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Fig. 1. Ras-dva1 is expressed in the non-neural anterior ectoderm and regulates forebrain development by a cell non-autonomous mechanism.
(A) In situ hybridization with dig-labeled probes to Ras-dva1 and FoxG1 on the left and right halves of the same embryo. Upon completing the in situ hybridization procedure, the two halves of the embryo were stacked together and photographed from the anterior with the dorsal side upward. The dotted line corresponds to the dotted lines in panels A1-A4. (A1,A2) Adjacent vibratome medial sagittal sections of the same embryo were hybridized separately with Ras-dva1 (left section) or FoxG1 (right section) probe. Anterior sides face each other, dorsal sides up. "1" indicates region of the inner layer in which only FoxG1 is expressed. "2" indicates region of the outer layer in which Ras-dva1 and FoxG1 are co-expressed. (A3,A4) Enlarged images of fragments squared in panels A1 and A2. (B) In situ hybridization with dig- and fluorescein-labeled probes to Ras-dva1 and FoxG1 on the vibratom medial sagittal section of the midneurula (stage 15) embryo. (C) Schemas of DNA constructs used to generate transgenic embryos shown in panels C-J. (D-E′) Whereas no cement gland inhibition is seen in control embryo of transgenic line bearing proXag2-EGFP construct and transfected by proXag2-wtRas-dva1-proCA-DsRed (D,D′), the embryo of the same line but transfected by proXAG2-dnRas-dva1-proCA-DsRed construct has no cement gland (E,E′). (F) No inhibition of FoxG1 expression is seen in the early neurula embryo bearing the control transgene (proXag2-wtRas-dva1-proCA-DsRed) (a). In contrast, a decrease of FoxG1 expression is observed in the embryo transfected with proXAG2-dnRas-dva1-proCA-DsRed (b). Whole-mount in situ hybridization with probes to both FoxG1 and DsRed. (G) Transgenic tadpole bearing the control proXag2-wtRas-dva1-proCA-DsRed construct has normal sized telencephalon marked by FoxG1 expression. At the same time, a reduction of the telencephalon and FoxG1 is seen in embryo bearing proXAG2-dnRas-dva1-proCA-DsRed construct. Transgenic tadpoles were selected by revealing DsRed fluorescence and hybridized in whole-mount with the probe to FoxG1. (H-K) The telencephalons (upper row) and whole heads (bottom row) of the 5-day tadpoles bearing transgenic constructs indicated on the top. Scale bars: 200 µm (A-A2,B), 40 µm (A3,A4). |
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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. 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. |
