Matsuo-Takasaki M et al. (2005), An essential role of Xenopus Foxi1a for ventral...

XB-ART-1549

Development 2005 Sep 01;13217:3885-94. doi: 10.1242/dev.01959.

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

Matsuo-Takasaki M , Matsumura M , Sasai Y .

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During gastrulation in Xenopus, the head ectoderm is subdivided into the central nervous system (CNS) anlage (neural plate) and the non-CNS ectoderm (i.e. epidermis, placodes and neural crest). The winged-helix transcription factor Xfoxi1a is one of the earliest markers for the preplacodal region at the mid-neurula stage. Interestingly, before the establishment of the preplacodal region, Xfoxi1a expression is detected in the entire cephalic non-neural ectoderm at the mid- and late gastrula stages. The present study focuses on the role of Xfoxi1a particularly at the gastrula stages. The early Xfoxi1a expression in the anteroventral ectoderm is dependent on Bmp signals and suppressed by Wnt signals. Inhibition of Xfoxi1a activities by injection of antisense oligonucleotides leads to suppression of non-CNS ectodermal markers (e.g. keratin) and expansion of the anterior expression domain of the CNS marker Sox2. Conversely, misexpression of Xfoxi1a suppresses Sox2 and induces keratin in the anterior neural plate. In the animal cap, Xfoxi1a overexpression antagonizes the neuralizing activity of Chordin (Chd). Studies using an inducible Xfoxi1a construct (GR-Xfoxi1a) show that the ventralizing function of Xfoxi1a is confined to the gastrula stage. Thus, Xfoxi1a is an essential regulator of ventral specification of the early head ectoderm during gastrulation.

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Species referenced: Xenopus
Genes referenced: ag1 bmp4 chdh chrd dkk1 dlx3 dlx5 foxd3 foxi4 foxi4.2 hsp70 hspa1l krt12.4 krt12.5 msx1 myod1 six1 sox2 wnt1
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	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.
	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.
	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 δ5′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.δ 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.
	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.
	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.
	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.
	foxi4.2 (forkhead box I4, gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, anterior left, dorsal up.
	foxi4.2 (forkhead box I4, gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.
	foxi4.2 (forkhead box I4, gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	foxi4.2 (forkhead box I4, gene 2) red, and six1 (SIX homeobox 1) blue, gene expression in Xenopus laevis embryo, assayed via double in situ hybridization, NF stage 15, anterior view, dorsal up.
	Whole-mount in situ hybridization using albino embryos was performed with foxi4.2. Stage 15.