Regulation of Otx2 expression and its functions in mouse epiblast and anterior neuroectoderm

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.     

A novel fork head gene mediates early steps during Xenopus lens formation.

Xlens1 is a novel Xenopus member of the fork head gene family, named for its nearly restricted expression in the anterior ectodermal placode, presumptive lens ectoderm (PLE), and anterior epithelium of the differentiated lens. The temporal and spatial restriction of its expression suggests that: (1) Xlens1 is transcribed initially at neural plate stages in response to putative signals from the anterior neural plate that transform lens-competent ectoderm to lens-biased ectoderm; (2) further steps in the process of lens-forming bias restrict Xlens1 expression to the presumptive lens ectoderm (PLE) during later neural plate stages; (3) interactions with the optic vesicle maintain Xlens1 expression in the lens placode; and (4) Xlens1 expression is downregulated as committed lens cells undergo terminal differentiation. Induction assays demonstrate that pax6 induces Xlens1 expression, but unlike pax6, Xlens1 cannot induce the expression of the lens differentiation marker beta-crystallin. In the whole embryo, overexpression of Xlens1 in the lens ectoderm causes it to thicken and maintain gene expression characteristics of the PLE. Also, this overexpression suppresses differentiation in the lens ectoderm, suggesting that Xlens1 functions to maintain specified lens ectoderm in an undifferentiated state. Misexpression of Xlens1 in other regions causes hypertrophy of restricted tissues but only occasionally leads ectopic sites of gamma-crystallin protein expression in select anterior head regions. These results indicate that Xlens1 expression alone does not specify lens ectoderm. Lens specification and differentiation likely depends on a combination of other gene products and an appropriate level of Xlens1 activity.     

Xenopus laevis FoxE1 is primarily expressed in the developing pituitary and thyroid

The members of the FoxE subfamily of Fox (forkhead) genes are expressed in the developing pituitary, thyroid and lens. Mammalian Foxe1 is expressed primarily in the developing pituitary and thyroid gland, Foxe3 is expressed in the developing lens, while Xenopus FoxE4 is expressed in the developing lens and thyroid. Here we report the identification of Xenopus FoxE1, a gene that is primarily expressed in the developing pituitary and thyroid.     

Defining progressive stages in the commitment process leading to embryonic lens formation

The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well‐studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e., when presumptive lens ectoderm (PLE) can be isolated, cultured, and will differentiate into a lens that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while ～ 3 h later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Furthermore, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g., foxe3, mafB) in an ectopic environment, while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment. genesis 50:728–740, 2012. © 2012 Wiley Periodicals, Inc.     

A lineage specific enhancer drives Otx2 expression in teleost organizer tissues

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 (Kurokawa et al., 2010). This would make doubtful an earlier proposal of ours that a 1.1 kb fragment located at +14.4 to +15.5 kb 3' (3'En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (Kimura-Yoshida et al., 2007). 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 -800 bp, which is uniquely conserved among teleost Otx2b orthologues.     

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.     

Correct Expression of spec2a in the sea urchin embryo requires both Otx and other cis-regulatory elements

Strongylocentrotus purpuratus Otx (SpOtx) is required simultaneously in sea urchin development for the activation of endo16 in the vegetal plate and for the activation of spec2a in the aboral ectoderm. Because Otx binding sites alone do not appear to be responsible for the spatially restricted expression of spec2a, additional DNA elements were sought. We show here that consensus Otx binding sites fused to basal promoters are sufficient to activate CAT reporter gene expression in all cell types, although expression in endomesoderm progenitors is enhanced. On the other hand, three non-Otx elements derived from the spec2a enhancer are needed together with Otx sites for specifically aboral ectoderm expression. A DNA element termed Y/CBF, lying just downstream from an Otx site within the spec2a enhancer, mediates general activation in the ectoderm. A second element lying between the Otx and Y/CBF sites, called OER, functions to prevent expression in the oral ectoderm. A third site, called ENR, overlapping another Otx site, is required to repress endoderm expression. Three distinct DNA binding proteins interact sequence specifically at the Y/CBF, OER, and ENR elements. The spec2a enhancer thus consists of closely linked activator and repressor elements that function collectively to cause expression of the spec2a gene in the aboral ectoderm.     

Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart

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 Otx2(DeltaAN/-) 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.     

Otx2 expression in anterior neuroectoderm and forebrain/midbrain is directed by more than six enhancers

Otx2 plays essential roles in each site at each step of head development. We previously identified the AN1 enhancer at 91 kb 5' upstream for the Otx2 expressions in anterior neuroectoderm (AN) at neural plate stage before E8.5, and the FM1 enhancer at 75 kb 5' upstream and the FM2 enhancer at 122 kb 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 88 kb 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 153 kb 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.     

XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos

Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.