Regulation ofOtx2expression and its functions in mouse epiblast and anterior neuroectoderm

Abstract

We have identified cis-regulatory sequences acting on Otx2expression 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 Otx2ΔAN/– mutants, in which one Otx2allele 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.

Evolutionary constraint on Otx2 neuroectoderm enhancers-deep conservation from skate to mouse and unique divergence in teleost

“…Diversity in the animal body plan might have been brought about by changes in expression of a relatively limited number of key developmental regulators such as Hox genes in the trunk (2,3). The Otx family of genes plays essential roles in head development (4)(5)(6)(7)(8)(9). Otx genes encode a paired-type of homeoprotein homologous to a Drosophila head gap gene, otd.…”

“…Gnathostomes possess three paralogues, Otx1, Otx2, and Otx5, whereas teleosts that underwent genome duplication could possess extra copies. In mouse, Otx2 plays major roles in each site of head development (5)(6)(7)(8)(9)(10)(11): epiblast, anterior visceral endoderm, anterior mesendoderm, anterior neuroectoderm, forebrain/midbrain, and cephalic neural crest cells. Fugu has two Otx2, but zebrafish has only one (9).…”

“…To elucidate the molecular mechanisms regulating Otx2 expression during mouse brain development, we have identified three enhancers: anterior neuroectoderm enhancer (AN), forebrain/midbrain enhancer (FM), and forebrain/midbrain enhancer 2 (FM2) at 90 kb and 75 kb upstream and 115 kb downstream of the Otx2 translational start site, respectively (8,9). The AN enhancer is not active in the epiblast but becomes active at embryonic day (E) 7.0 in the entire anterior neuroectoderm.…”

“…FM and FM2 enhancers also do not have activity in the most rostral part of forebrain that corresponds to the future telencephalon and hypothalamus. In the mutant, Otx2 ⌬AN/Ϫ , which lacks the AN enhancer, the entire anterior neuroectoderm is unable to be maintained and is caudalized into Gbx2-positive metencephalon; the Emx2-positive telencephalon is not formed (8). The Otx2 expression under FM and FM2 enhancers overlaps with Otx1 expression, and, in the compound mutant that lacks the FM and FM2 enhancers and Otx1, Otx1 Ϫ/Ϫ Otx2 ⌬FM⌬FM2/⌬FM⌬FM2 (Y. Sakurai, D.K., and S.A., unpublished data; and ref.…”

“…To test whether the FM region in zebrafish (zfFM) has the same enhancer activity as mouse FM (mFM) enhancer, transgenic mouse lines were established with zfFM-lacZ and mFM-lacZ constructs. Throughout this study the enhancer analysis with transgenic mouse embryos was conducted by using the 1.8-kb mouse Otx2 promoter region (8). The 1.8-kb region harbors enhancer activities in the anterior visceral endoderm, anterior mesoendoderm, and cephalic mesenchyme cells; these activities serve as a positive control for proper transgene integration and are indicated by arrows in each figure.…”