (D,I) or Dkk1 (125 pg RNA/cell) (E,J) into all the animal blastomeres of eight-cell embryos. The embryos were fixed at stage 12 or 15, then whole-mount in situ hybridization was performed with a probe for Xfoxi1a. Control embryos are shown in A and F. (K) Gene expression in animal caps injected with RNAs encoding Chd (200 pg) or Chd (200 pg) +Bmp4 (20 pg) was analyzed by RT-PCR. (L-Q) Animal caps given a Chd mRNA (200 pg; M and P) or pCS2-Wnt1 (10 pg; N and Q) injection were excised at stage 9, and then cultured in LCMR until sibling embryos reached stage 12. The Xfoxi1a (L-N) or Sox2 (O-Q) probes were used for whole-mount in situ hybridization.

View larger version (54K): [in a new window]   Fig. 3. Loss of Xfoxi1a/b function results in an expansion of the neural plate and reduction of non-neural ectodermal tissues. (A) The Xfoxi1a/b-MO binding sites are shown with the underlines. The red box indicates the start codon. Identical nucleotides are marked with asterisks. (B) Flag-tagged Xfoxi1a mRNA (50 pg/cell) was injected with Xfoxi1a-MO (2.5 ng/cell), Xfoxi1b-MO (2.5 ng/cell) or the control five-base mismatched Xfoxi1a MO into animal cells of eight-cell embryos. (C) Flag-tagged Xfoxi1b mRNA (50 pg/cell) was injected with Xfoxi1b-MO (2.5 ng/cell), Xfoxi1a-MO (2.5 ng/cell) or control five-base mismatched Xfoxi1b MO into animal cells of eight-cell embryos. (D) Flag-tagged 5'UTR-Xfoxi1a mRNA (50 pg/cell) was injected with Xfoxi1a-MO (2.5 ng/cell) or Xfoxi1b-MO (2.5 ng/cell) into animal cells of eight-cell embryos. Animal caps were excised at stage 9 and cultured until stage 11. Xfoxi1a-flag (B,D) or Xfoxi1b-flag (C) proteins were detected by western blot analysis using an anti-flag antibody. Hsp70 was used as the loading control. 5'UTR means that the synthetic mRNA contains only the coding sequence and not the target sequence of Xfoxi1a/b-MO. (E-U) Xfoxi1a-MO (12.5 ng/cell; i1aMO; E-M), five-base mismatched control MO of Xfoxi1a (12.5 ng/cell; 5mis; N-P), Xfoxi1a-MO (12.5 ng/cell) ± Xfoxi1a mRNA (25 pg/cell; Q,R) or Xfoxi1b-MO (12.5 ng/cell; i1bMO; S-U) was injected into two unilateral blastomeres of eight-cell embryos. Embryos were harvested at stage 14-15 (E-J,L-U) or stage 24 (K) and analyzed by whole-mount in situ hybridization with the probes indicated in each panel. (E-H,J,L-O,Q-T) Dorsal view (anterior towards the top); (I,K,P,U) anterior view (dorsal towards the top). Injected sides are marked with arrowheads. Dashes indicate the midline. NP, nasal placode. Double-headed arrows in G,Q show the expansion of Dlx3– and Six2+ regions, respectively.

View larger version (59K): [in a new window]   Fig. 4. Loss of Xfoxi1a/b function leads to reduction of epidermal tissue in naïve ectodermal cells. Animal caps given injection of Xfoxi1a-MO (50 ng), Xfoxi1b-MO (50 ng), Xfoxi1a-MO+Xfoxi1b-MO (25 ng each) or five-base mismatched control MOs for Xfoxi1a and Xfoxi1b (25 ng each) were excised from stage 9 embryos, and then cultured until sibling embryos reached stage 14. RT-PCR was performed using primers to detect the neural marker Sox2, non-neural ectodermal markers (XK81, Dlx3, Msx1) and the mesodermal marker MyoD. H4 (histone H4) was used as the loading control.

View larger version (67K): [in a new window]   Fig. 5. Microinjection of Xfoxi1a mRNA induces epidermal differentiation and suppresses neural induction in both in vivo and in vitro. Xfoxi1a mRNA (25 pg/cell) was injected into two unilateral blastomeres of eight-cell embryos. Embryos were fixed at stage 14-15 and then whole-mount in situ hybridization was performed with the following probes. (A) Sox2, (B) XK81, (C) Dlx3, (D) FoxD3 and (E) Six1. (A-D) Dorsal view (anterior towards top); (E) anterior view (dorsal towards the top). Dashes indicate the midline. White arrows indicate the injected side. The activity of Xfoxi1a (12.5 pg/cell) was assessed by RT-PCRs in Chd (50 pg/cell)-injected animal caps (F) or dominant-negative Bmp receptor (dnBMPR) (100 pg/cell)-injected animal cap (G). RNAs were injected into all the animal blastomeres of eight-cell embryos. The animal caps were excised at stage 9 and cultured until sibling embryos reached stage 15. The expression patterns of the neural marker Sox2, the non-neural ectodermal markers (XK81, Dlx3, Msx1, Xfoxi1a, Bmp4), the mesodermal marker MyoD were analyzed. H4 was used as the loading control.

View larger version (78K): [in a new window]   Fig. 6. Crucial time window of Xfoxi1a. GR-Xfoxi1a mRNA (50 pg/cell) was injected into two unilateral animal blastomeres of eight-cell embryos. Dex was added at stage 11 (B,E,H,K) or stage 13 (C,F,I,L). Embryos were harvested at stage 15 and used for whole-mount in situ hybridization with a probe for Sox2 (A-C), XK81 (D-F), FoxD3 (G-I) or Six1 (J-L). Embryos without Dex treatment (A,D,G,J) were used as the negative control. Arrowheads indicate the injected side. (M) Flag-tagged GR-Xfoxi1a mRNA (50 pg/cell) was injected into all the animal blastomeres of eight-cell embryos. Animal caps were excised at stage 9 and cultured until stage 11, 13 or 15. The intact form of flag-GR-Xfoxi1a was detected by western blot analysis. Hsp70 was used as the loading control.