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Abstract: Otx2 plays essential roles in each site at each step of head development. We previously identified the AN1 enhancer at 91kb 5' upstream for the Otx2 expressions in anterior neuroectoderm (AN) at neural plate stage before E8.5, and the FM1 enhancer at 75kb 5' upstream and the FM2 enhancer at 122kb 3' downstream for the expression in forebrain/midbrain (FM) at brain vesicle stage after E8.5. The present study identified a second AN enhancer (AN2) at 88kb 5' upstream; the AN2 enhancer also recapitulates the endogenous Otx2 expression in choroid plexus, cortical hem and choroidal roof. However, the enhancer mutants indicated the presence of another AN enhancer. The study also identified a third FM enhancer (FM3) at 153kb 5' upstream. Thus, the Otx2 expressions in anterior neuroectoderm and forebrain/midbrain are regulated by more than six enhancers located far from the coding region. The enhancers identified are differentially conserved among vertebrates; none of the AN enhancers has activities in caudal forebrain and midbrain at brain vesicle stage after E8.5, nor do any of the FM enhancers in anterior neuroectoderm at neural plate stage before E8.5. [Copyright &y& Elsevier]

Abstract: In mouse Otx2 plays essential roles in anterior–posterior axis formation and head development in anterior visceral endoderm and anterior mesendoderm. The Otx2 expression in these sites is regulated by VE and CM enhancers at the 5′ proximal to the translation start site, and we proposed that these enhancers would have been established in ancestral sarcoptergians after divergence from actinopterigians for the use of Otx2 as the head organizer gene (). This would make doubtful an earlier proposal of ours that a 1.1kb fragment located at +14.4 to +15.5kb 3′ (3′En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (). In the present study, we demonstrate that fugu Otx2a is not expressed in the dorsal margin of blastoderm, shield and early anterior mesendoderm, and that the fugu Otx2a 3′En do not exhibit activities at these sites of fugu embryos. We conclude that the fugu Otx2a 3′En does not harbor an organizer enhancer, but encodes an enhancer for the expression in later anterior mesendodermal tissues. Instead, in fugu embryos Otx2b is expressed in the dorsal margin of blastoderm at blastula stage and shield at 50% epiboly, and this expression is directed by an enhancer, 5′En, located at −1000 to −800bp, which is uniquely conserved among teleost Otx2b orthologues. [Copyright &y& Elsevier]

Abstract: Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2 ΔFM1ΔFM2/ΔFM1ΔFM2 ) and examined the TKO (Otx1 −/− Otx2 ΔFM1ΔFM2/ΔFM1ΔFM2 ) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers. [ABSTRACT FROM AUTHOR]

The mouse homeobox gene Otx2 plays essential roles at each step and in every tissue during head development. We have previously identified a series of enhancers that are responsible for driving the Otx2 expression in these contexts. Among them the AN enhancer, existing 92 kb 5′ upstream, directs Otx2 expression in anterior neuroectoderm (AN) at the headfold stage. Analysis of the enhancer mutant Otx2ΔAN/− indicated that Otx2 expression under the control of this enhancer is essential to the development of AN. This study demonstrates that the AN enhancer is promoter-dependent and regulated by acetylated YY1. YY1 binds to both the AN enhancer and promoter region. YY1 is acetylated in the anterior head, and only acetylated YY1 can bind to the sequence in the enhancer. Moreover, YY1 binding to both of these two sites is essential to Otx2 expression in AN. These YY1 binding sites are highly conserved in AN enhancers in tetrapods, coelacanth and skate, suggesting that establishment of the YY1 regulation coincides with that of OTX2 function in AN development in an ancestral gnathostome. [ABSTRACT FROM AUTHOR]

Otx2 expression in the forebrain and midbrain was found to be regulated by two distinct enhancers (FM and FM2) located at 75 kb 5′ upstream and 115 kb 3′ downstream. The activities of these two enhancers were absent in anterior neuroectoderm earlier than E8.0; however, at E9.5 their regions of activity spanned the entire mesencephalon and diencephalon with their caudal limits at the boundary with the metencephalon or isthmus. In telencephalon, activities were found only in the dorsomedial aspect. Potential binding sites of OTX and TCF were essential to FM activity, and TCF sites were also essential to FM2 activity. The FM2 enhancer appears to be unique to rodent; however, the FM enhancer region is deeply conserved in gnathostomes. Studies of mutants lacking FM or FM2 enhancer demonstrated that these enhancers indeed regulate Otx2 expression in forebrain and midbrain. Development of mesencephalic and diencephalic regions was differentially regulated in a dose-dependent manner by the cooperation between Otx1 and Otx2 under FM and FM2 enhancers: the more caudal the structure the higher the OTX dose requirement. At E10.5 Otx1-/-Otx2ΔFM/ΔFM mutants, in which Otx2 expression under the FM2 enhancer remained, exhibited almost complete loss of the entire diencephalon and mesencephalon; the telencephalon did, however, develop. [ABSTRACT FROM AUTHOR]

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 nrequired 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 Otx2ΔAN/- 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. [ABSTRACT FROM AUTHOR]