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Kurokawa D
,
Takasaki N
,
Kiyonari H
,
Nakayama R
,
Kimura-Yoshida C
,
Matsuo I
,
Aizawa S
.
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We have identified cis-regulatory sequences acting on Otx2 expression in epiblast (EP) and anterior neuroectoderm (AN) at about 90 kb 5' upstream. The activity of the EP enhancer is found in the inner cell mass at E3.5 and the entire epiblast at E5.5. The AN enhancer activity is detected initially at E7.0 and ceases by E8.5; it is found later in the dorsomedial aspect of the telencephalon at E10.5. The EP enhancer includes multiple required domains over 2.3 kb, and the AN enhancer is an essential component of the EP enhancer. Mutants lacking the AN enhancer have demonstrated that these cis-sequences indeed regulate Otx2 expression in EP and AN. At the same time, our analysis indicates that another EP and AN enhancer must exist outside of the -170 kb to +120 kb range. In Otx2DeltaAN/- mutants, in which one Otx2 allele lacks the AN enhancer and the other allele is null, anteroposterior axis forms normally and anterior neuroectoderm is normally induced. Subsequently, however, forebrain and midbrain are lost, indicating that Otx2 expression under the AN enhancer functions to maintain anterior neuroectoderm once induced. Furthermore, Otx2 under the AN enhancer cooperates with Emx2 in diencephalon development. The AN enhancer region is conserved among mouse, human and Xenopus; moreover, the counterpart region in Xenopus exhibited an enhancer activity in mouse anterior neuroectoderm.
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) β-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.
Fig. 3. Deletion analysis of EP enhancer. (A) Dissection of the #12 genomic fragment for EP and AN enhancers. The number of β-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) β-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 β-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 anteriormesendoderm with hsp68 promoter (an arrow in h, compare with Fig. 2B, part d). Scale bars: 100 μm.
Fig. 6. AN enhancer mutant phenotype. (A) Histological features of forebrain defects in Otx2δAN/– (b) and Emx2–/–Otx2δAN/δAN (c) mutants at E12.5; (a) wild-type brain. These phenotypes were examined with both the Otx2δAN mutant in which the neo insert remained and the Otx2δAN 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 Otx2δAN/– 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 Otx2δAN/– 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–/–Otx2δAN/δAN 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.
