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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
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.
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. Development 124, 693-702.
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 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. (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′).
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. Development 131, 2329-2338.
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