Ogino H , Fisher M , Grainger RM .

EY06675 NEI NIH HHS , EY10283 NEI NIH HHS , EY17400 NEI NIH HHS , RR13221 NCRR NIH HHS , R01 EY006675 NEI NIH HHS , R01 EY010283 NEI NIH HHS , R01 EY017400 NEI NIH HHS , R01 RR013221 NCRR NIH HHS

	Fig. 1. In vivo deletion analysis identifies a 901 bp enhancer that directs PLE-specific expression of FoxE3. (A) FoxE3 expression in X. tropicalis embryos (stage 23) detected by in situ hybridization. (B-G) GFP expression detected by in situ hybridization in representative transgenic embryos (stages 22-24) generated with the reporter constructs shown to the left. White and black arrows in A-G indicate the PLE. The arrowhead in B indicates ectopic GFP expression in the presumptive oral ectoderm. Numbers of embryos with GFP expression in the PLE and the total number of normally (or near normally) developing embryos injected with each construct are indicated to the right, along with the percentage of GFP-positive cases. The 901 bp element necessary for PLE-specific expression is boxed with a dotted red line. *The expression in D was positive in the PLE but very spotty and broad, as shown in the left-hand panel.
	Fig. 4. Comparative expression analysis of Notch signaling components and FoxE3 in X. laevis embryos, showing that Notch2 and FoxE3 are expressed in PLE, while Delta1 and Delta2 are expressed in presumptive retina. Expression of Notch2 (A-B′), Delta1 (C-E′), Delta2 (F-H′), and FoxE3 (I-K′) was detected by in situ hybridization from neural plate stages to early tailbud stages. Regions circled with black dotted lines in C and F are the approximate presumptive retina fields, where neither Delta1 nor Delta2 is expressed. Arrows in B,B′, D-E′, G-H′ and J-K′ indicate expression of Notch2, Delta1, Delta2 and FoxE3, respectively. The black arrowhead in C indicates Delta1 expression in the anterior neural ridge, and white arrowheads in A and I indicate Notch2 and FoxE3 expression in the pre-placodal ectoderm, respectively. White lines in E,H,K indicate the planes of transverse eye sections shown in E′,H′,K′.
	Fig. 5. Effects of manipulation of Notch signaling on FoxE3 expression and subsequent lens placode formation. (A,B) Frontal view of Xenopus embryos injected with mRNA encoding Delta2Tr (500 pg), fixed at stage 23, and subjected to lacZ staining (magenta) and in situ hybridization with FoxE3 or Rx probe (purple or deep purple staining). White and black arrowheads in A-H indicate in situ hybridization signals on injected and uninjected sides of embryos, respectively. (C,D,F,G) The injected and uninjected sides of embryos injected with Delta2Tr mRNA, fixed at stages 29/30, and hybridized with γ1-crystallin or Rx probe. (H) A transverse head section of the embryo shown in F,G. (E) The injected side of an embryo injected with both Delta2Tr mRNA (500 pg) and wild-type Delta2 mRNA (500 pg), fixed at stage 29, and hybridized with γ1-crystallin probe. (I) Summary of Delta2Tr mRNA injection experiments. GFP mRNA (1000 pg) was injected as a control. (J,K) The injected and uninjected sides, respectively, of an embryo injected with GR-Su(H)DBM mRNA (1000 pg) and induced with Dex. Arrows in J-O indicate endogenous FoxE3 expression in the PLE. (L) The injected side of an embryo injected with GR-Su(H)DBM but not induced with Dex. (M,N) The injected and uninjected sides, respectively, of an embryo injected with GR-Su(H)VP16 mRNA (1000 pg) and induced with Dex. Black and white arrowheads in M indicate ectopic FoxE3 expression in the ectoderm overlying the anterior brain and that surrounding the cement gland, respectively. (O) The injected side of an embryo injected with GR-Su(H)VP16 but not induced with Dex.
	Fig. 6. Otx2 confers the ability to activate FoxE3 in response to Notch signaling. (A-C′) Expression of Otx2 in the anterior ectoderm detected by in situ hybridization. At the neural plate stages, Otx2 is expressed in the anterior ectoderm including the presumptive lens-fields, which are circled with white dotted lines in A. Arrows in B indicate broad expression in the ectoderm that overlies the optic vesicles (ov) and surrounds the cement gland primordium (cg). Arrows in C indicate the border of ectodermal Otx2 expression. White lines in C indicate the planes of transverse sections shown in C′ and C′. Arrows in C′ and C′, respectively, indicate expression in the PLE overlying the optic vesicle and in the ectoderm overlying the forebrain. (D-H) Notch-Otx2 combination activates FoxE3 in the trunk ectoderm. Xenopus embryos injected with the mRNAs indicated in each panel were induced with Dex, and then subjected to lacZ staining and in situ hybridization with FoxE3 probe. Ectopic FoxE3 expression was not detected in embryos injected with mRNA encoding GR-Su(H)VP16 (1000 pg) (D), Otx2-GR (250 pg) (E), or NICD (1000 pg) (G), whereas it was detected in embryos injected with both GR-Su(H)VP16 (750 pg) and Otx2-GR (250 pg) (F), or both NICD (750 pg) and Otx2-GR (250 pg) (H). Arrowheads in F and H indicate ectopic FoxE3 expression. Arrows indicate endogenous FoxE3 expression in the PLE. The white line in F indicates the plane of the transverse section shown in the inset. Black arrowheads in the inset indicate the overlap of FoxE3 expression and nuclear lacZ staining in the ectodermal cells, and white arrowheads indicate cells in the underlying mesoderm layer showing nuclear lacZ staining but no FoxE3 expression. (I,J) The injected and uninjected sides, respectively, of an embryo injected with mRNA encoding GR-Otx2-En (250 pg), induced with Dex from stage 18, and then subjected to lacZ staining and in situ hybridization with FoxE3 probe at stage 22. Arrows indicate the PLE. (K) Transgenic experiments using Otx-Su(H) reporter constructs. Numbers of embryos with GFP expression in the PLE and the total number of normally (or near normally) developing embryos injected with the constructs shown on the left are indicated on the right-hand side with percentages of the GFP-positive cases. Gray and red boxes indicate Otx- and Su(H)-binding motifs, respectively, in the constructs, and crosses indicate base-substitution mutations introduced there. (L,L′) A representative transgenic embryo generated with Otx-Su(H)-βGFP. Black and white arrows indicate GFP expression in the eye and spinal cord, respectively. The white line indicates the plane of the transverse eye section shown in L′. Black and white arrowheads indicate GFP expression in the PLE and optic vesicle, respectively.
	Fig. 3. Mapping of regulatory motifs essential for PLE-specific activity of the FoxE3 enhancer. (A-C) Representative transgenic embryos (stages 22-24) generated with the GFP reporter constructs shown on the left. Black arrows indicate the PLE. Numbers of embryos with GFP expression in the PLE and the total number of normally (or near normally) developing embryos injected with each construct are indicated on the right-hand side with percentages of the GFP-positive cases. The white line in A indicates the plane of the transverse section shown in the inset. The black arrow in the inset indicates GFP expression in the PLE overlying the optic vesicle. The embryo shown in C was generated by co-transgenesis, i.e. co-injection of the 462 bp enhancer of Xenopus FoxE3 amplified by PCR along with the βGFP cassette. (D) Identification of transcription factor-binding motifs essential for PLE-specific expression by mutation analysis. wt is the construct used in Fig. 3A (Xt462-βGFP). mt1-mt9 were generated from wt/Xt462-βGFP by introducing a base-substitution mutation (cross) into each of the conserved transcription factor-binding motifs. The bar chart shows the percentage of the embryos that showed GFP expression in the PLE among total developed embryos injected with the constructs shown on the left. Actual numbers of GFP-positive cases and total numbers of scored embryos are indicated in parentheses.
	foxe3 (forkhead box E3) gene expression in X. tropicalis embryo, NF stage 23, assayed via in situ hybrodization, lateral view (left) and anterior view (right), dorsal up.
	Supplemental Figure S1 (Adobe PDF) Fig. S1. GFP expression driven by a 10.6 kb FoxE3 promoter closely recapitulates PLE-specific expression of endogenous FoxE3. GFP expression in Xenopus embryos injected with −10.6kGFP was examined by in situ hybridization (E-H). At neural plate stages (stages 13-15), GFP expression was detected in the pre-placodal ectoderm adjacent to the anterior margin of the neural plate (39%, n=44, E). After neural tube formation (stages 22-24), the expression was confined to the PLE overlying the developing optic vesicle and to the presumptive oral ectoderm (32%, n=135, F). In the late-tailbud embryos (stages 31-33), GFP expression was observed in the developing lens placode derived from the PLE and in the oral ectoderm, and additionally in cranial ganglia (57%, n=61, G and H). Comparison of the GFP expression with endogenous FoxE3 expression in X. tropicalis embryos detected by in situ hybridization (A-D) identified minor differences in the oral ectoderm, cranial ganglia and thyroid primordium (compare B-D and F-H). However, the GFP expression closely recapitulates the endogenous FoxE3 expression in the pre-placodal ectoderm, PLE and lens placode, indicating that the −10.6 kb region tested here contains all the information necessary for the expression in the lens lineage. Comparison of the GFP expression detected by in situ hybridization with that by epifluorescent microscopy (I-L) revealed a significant delay between its transcription and fluorescence development, which was likely to be caused by the time required for maturation and/or accumulation of GFP proteins (Tsien, 1998) (compare E-H and I-L). Triangles indicate expression in the pre-placodal ectoderm (A,E,I); red arrows indicate the PLE (B,F,J,K) and the lens placode (C,D,G,H, L); blue arrows indicate the presumptive oral ectoderm (B,F,G,J,K). tp, thyroid primordium; cg, cranial ganglia. Tsien, R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem.67, 509-544. Supplemental Figure 2
	Supplemental Figure S2 (Adobe PDF) Fig. S2. The 462 bp enhancer of Xenopus FoxE3 and the orthologous mouse element drive indistinguishable PLE-specific expression in Xenopus embryos. (A) A representative transgenic embryo (stage 24) generated with a GFP reporter construct where the 462 bp Xenopus enhancer is directly linked to the FoxE3 basal promoter (−640 promoter used in Fig. 1E). (B) A representative transgenic embryo (stage 24) generated with a GFP reporter construct where the orthologous mouse element (423 bp) is directly linked to the FoxE3 basal promoter. Black arrows indicate GFP expression in the PLE detected by in situ hybridization. Numbers of embryos with GFP expression in the PLE and the total number of normally (or near normally) developing embryos injected with the constructs shown on the left are indicated on the right side with percentages of the GFP-positive cases.
	Supplemental Figure S3 (Adobe PDF) Fig. S3. Gel retardation assay showing in vitro binding of Su(H) and Otx2 proteins to the FoxE3 enhancer. The actual probe length was 33 bp, but only sequences around the Su(H) or Otx motif are shown at the top. (A) Labeled probe DNAs were incubated with either glutathione S-transferase (GST, indicated as −) or Xenopus Su(H) protein fused to GST (indicated as +) (Wettstein et al., 1997). A DNA-protein complex (c) was formed with a probe containing the putative Su(H) motif of the FoxE3 enhancer probe FoxE3-Su(H), as well as with a probe containing the previously identified high-affinity Su(H) site of the m8 gene in the Drosophila Enhancer of split cluster probe E(spl) m8 (Tun et al., 1994). The complex was not formed with a mutated FoxE3 probe whose Su(H) site has the same four-base substitution probe FoxE3-Su(H) mt, mutated sequences are underlined as the mutant reporter construct used in the transgenic assay (Fig. 3D, mt7). The m8 probe and Su(H) consensus sequences shown here are the reverse strands of those originally reported (Tun et al., 1994). (B) Labeled probe DNAs were incubated with either the rabbit reticulocyte lysate (TNT Quick Coupled Transcription/Translation System, Promega) programmed with an empty vector (pCS2+) (Turner and Weintraub, 1994) (indicated as −) or that programmed with Xenopus Otx2 expression vector (pCS2+XOtx2; indicated as +). The Otx2 protein formed two shifted complexes (c) with a probe containing the 3′ Otx motif of the FoxE3 enhancer (probe FoxE3-Otx), as well as with a probe containing the previously identified Otx2-binding site of rat gonadotropin-releasing hormone (rGnRH) gene (probe rGnRH-Otx) (Kelley et al., 2000). The shifted complexes were not formed with a mutated FoxE3 probe whose Otx site has the same four-base substitution (probe FoxE3-Otx mt, mutated sequences are underlined) as the mutant reporter construct used in the transgenic assay (Fig. 3D, mt5). The two shifted bands observed are likely to be due to the monomeric and dimeric DNA-binding activity of Otx2 (Briata et al., 1999). Briata, P., Ilengo, C., Bobola, N. and Corte, G. (1999). Binding properties of the human homeodomain protein OTX2 to a DNA target sequence. FEBS Lett.445, 160-164. Kelley, C. G., Lavorgna, G., Clark, M. E., Boncinelli, E. and Mellon, P. L. (2000). The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter. Mol. Endocrinol.14, 1246-1256. Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev.8, 1434-1447. Wettstein, D. A., Turner, D. L. and Kintner, C. (1997). The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development124, 693-702.
	Supplemental Figure S4 (Adobe PDF) Fig. S4. Chromatin immunoprecipitation (ChIP) assay showing in vivo binding of Su(H) and Otx2 to FoxE3 enhancer. (A) Sheared crosslinked chromatin was prepared from stage 22-24 embryos injected with mRNA encoding either myc-Su(H) (Wettstein et al., 1997) (200 pg per embryo) or myc-tag alone, and immunoprecipitated with 5 µg of anti-myc antibody (Santa Cruz, sc-40). The FoxE3 enhancer is enriched approximately 4.5-fold in the chromatin sample prepared from embryos expressing myc-Su(H) as compared with the sample from embryos expressing myc-tag alone, whereas exon sequences of FoxE3 and EF-1α are not siginificantly enriched. Consistent with this result, anti-human Su(H)/RBP-Jκ antibody (Santa Cruz, sc-8213), which weakly binds to native Xenopus Su(H) protein, enriched the FoxE3 enhancer in chromatin samples prepared from uninjected embryos, although the enrichment level was lower (1.8-fold compared with control IgG, not shown). (B) Chromatin samples were prepared from heads of uninjected stage 22-24 embryos, and imunoprecipitated with either 5 µg of control IgG or anti-Otx2 antibody (Abcam, ab21990). The strong reactivity of this anti-Otx2 antibody to Xenopus Otx2 protein was confirmed by western blotting (not shown). The FoxE3 enhancer is enriched approximately 10-fold in the anti-Otx2 chromatin sample compared with the sample treated with control IgG, whereas the exon sequences of FoxE3 and EF-1α are not siginificantly enriched. In A and B, the size of the sheared chromatin was 200-1000 bp (not shown), and the EZ ChIP Kit (Upstate Biotechnology) was used with some modifications (our detailed protocol is available upon request). Relative enrichment of imunoprecipitated DNA sequences was analyzed by real-time PCR (MyiQ system, Bio-Rad) using a dilution series of the input chromatin as standard curve templates, and means of three independent experiments are shown with standard errors. The primers used were as follows: for FoxE3 enhancer, 5′- AGGTTTAAGGGTGACCTGCTC-3′ and 5′-TGTGTGGGATTTCTGCAGTC-3′; for FoxE3 exon, 5′-TGAATGGGAACCTAGGGAAC-3′ and 5′-AGGTTAGGTTGGAAGACACAGC-3′; for EF-1α exon, 5′-ATGCACCATGAAGCCCTTAC-3′ and 5′- CTTCCATTGGTGGGTCATTC-3′. These primers were designed for X. laevis DNA sequences. X. laevis FoxE3 enhancer sequence has 95% identity with the X. tropicalis sequence in Fig. 2B (not shown).
	Fig. S5. [A, B] A Xenopus laevis EST clone (NCBI accession BX855333) was identified as a partial cDNA clone of Xenopus Notch2. (A) An amino acid sequence encoded by BX855333 (indicated as XNotch2) is aligned with partial amino acid sequences of mouse Notch proteins and Xenopus Notch1 (mNotch2, mNotch3, mNotch4, mNotch1 and XNotch1; NCBI accession NP035058, NP032742, NP035059, NP032740 and AAB02039). This alignment and the phylogenetic tree (B) were both generated on the ClustalW website (http://www.ebi.ac.uk/clustalw/). XNotch1 is the only Notch family member previously reported in Xenopus (Chitnis et al., 1995). (B) A phylogenetic tree showing evolutionary distances between the protein encoded by BX855333 and other Notch family proteins. Note that BX855333 is closest to mouse Notch2. (
	Supplemental Figure S5 (C,C′) In situ hybridization analysis of XNotch2 (BX855333) expression in late-tailbud embryos. Expression is evident in the lens vesicle (black arrow), olfactory placode (white arrowhead) and otic vesicle (black arrowhead) (C). A transverse eye section shows the localized expression in the lens epithelium (white arrow, C′).
	Supplemental Figure S6 (Adobe PDF) Fig. S6. Activation of FoxE3 by hormone-inducible versions of Su(H) and Otx2 under conditions of inhibition of protein synthesis by cycloheximide. We injected mRNAs encoding GR-Su(H)VP16 (750 pg) and Otx2-GR (250 pg) into a ventral blastomere of 4-cell stage embryos to target expression in the trunk ectoderm. When the injected embryos reached stages 22-24, they were placed for 1 hour into medium containing cycloheximide (10 µg/ml). According to our previous data (Martynova et al., 2004), this period of time is sufficient to block total protein synthesis by more than 90%. After this period, dexamethasone (Dex) was added to the same incubation medium at a 10 µM final concentration to release activities of the previously accumulated GR-Su(H)VP16 and Otx2-GR proteins. Under these conditions, only direct targets of Su(H) and Otx2 should be activated because mRNA translation is blocked by cycloheximide. After 2 hours of incubation with Dex, the embryos were processed for in situ hybridization with FoxE3 probe. Ectopic FoxE3 expression was detected in the trunk ectoderm of 60% of these embryos (n=48, A, arrowheads), whereas it was not detected in any sibling embryos incubated in the cycloheximide-containing medium but without Dex (n=53, B). Arrows in A and B indicate endogenous FoxE3 expression in the PLE. Cycloheximide (Sigma) was prepared as a 10 mg/ml stock solution in ethanol and stored at −20°C; this stock solution was freshly diluted to 10 µg/ml in 0.3×MMR for culturing embryos. Martynova, N., Eroshkin, F., Ermakova, G., Bayramov, A., Gray, J., Grainger, R. and Zaraisky, A. (2004). Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate. Development131, 2329-2338.

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