The role of Otx2 in organizing the anterior patterning in mouse

Understanding the molecular mechanism controlling induction and maintenance of signals required for specifying anterior territory (forebrain and midbrain) of the central nervous system is a major task of molecular embryology. The current view indicates that in mouse, early specification of the anterior patterning is established at the beginning of gastrulation by the anterior visceral endoderm, while maintenance and refinement of the early specification is under the control of epiblast-derived tissues corresponding to the axial mesendoderm and rostral neuroectoderm. In vertebrates a remarkable amount of data has been collected on the role of genes contributing to brain morphogenesis. Among these genes,the orthodenticle group is defined bythe Drosophila orthodenticle and the vertebrate Otx1 and Otx2 genes, which contain a bicoid-like homeodomain. Mouse models and chimera experiments have provided strong evidence that Otx2 plays an important role in the specification and maintenance of the rostral neuroectoderm destined to become forebrain and midbrain. In evolutionary terms, some of these findings lead us to hypothesize a fascinating and crucial contribution of the Otx genes to the genetic program underlying the establishment of the mammalian brain.     

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.     

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.     

Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression.

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2(-/-) embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes.     

The zinc-finger protein CNBP is required for forebrain formation in the mouse

Mouse mutants have allowed us to gain significant insight into axis development. However, much remains to be learned about the cellular and molecular basis of early forebrain patterning. We describe a lethal mutation mouse strain generated using promoter-trap mutagenesis. The mutants exhibit severe forebrain truncation in homozygous mouse embryos and various craniofacial defects in heterozygotes. We show that the defects are caused by disruption of the gene encoding cellular nucleic acid binding protein (CNBP); Cnbp transgenic mice were able to rescue fully the mutant phenotype. Cnbp is first expressed in the anterior visceral endoderm (AVE) and, subsequently, in the anterior definitive endoderm (ADE), anterior neuroectoderm (ANE), anterior mesendoderm (AME), headfolds and forebrain. In Cnbp(-/-) embryos, the visceral endoderm remains in the distal tip of the conceptus and the ADE fails to form, whereas the node and notochord form normally. A substantial reduction in cell proliferation was observed in the anterior regions of Cnbp(-/-) embryos at gastrulation and neural-fold stages. In these regions, Myc expression was absent, indicating CNBP targets Myc in rostral head formation. Our findings demonstrate that Cnbp is essential for the forebrain induction and specification.     

Evolution of Otx paralogue usages in early patterning of the vertebrate head

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.     

Differential transcriptional control as the major molecular event in generating Otx1-/- and Otx2-/- divergent phenotypes

Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, show a limited amino acid sequence divergence. Their embryonic expression patterns overlap in spatial and temporal profiles with two major exceptions: until 8 days post coitum (d.p.c. ) only Otx2 is expressed in gastrulating embryos, and from 11 d.p.c. onwards only Otx1 is transcribed within the dorsal telencephalon. Otx1 null mice exhibit spontaneous epileptic seizures and multiple abnormalities affecting primarily the dorsal telencephalic cortex and components of the acoustic and visual sense organs. Otx2 null mice show heavy gastrulation abnormalities and lack the rostral neuroectoderm corresponding to the forebrain, midbrain and rostral hindbrain. In order to define whether these contrasting phenotypes reflect differences in expression pattern or coding sequence of Otx1 and Otx2 genes, we replaced Otx1 with a human Otx2 (hOtx2) full-coding cDNA. Interestingly, homozygous mutant mice (hOtx2(1)/hOtx2(1)) fully rescued epilepsy and corticogenesis abnormalities and showed a significant improvement of mesencephalon, cerebellum, eye and lachrymal gland defects. In contrast, the lateral semicircular canal of the inner ear was never recovered, strongly supporting an Otx1-specific requirement for the specification of this structure. These data indicate an extended functional homology between OTX1 and OTX2 proteins and provide evidence that, with the exception of the inner ear, in Otx1 and Otx2 null mice contrasting phenotypes stem from differences in expression patterns rather than in amino acid sequences.     

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.     

Visceral endoderm mediates forebrain development by suppressing posteriorizing signals

The anterior visceral endoderm (AVE) has attracted recent attention as a critical player in mouse forebrain development and has been proposed to act as "head organizer" in mammals. However, the precise role of the AVE in induction and patterning of the anterior neuroectoderm is not yet known. Here we identified a 5'-flanking region of the mouse Otx2 gene (VEcis) that governs the transgene expression in the visceral endoderm. In transgenic embryos, VEcis-active cells were found in the distal visceral endoderm at 5.5 days postcoitus (dpc), had begun to move anteriorly at 5.75 dpc, and then became restricted to the AVE prior to gastrulation. The VEcis-active visceral endoderm cells exhibited ectodermal morphology distinct from that of the other endoderm cells and consisted of two cell layers at 5.75 dpc. In the Otx2(-/-) background, the VEcis-active endoderm cells remained distal even at 6.5 dpc when a primitive streak was formed; anterior definitive endoderm was not formed nor were any markers of anterior neuroectoderm ever induced. The Otx2 cDNA transgene under the control of the VEcis restored these Otx2(-/-) defects, demonstrating that Otx2 is essential to the anterior movement of distal visceral endoderm cells. In germ-layer explant assays between ectoderm and visceral endoderm, the AVE did not induce anterior neuroectoderm markers, but instead suppressed posterior markers in the ectoderm; Otx2(-/-) visceral endoderm lacked this activity. Thus Otx2 is also essential for the AVE to repress the posterior character. These results suggest that distal visceral endoderm cells move to the future anterior side to generate a prospective forebrain territory indirectly, by preventing posteriorizing signals.     

Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm

Previous studies of head induction in the chick have failed to demonstrate a clear role for the hypoblast and anterior definitive endoderm (ADE) in patterning the overlying ectoderm, whereas data from both mouse and rabbit suggest patterning roles for anterior visceral endoderm (AVE) and ADE. Based on similarity of gene expression patterns, fate and a dual role in 'protecting' the prospective forebrain from caudalising influences of the organiser, the chick hypoblast has been suggested to be the homologue of the mouse anterior visceral endoderm. In support of this, when transplanted to chick embryos, the rabbit AVE induces anterior markers in the chick epiblast. To reevaluate the role of the hypoblast/ADE (lower layer) in patterning the chick ectoderm, we used rostral blastoderm isolates (RBIs) as an assay, that is, rostral regions of blastoderms transected at levels rostral to the node. RBIs are, therefore, free from the influences of Hensen's node and ingressing axial mesoderm - tissues that are able to induce Ganf, the earliest specific marker of anterior neural plate. We demonstrate, using such RBIs (or RBIs dissected to remove the lower layer with or without tissue replacement), that the hypoblast/ADE (lower layer) is required and sufficient for patterning anterior positional identity in the overlying ectoderm, leading to expression of Ganf in neuroectoderm. Our results suggest that patterning of anterior positional identity and specification of neural identity are separable events operating to pattern the rostral end of the early chick embryo. Based on this new evidence we propose a revised model for establishing anteroposterior polarity, neural specification and head patterning in the early chick that is consonant with that occurring in other vertebrates.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization

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.     

Mouse homologues of Shisa antagonistic to Wnt and Fgf signalings

In an effort to identify Otx2 targets in mouse anterior neuroectoderm we identified a gene, mShisa, which is homologous to xShisa1 that we previously reported as a head inducer in Xenopus. mShisa encodes an antagonist against both Wnt and Fgf signalings; it inhibits these signalings cell-autonomously as xShisa1 does. The mShisa expression is lost or greatly reduced in Otx2 mutant visceral endoderm, anterior mesendoderm and anterior neuroectoderm. However, mShisa mutants exhibited no defects in head development. Shisa is composed of five subfamilies, but normal head development in mShisa mutants is unlikely to be explained in terms of the compensation of mShisa deficiency by its paralogues or by known Wnt antagonists in anterior visceral endoderm and/or anterior mesendoderm.     

Characterization of cis-regulatory elements of the homeobox gene Xanf-1

Investigation of molecular mechanisms underlying early patterning of the nervous system is an important task of modern developmental biology. Previously, we identified a novel homeobox gene, Anf, that is expressed in the most anterior zone at the beginning of neuroectoderm specification. The expression pattern of Anf corresponds to primordia of the telencephalon and the rostral part of the diencephalon. In the present work, we investigated cis-regulation of expression of the Xenopus laevis Anf, Xanf-1. Two elements, highly conserved in Xenopus, chick and human, were identified within the Xanf-1 promoter region. The first element, located near position -500, is necessary for overall enhancement of the Xanf-1 expression. The second element, near position -200, is crucial for maintenance of the Xanf-1 expression at moderate levels and also for specific localization of the expression in the anterior neuroectoderm. Thus, the distal part of this element is responsible for suppression of Xanf-1 posterior to the normal expression domain of this gene. The data obtained corroborate with the Nieuwkoop two-signal model of neural induction. This model states that at the first step of induction, all neuroectoderm acquires potencies to develop toward forebrain structures, but later these potencies are suppressed in posterior regions.     

Requirement of mesodermal retinoic acid generated by Raldh2 for posterior neural transformation

Studies in amphibian embryos have suggested that retinoic acid (RA) may function as a signal that stimulates posterior differentiation of the nervous system as postulated by the activation-transformation model for anteroposterior patterning of the nervous system. We have tested this hypothesis in retinaldehyde dehydrogenase-2 (Raldh2) null mutant mice lacking RA synthesis in the somitic mesoderm. Raldh2^−/− embryos exhibited neural induction (activation) as evidenced by expression of Sox1 and Sox2 along the neural plate, but differentiation of spinal cord neuroectodermal progenitor cells (posterior transformation) did not occur as demonstrated by a loss of Pax6 and Olig2 expression along the posterior neural plate. Spinal cord differentiation in Raldh2^−/− embryos was rescued by maternal RA administration, and during the rescue RA was found to act directly in the neuroectoderm but not the somitic mesoderm. RA generated by Raldh2 in the somitic mesoderm was found to normally travel as a signal throughout the mesoderm and neuroectoderm of the trunk and into tailbud neuroectoderm, but not into tailbud mesoderm. Raldh2^−/− embryos also exhibited increased Fgf8 expression in the tailbud, and decreased cell proliferation in tailbud neuroectoderm. Our findings demonstrate that RA synthesized in the somitic mesoderm is necessary for posterior neural transformation in the mouse and that Raldh2 provides the only source of RA for posterior development. An important concept to emerge from our studies is that the somitic mesodermal RA signal acts in the neuroectoderm but not mesoderm to generate a spinal cord fate.