Tereshina MB , Ermakova GV , Ivanova AS , Zaraisky AG .

	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).
	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).
	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′.
	Fig. 4. Fgf8 regulates the expression of FoxG1, Ras-dva1, Xag and Xagr2. (A-D) Injections of Fgf8a mRNA elicit ectopic expression of FoxG1, Ras-dva1, Xag and Xagr2 in the anterior ectoderm of the midneurula stage embryos. Fgf8a mRNA mixed with FLD tracer was injected at concentration of 20 pg/blastomere in a pair of adjacent animal dorsal and ventral blastomeres on the left sides of 16- to 32-cell stage embryos. Whole-mount in situ hybridization was performed at the midneurula stage. Anterior view with the dorsal side upward. (A′-D′) Overlays of the white light and fluorescent images of embryos shown in panels A-D. Red arrowheads in panels A and C indicate the borders of ectopic expression, which correspond to the borders of cell clones with strong fluorescence. (E-H) Vibratome sections of embryos injected with Fgf8a mRNA. Note that whereas Ras-dva1, Xag and Xagr2 are activated only in the outer layer of the ectoderm (E-G, overlays of bright light and fluorescent images), ectopic FoxG1 is induced exclusively in the inner layer (H,H′). (I-L) Injection of Fgf8a MO leads to inhibition of FoxG1, Ras-dva1, Xag and Xagr2 expression. An Fgf8a MO (1-2 pmol/blastomere) was injected with a FLD tracer in a pair of adjacent animal dorsal and ventral blastomeres on the left sides of 16- to 32-cell stage embryos. (M,N) Co-injection of Fgf8 mRNA is unable to rescue inhibition of Xag and Xagr2 expression elicited by Ras-dva1 MO. The black arrowheads indicate sites of expression inhibition.
	Fig. 5. Inhibition of Xag and Xagr2 mRNA translation by anti-sense morpholinos elicits brain abnormalities similar to those observed when Ras-dva1 and Fgf8 were inhibited. (A,C) Inhibition of Xag (A) and Xagr2 (C) mRNA translation by anti-sense morpholinos elicits reduction of the telencephalon (see enlarged images in the upper row, black arrow), otic vesicles (yellow arrowhead) and eyes (red arrowhead for Xag downregulation). (A′,C′) Overlays of the white light and fluorescent images of embryos shown in panels A and C demonstrate the distribution of cells containing injected MO mixed with a FLD tracer. (B,D) Rescue of anatomical abnormalities by co-injection of Xag and Xagr2 mRNAs with Xag and Xagr2 morpholinos to these genes. (B′,D′) Overlays of the white light and fluorescent images of embryos shown in panels B and D demonstrate the distribution of cells containing the injected MO mixed with a FLD tracer.
	Fig. 6. Agrs, Fgf8 and Ras-dva1 regulate expression of each other. (A-C) Injections of Xag and Xagr2 MO elicit inhibition of FoxG1, Fgf8 and Ras-dva1 expression in the anterior ectoderm of midneurula embryos. (D) Injections of Ras-dva MO elicit inhibition of Fgf8 expression in the anterior ectoderm of midneurula embryos. (E,F) Co-injection of Agr mRNA (equimolar mixture of Xag1, Xag2, Xagr2A and Xagr2B mRNAs) or Ras-dva1 mRNA is unable to prevent the inhibitory influence of Fgf8 MO on FoxG1 expression. (G) Injection of Fgf8a mRNA elicits massive ectopic expression of FoxG1 in the anterior ectoderm. (H) Induction of FoxG1 expression elicited by ectopic Fgf8a is suppressed by co-injection of Agrs MO (a mixture of Xag and Xagr2 MO). (A′-H′) Fluorescent image of the embryo shown in panels A-H demonstrates the distribution of injected cells labeled by the co-injected FLD tracer.
	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.
	Fig. 8. Model for the Fgf8- and Agrs-based signal exchange between neural and non-neural cells at the anterior neural plate border. Fgf8 produced by the ANB cells induces the expression of Agrs in cells adjacent to the anterior non-neural ectoderm by mediating Ras-dva1 and Otx2. In turn, Agrs secreted by cells in the anterior non-neural ectoderm promote forebrain development through stimulation of both Fgf8 gene expression and Fgf8 protein signaling in the ANB cells.
	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).
	Fig. S2. Analysis by transgenic embryos of the effects of Ras-dva1 downregulation. (A-A2) Whereas no cement gland inhibition is revealed in control transgenic embryos expressing wild-type Ras-dva1 under the control of Xag2 promoter (proXag2-wtRas-dva1-proCA-DsRed transgene), the cement glands are inhibited in embryos expressing Xanf1 under the same promoter (proXag2-Xanf1-proCA-DsRed transgene). (A) Bright light, (A1) DsRed fluorescence, (A2) overlay of panels A and A1. (B�C4) 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 (B�B4), the embryo of the same line but transfected by proXAG2-dnRas-dva1-proCA-DsRed construct has no cement gland (C�C4). (D) No inhibition of FoxG1 expression is seen in the early neurula embryos bearing the control transgene proXag2-wtRas-dva1-proCA-DsRed (a). In contrast, a decrease of FoxG1 expression is observed in embryos transfected with proXAG2-Xanf1-proCA-DsRed. Whole-mount in situ hybridization with probes to both FoxG1 and DsRed. (E) Transgenic tadpole bearing the control proXag2-wtRas-dva1-proCA-DsRed construct has normal sized telencephalon marked by FoxG1 expression (a). At the same time, a reduction of the telencephalon and FoxG1 expression is seen in embryo bearing proXAG2-Xanf1-proCA-DsRed construct (b). Transgenic tadpoles were selected by revealing DsRed fluorescence and hybridized in whole-mount with the probe to FoxG1.
	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.
	Fig. S4. Effects elicited by misexpression of Fgf8, Otx2 and Xag. (A,B,C,D) Inhibition of Fgf8a and Xag mRNA translation by antisense morpholino oligonucleotides inhibit cement gland differentiation and cause a reduction in the telencephalon and eyes. (E,F) Examples of the midneurula stage embryos (anterior view with the dorsal side upward) injected with Fgf8a mRNA and hybridized in whole mount with a dig-labeled probe to Otx2. Note the expansion of the domains with low Otx2 expression that are bordered along the injected areas by stripes of enhanced expression. Black and white arrowheads indicate the borders of the injected cell clones. (G,H) Whereas Fgf8 MO injected alone results in the inhibition of Xag expression, coinjection of Otx2 mRNA elicits rescue of Xag expression. (I) Ras-dva1 mRNA co-injected with Fgf8 MO cannot rescue Xag expression. (J,K) In contrast to Xag, co-injection of Otx2 mRNA is unable to rescue FoxG1 expression inhibited by Fgf8 MO.

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