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First published online 16 June 2004
doi: 10.1242/dev.01219

Development 131, 3307-3317 (2004)
Published by The Company of Biologists 2004
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Regulation of Otx2 expression and its functions in mouse epiblast and anterior neuroectoderm

Daisuke Kurokawa1, Nobuyoshi Takasaki1, Hiroshi Kiyonari2, Rika Nakayama2, Chiharu Kimura-Yoshida1, Isao Matsuo1 and Shinichi Aizawa1,2,*

1 Laboratory for Vertebrate Body Plan, Center for Developmental Biology (CDB), RIKEN Kobe, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0046, Japan
2 Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology (CDB), RIKEN Kobe, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0046, Japan



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  		Fig. 2. Dissection of ectodermal enhancers at 5' upstream. (A) Schematic diagram depicting the 15 fragments assayed for enhancer activity. Number of lines exhibiting ß-gal expression among permanent transgenic lines established is indicated in parenthesis. Fragments displaying enhancer activity are indicated in red. (B) ß-Gal expression driven by #12 (a-f), by endogenous enhancers in Otx2+/lacZ knock-in embryos (g-k) and by #4 (l). The ß-gal expression indicated by arrows represents the expression in anterior visceral endoderm (b), anterior mesendoderm (d) and cephalic mesenchyme (e,l) by the 1.8 kb promoter (Kimura et al., 1997Go). The expression in e is solely due to the activity of the 1.8 kb promoter (compare with Fig. 1B, part d). Compare the expression in d with the 1.8 kb promoter with that in Fig. 3B, part h with the hsp68 promoter. The #4 fragment exhibited ß-gal expression in a region of the ventral diencephalon and dorsal mesencephalon. In lateral views (a,c-e,g-j,l), anterior is leftwards; (b) cross-section in the plane shown in a; (f,k) frontal views. Arrowheads and double arrows indicate the position of the isthmus and the expression in the eyes, respectively. a, anterior; p, posterior. Scale bars: 100 µm in a-d,g,h; 400 µm in e,f,i-l.

 


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  		Fig. 7. Genome organization in tetrapod Otx2 locus. Locations of the 22 domains in Otx2 locus conserved among mouse, human and Xenopus. Black bars in mouse and human Otx2 locus define the locations of the nearest genes at the 5' and 3' sides, respectively. Domains that exhibited enhancer activity are shown by red bars [see Kurokawa et al. (Kurokawa et al., 2004Go) for details of the analysis of the 3' region]. The contiguous mapping is partial in the Xenopus Otx2 locus; however, domains {kappa}–{pi} and {tau}–{chi} exist in the genome. White boxes indicate the Otx2-coding regions.

 


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  		Fig. 1. Search for enhancers of Otx2 expression in ectoderm at 5' upstream by BAC transgenesis. (A) BAC #1/lacZ reporter gene. A of the ATG translation start codon is taken as +1 bp. (B) ß-Gal expression in ectoderm at each stage by BAC #1 (a-c) and expression in cephalic mesenchyme (d) by the 1.8 kb promoter. Arrows indicate expression by the 1.8 kb promoter. Scale bars: 100 µm in a,b; 400 µm in c,d.

 


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  		Fig. 4. Deletion analysis of AN enhancer. (A) Fine mapping of the AN enhancer. (11-7)x2 is a duplicate of the #11-#7 sequences (see B), whereas (11-10)x5 is a quintuplet of the #11-#10 sequences. Percentages indicate the sequence identity of the fragment with the counterpart region of human or Xenopus. (B) Nucleotide sequences of the mouse, human and Xenopus EB 165 bp region. Linker-scanner mutations were introduced such that each 15 bp block was replaced with a transcriptionally inert 15 bp linker. The number of ß-gal-positive embryos among transient transgenic embryos in each block is provided in parenthesis; mutations affecting enhancer activity are indicated in red. Asterisks in #8, #9 and #11 represent residual expression as shown in C, part c. (C) ß-gal expression driven by the EcoT22I/BglII(EB)165 bp subfragment (a), by the EB165 bp with the mutation at #10 (b), by the EB165 bp with the mutation at #11 (c), by the (11-7)x2 fragment (d) and by a 1.6 kb Xotx2 region (e) at E7.75. (a-d) Lateral views, anterior is leftwards; (e) a frontal view. Note the loss of expression by the #10 mutation (b), residual expression by the #11 mutation (c) and significant expression with the (11-7)x2 fragment (d) in the anterior neuroectoderm (arrowheads). Arrows indicate the expression in anterior mesoderm and endoderm by the 1.8 kb promoter. Scale bars: 100 µm.

 


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  		Fig. 3. Deletion analysis of EP enhancer. (A) Dissection of the #12 genomic fragment for EP and AN enhancers. The number of ß-gal-positive embryos among transient transgenic embryos obtained is indicated on the right (ND, not determined). Blue boxes indicate regions demonstrating in excess of 80% identity over more than 100 bp between mouse and human; yellow boxes indicate the regions demonstrating in excess of 80% identity over more than 100 bp between mouse and Xenopus. (B) ß-Gal expression driven by the EcoRV/BglII 2.3 kb fragment with 1.8 kb promoter (a-d) and with hsp68 promoter (e-h) at E3.5 (a), E5.5 (b), E6.5 (c-f) and E7.75 (g,h). In lateral views (c,e,g), anterior is leftwards; (d,f) cross-sections at the level indicated in b,d, respectively; (h) a sagittal section. Arrows in d indicate ß-gal expression in anterior visceral endoderm by the 1.8 kb mouse Otx2 promoter; this expression is absent with hsp68 promoter (f). The expression is absent in definitive anterior mesendoderm with hsp68 promoter (an arrow in h, compare with Fig. 2B, part d). Scale bars: 100 µm.

 


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  		Fig. 5. Targeted disruption of the EP/AN enhancer. (A) Wild-type Otx2 allele, targeting vector and recombinant allele. The black box indicates the SpeI-BglII 559 bp region (AN) that is replaced with a neomycin-resistant gene (Neo, white boxes) flanked by loxP sequences (black triangles). DT-A is the diphtheria toxin-A fragment gene with MCI promoter, which is used for negative selection of homologous recombinants (Yagi et al., 1993bGo). Thick and thin lines indicate genomic and vector-derived sequences, respectively. Probe A is the Southern blotting probe used for identification of homologous recombinant ES cells displayed in the right panel. (B) Otx2 expression at E6.5 by whole-mount in situ hybridization (a,b) and by quantitative RT-PCR (c). +/+ and {Delta}AN/{Delta}AN denote wild-type embryos and homozygous mutants lacking the SpeI/BglII region, respectively. (C) Otx2{Delta}AN/– mutant phenotype at E6.5. Normal expression of cerberus-like in anterior visceral endoderm (a) and of T in primitive streak (b). Scale bars: 100 µm.

 


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  		Fig. 6. AN enhancer mutant phenotype. (A) Histological features of forebrain defects in Otx2{Delta}AN/– (b) and Emx2–/–Otx2{Delta}AN/{Delta}AN (c) mutants at E12.5; (a) wild-type brain. These phenotypes were examined with both the Otx2{Delta}AN mutant in which the neo insert remained and the Otx2{Delta}AN mutant in which the insert was deleted by Cre recombination. No differences were found, and the following marker analyses were performed with the mutant that retained the neo insert. The E12.5 Otx2{Delta}AN/– phenotype was variable, and a severe phenotype is shown in b. Scale bars: 400 µm. (B) Marker analyses of wild-type (a,c,e,g,i,k) and Otx2{Delta}AN/– mutant (b,d,f,h,j,l) embryos at E7.5 (a,b), E8.5 (c-h) and E9.5 (i-l); expression of Six3 (a-d), Fgf8 (e,f), Gbx2 (g-j) and Emx2 (k,l). The E9.5 phenotype was variable; severe examples are shown. Scale bars: 100 µm in a,b; 150 µm in c-h; 400 µm in i-l. (C) Marker analyses of diencephalon in E11.5 wild-type (a,c,e,g,i) and Emx2–/–Otx2{Delta}AN/{Delta}AN mutant (b,d,f,h,j) embryos; expression of Pax6 (a,b), Dlx1 (c,d), Gbx2 (e,f), Tcf4 (g,h) and Lhx1 (i,j). Scale bars: 400 µm.

 

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© The Company of Biologists Ltd 2004