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Fig. S1. Testing of Xag and Xagr2 MOs efficiency and analysis of Ras-dva1, Xag, FoxG1 and Fgf8 expression. (A) Xag2-TagRFP and Xagr2-TagRFP mRNA were injected in dorsal blastomeres of 8-cell embryos (100 pg/blastomere) either alone or in a mixture with the corresponding MO (8 nl of 0.4 mM water solution). The injected embryos were collected at the midneurula stage and analyzed for presence of Xag2-TagRFP and Xagr2-TagRFP proteins by Western blotting with an anti-tRFP antibody (see Materials and Methods for details). (B1,B2) At the late gastrula (stage 12), Ras-dva1 and Xag are expressed exclusively in the outer layer of the anterior ectoderm. No expression is in the inner layer, in which FoxG1 and Fgf8 begin to be expressed with the onset of neurulation. Adjacent vibratome sagittal sections of the same embryo were hybridized separately with Ras-dva1 (left section) or Xag (right section) probe. Anterior sides face each other, dorsal sides up. (C,D) Embryos at the tailbud stage (stage 23) hybridized in whole-mount with probes to Ras-dva1 and FoxG1, respectively. Dashed lines indicate approximate levels of sections shown in panels E1 and E2. Anterior view, dorsal sides up. (E1,E2) Adjacent vibratom frontal sections of the same stage 23 embryo hybridized separately with Ras-dva1 or FoxG1 probe (see approximate levels of sections in panels C and D). Anterior up. (F,G) Embryos at the tailbud stage (stage 20) hybridized in whole-mount with probes to Xag and FoxG1, respectively. Dashed lines indicate approximate levels of sections shown in panels H1 and H2. Anterior view, dorsal sides up. (H1,H2) Adjacent frontal sections of the same stage 20 embryo hybridized separately with Xag or FoxG1 probe (see approximate levels of sections in panels C and D). Anterior up. (I,J) Expression of FoxG1 and Fgf8 in presumptive telencephalic (inner layer, zone 1) and non-telencephalic (outer layer, zone 2) cells as it is seen in the midneurula embryos hybridized in whole-mount. Anterior view, dorsal side up. (K1-K4) Expression of FoxG1 and Fgf8 revealed on adjacent sagittal sections of the same embryo at midneurula stage. Vibratom sections of the same embryo were hybridized separately with FoxG1 or Fgf8 probe. Anterior sides face each other, dorsal sides up. (K5) Expression of FoxG1 in the same region as shown in panel K3 but at stage 18 (late neurula). Note that no expression is seen in the outer layer except few cells in posterior region of the expression spot. This region is in the internal surface of the anterior neural fold and further hives rise to the diencephalon. Scale bars: 200 mm (B1-K2), 40 mm (K3-K5). |
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Fig. 2. Pairwise comparison of expression patterns of genes expressed in the anterior ectoderm at the midneurula stage.
(A-D) Whole-mount in situ hybridization with probes to transcripts of the indicated pairs of genes on the left and right halves of individual embryos as it is described in Fig. 1A. (A1-D1,A2-D2) In situ hybridization on adjacent vibratome sagittal sections of individual embryos with probes to indicated pairs of transcripts. Note that panels C1,D1,C2,D2 show results of hybridization made on two pairs of adjacent sections of the same embryo. (A3-D3,A4-D4) Enlarged images of fragments framed in panels A1-D1 and A2-D2. For abbreviations, see Fig. 1A-A4. Scale bars: 200 µm (A-D2), 40 µm (A3-D4). |
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Fig. 3. Inhibition of Ras-dva1 mRNA translation by the Ras-dva1 morpholino elicits the downregulation of Agrs and a reduction of the forebrain.
(A,C) Whole-mount in situ hybridization of midneurula control embryos with dig-labeled probes to Xag and Xagr2, respectively. Anterior view with dorsal side upward. (B,B′,D,D′) Whole-mount in situ hybridization of the Ras-dva1 MO-injected midneurula embryos with dig-labeled probes to Xag and Xagr2, respectively. Fluorescent images in panels B′ and D′ demonstrate distribution of cell clones containing the co-injected FLD fluorescent tracer. (E) The telencephalon (upper row) and whole head of the control 5-day tadpole. Dorsal view, anterior to the top. (F,F′) The telencephalon (upper row) and whole head of the 5-day tadpole developed from the embryos injected with Ras-dva1 MO into the right dorsal blastomere at the 8-cell stage. Note the reduced telencephalon and eye on the injected side. The fluorescent image in panel F demonstrates the distribution of cell clones containing the co-injected FLD fluorescent tracer. (G,G′) Rescue of the Ras-dva1 MO-induced abnormalities by the co-injection of Ras-dva1 mRNA. Note the normal telencephalon and eye on the injected side (see distribution of the injected cells in panel G′). (H,H′) Whole-mount in situ hybridization of the Ras-dva1 MO-injected midneurula embryos with dig-labeled probes to FoxG1. Note the inhibition of FoxG1 expression on the injected side. See the distribution of cell clones containing the co-injected FLD fluorescent tracer in panel H′. (I,I′) Rescue of the Ras-dva1 MO-induced inhibition of FoxG1 expression by the co-injection of Ras-dva1 mRNA. See the distribution of cell clones containing the co-injected FLD fluorescent tracer in panel I′. |
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Fig. 7. Ras-dva1-mediated Fgf8 signaling induces expression of Agrs by upregulating Otx2.
(A-C) Otx2 is co-expressed with Ras-dva1 in cells of the outer layer of the anterior ectoderm. In situ hybridization on the left and right halves of the entire midneurula embryo and on sagittal vibratome sections was performed as described in the legends to Figs 1 and 2. (D,E) Inhibition of Fgf8 mRNA translation by an Fgf8 MO elicits partial inhibition of Otx2 expression and lateral and posterior expansion of the expression domain (red arrowheads). Yellow and black arrowheads indicate a reduction of high Otx2 expression in the presumptive midbrain and cement gland, respectively. (F,G,H,I) Inhibition of Otx2 mRNA translation by an Otx2 MO inhibits Xag and Ras-dva1 expression. (J,K,L,M) Co-injection of Fgf8a mRNA is unable to prevent the inhibitory influence of the Otx2 MO on Xag and Ras-dva1 expression. (N,O) Inhibition of Otx2 mRNA translation by the Otx2 MO inhibits Fgf8 expression (black arrows). (E′,G′,I′,K′,M′,O′) Fluorescent images of embryos shown in panels E,G,I,K,M,O demonstrate distribution of injected cells labeled by the co-injected FLD tracer. |
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Fig. S3. Lack of effects of control morpholino oligonucleotides on expression of all genes studied in this work. Control MO (supplementary material Table S1) to all genes whose expression was inhibited by active MO were injected in concentration 1 mM (3-5 nl/blastomere) in one of the animal dorsal blastomeres at 8 blastomere stage in mixture with living tracer FLD. Injected embryos were collected at midneurula stage and hybridized in whole-mount with probes to all genes analyzed in this work. |
