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First published online 16 June 2004
doi: 10.1242/dev.01220

Development 131, 3319-3331 (2004)
Published by The Company of Biologists 2004
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Regulation of Otx2 expression and its functions in mouse forebrain and midbrain

Daisuke Kurokawa1, 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. 4. Search for enhancers of Otx2 expression in ectoderm at 3' downstream. (A) The 3' BAC #2/lacZ transgene and the 14 fragments assayed for enhancer activity. (B) ß-Gal expression driven by the 3' BAC #2/lacZ transgene (a), by #17 in nasal placode (b), by #20 in nasal cavity (c) and by #29 in forebrain and midbrain (d-i) at indicated stages. Open arrows, ß-gal expression in eyes; double arrows, ß-gal expression in nasal placode or cavity; arrowheads, the position of the isthmus. The activity of the 1.8 kb promoter in cephalic mesenchyme (indicated by an arrow in b) is suppressed by the 3' BAC #2 (a) and the #29 fragment (f,h); by contrast, promoter activity in anterior mesendoderm is not (an arrow in d). Open arrowheads in d indicate weak ß-gal expression in the future mid/hindbrain boundary region. With the exception of the nasal cavity, expression in c is ectopic. In lateral views (a-c,e-h), anterior is towards the left; (d,i), frontal views. Scale bars: 400 µm.

 


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  		Fig. 5. Deletion analysis of FM2 enhancer. (A) Fine mapping of the FM2 enhancer. Blue boxes indicate the domains conserved between human and mouse characterized by sequence identity exceeding 80% over more than 100 bp. Sequences in the boxes denoted by P were used as primers to isolate the counterpart 3.0 kb region in human genome by PCR. Asterisk in HB 0.9 kb indicates residual expression shown in (Be). Percentages on each bar provide the sequence identity of each domain with human or rat. (B) ß-Gal expression driven by the SB 2.8 kb fragment (a), by the BH 1.2 kb fragment (b), by the human 3.0 kb counterpart of the SB 2.8 kb region (c), by the BB 1.0 kb fragment (d), by the HB 0.9 kb fragment (e), by the BB 1.0 kb fragment with mutations in three TCF binding sites (f) and by the BP 200 bp fragment (g) at E10.5 (a-f) and at E11.5 (g). Arrowheads in a and e indicate faint ectopic expression in DRG and residual expression in dorsal midbrain, respectively. Arrows indicate ß-gal expression in cephalic mesenchyme by the 1.8 kb promoter. Scale bars: 400 µm. (C) Alignment of nucleotide sequences of BP 200 bp between mouse and rat.

 


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  		Fig. 8. Genome organization in fish Otx loci. (A) Locations of the 22 domains conserved among mouse, human and Xenopus in pufferfish and zebrafish Otx loci. No homologous region existed in the FrOtx1 locus. Domains that exhibited enhancer activity are shown by red bars. Regions homologous to the mouse FM2 enhancer region (indicated by a yellow bar) were also not found in fish genomes. White boxes indicate the coding regions. The numbers below each bar represent sequence identity with the mouse counterpart in percentage terms. ß-Gal staining in b yields expression directed by a 600 bp region covering domain ß in zebrafish Otx2. (B) Sequence alignment of domain ß in mouse, zebrafish and two pufferfish Otx2 loci. Red square indicates a sequence conserved throughout gnathostome Otx2 FM regions, the significance of which is currently unknown. Scale bars: 400 µm.

 


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  		Fig. 1. Enhancer of Otx2 expression in forebrain and midbrain at 5' upstream. (A) The location of the #13 fragment that exhibited the enhancer activity in forebrain midbrain. EP/AN indicates the position of the EP and AN enhancer at –92.5 to –90 kb. (B) ß-Gal expression driven by #13 (a-f,m,q) and by endogenous enhancers in Otx2+/{lambda}acZ knock-in embryos (g-j,l,p), at the stages indicated. Endogenous Otx2 mRNA expression is presented in k,n,o. (a,k-m) frontal views; (b-j) lateral views (anterior is towards the left); (n-q) frontal sections at the telencephalic level. Arrows and arrowheads indicate expression by the 1.8 kb promoter and the position of the isthmus, respectively. Double arrowheads in c indicate the absence of the ß-gal expression in the most anterior neural plate, which may correspond to future telencephalon. Scale bars: 100 µm in a,b,g; 400 µm in c-f,h-q.

 


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  		Fig. 2. Deletion analysis of FM enhancer. (A) Fine mapping of the FM enhancer. (B) Schematic organization of the AH 1.4 kb subfragment and sequence alignment of the CR region in mouse, human and Xenopus. (C) ß-Gal expression at E10.5 driven by the AH 1.4 kb fragment (a), by the CR domain of the fragment (b) and by the AH 1.4 kb fragment exhibiting a mutation in the OTX binding site (c), in both TCF-binding sites (d) and in the PAX-binding site (e). Expression in midbrain is lost in b; furthermore, only residual expression exists in midbrain (arrowhead). Arrows indicate ß-gal expression in cephalic mesenchyme driven by the 1.8 kb promoter. Scale bars: 400 µm.

 


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  		Fig. 3. Targeted disruption of FM enhancer. (A) Wild-type Otx2 allele, targeting vector and recombinant allele. The black box indicates the ApaI-HindIIII 1.4 kp region (FM) to be replaced with a neomycin-resistant gene (Neo, open boxes) flanked by loxP sequences (black triangles). DT-A is the diphtheria toxin A fragment gene with MC1 promoter, which is used for the negative selection of homologous recombinants (Matsuo et al., 1995Go). Thick and thin lines indicate genomic and vector-derived sequences, respectively. Probe A is the Southern blotting probe employed for identification of homologous recombinant ES cells displayed in the right panel. (B) FM enhancer mutant phenotype. Wild-type (a,b), Otx2{Delta}FM/{Delta}FM (c,d) and Otx2{Delta}FM/– (e,f) embryos at E12.5 (a,c,e) and E15.5 (b,d,f). A double arrowhead in f indicates the expanded cerebellum primordium. The phenotypes were examined with both the Otx2{Delta}FM mutant in which the neo insert remained and the Otx2{Delta}FM 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. (C) Marker analysis of Otx2{Delta}FM/– phenotype. Otx2 (a,b), Pax6 (c,d), Fgf8 (e,f) and Emx2 (g,h) expression in E10.5 wild-type (a,c,e,g) and Otx2{Delta}FM/– (b,d,f,h) embryos. Scale bars: 400 µm.

 


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  		Fig. 6. Targeted disruption of FM2 enhancer. (A) Diagrammatic representation of wild-type Otx2 allele, targeting vector and recombinant allele. The black box indicates the BsmI-BamHI 1.0 kb region (FM2) to be replaced with a neomycin-resistant gene (Neo) (see Fig. 3A for others). (B) Otx2{Delta}FM2/{Delta}FM2 (a,b) and Otx2{Delta}FM2/– (c,d) phenotype at E12.5 (a,c) and E15.5 (b,d). The phenotypes were examined with both the Otx2{Delta}FM2 mutant in which the neo insert remained and the Otx2{Delta}FM2 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. (C) Marker analysis of Otx2{Delta}FM2/– phenotype at E10.5; Otx2 (a), Pax6 (b), Fgf8 (c) and Emx2 (d) expression (see Fig. 3C for the expression in wild-type embryos). Scale bars: 400 µm.

 


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  		Fig. 7. Cooperation between Otx1 and Otx2 under FM and FM2 enhancers. (A) Histological features of a series of double mutants at E12.5; the genotype of each mutant is indicated at the left. Among mutants not shown, defects were either absent or subtle in Otx2{Delta}FM2/{Delta}FM2, Otx1+/–Otx2+/{Delta}FM2, Otx1–/–, Otx2+/–, Otx2{Delta}FM/{Delta}FM2, Otx1+/–Otx2+/{Delta}FM, Otx2{Delta}FM/{Delta}FM, Otx1+/–Otx2{Delta}FM2/{Delta}FM2 and Otx1–/–Otx2+/{Delta}FM2 mutants. Otx1+/–Otx2{Delta}FM2/– was affected to the same extent as Otx2{Delta}FM/– (f); furthermore, defects in Otx1+/–Otx2{Delta}FM/– were as severe as those in Otx1–/–Otx2{Delta}FM/{Delta}FM (h). Arrowheads indicate the position of the isthmus. Scale bars: 400 µm. (B) Marker analysis of the defects in Otx1–/–Otx2{Delta}FM/{Delta}FM at E10.5; Otx2 (a), Pax6 (b), Fgf8 (c) and Emx2 (d) expression. See Fig. 3C for the expression in wild-type embryos. Arrows in a and b indicate residual Otx2- and Pax6-positive domains, respectively. Scale bars: 400 µm.

 




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