Otx genes in brain morphogenesis - · PDF fileMarconi 12, 80125Naples, Italy b ... Otx1 transiently controls GH, FSH, and LH in the pituitary ... (Acampora et al., 1998c). 2.2. Otx1expression

Progress in Neurobiology 64 (2001) 69–95

Otx genes in brain morphogenesis

Dario Acampora a,b, Massimo Gulisano c, Vania Broccoli d, Antonio Simeone a,b,*a International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples, Italy

b MRC Centre for De6elopmental Neurobiology, 4th Floor, King’s College London, Guy’s Campus, New Hunts House, London Bridge,London SE1 9RT, UK

c Department of Physiological Sciences, Uni6ersity of Catania, Viale A. Doria 6, 95125 Catania, Italyd TIGEM-Telethon Institute of Genetics and Medicine, H.S. Raffaele Via Olgettina 58, 20132 Milan, Italy

Received 30 May 2000

Abstract

Most of the gene candidates for the control of developmental programmes that underlie brain morphogenesis in vertebrates arethe homologues of Drosophila genes coding for signalling molecules or transcription factors. Among these, the orthodenticle groupincludes the Drosophila orthodenticle (otd) and the vertebrate Otx1 and Otx2 genes, which are mostly involved in fundamentalprocesses of anterior neural patterning. These genes encode transcription factors that recognise specific target sequences throughthe DNA binding properties of the homeodomain. In Drosophila, mutations of otd cause the loss of the anteriormost headneuromere where the gene is transcribed, suggesting that it may act as a segmentation ‘gap’ gene. In mouse embryos, theexpression patterns of Otx1 and Otx2 have shown a remarkable similarity with the Drosophila counterpart. This suggested thatthey could be part of a conserved control system operating in the brain and different from that coded by the HOX complexescontrolling the hindbrain and spinal cord. To verify this hypothesis a series of mouse models have been generated in which thefunctions of the murine genes were: (i) fully inactivated, (ii) replaced with each others, (iii) replaced with the Drosophila otd gene.Otx1−/− mutants suffer from epilepsy and are affected by neurological, hormonal, and sense organ defects. Otx2−/− miceare embryonically lethal, they show gastrulation impairments and fail in specifying anterior neural plate. Analysis of theOtx1−/− ; Otx2+/− double mutants has shown that a minimal threshold level of the proteins they encode is required for thecorrect positioning of the midbrain-hindbrain boundary (MHB). In vivo otd/Otx reciprocal gene replacement experiments haveprovided evidence of a general functional equivalence among otd, Otx1 and Otx2 in fly and mouse. Altogether these datahighlight a crucial role for the Otx genes in specification, regionalization and terminal differentiation of rostral central nervoussystem (CNS) and lead to hypothesize that modification of their regulatory control may have influenced morphogenesis andevolution of the brain. © 2001 Elsevier Science Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702. Multiple roles of the Otx1 gene in the developing and adult mouse . . . . . . . . . . . . . . 71

2.1. Otx1 expression in early embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 712.2. Otx1 expression during corticogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

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Abbre6iations: ame, axial mesendoderm; AML, anterior midline; A/P, antero-posterior; AVE, anterior visceral endoderm; b-FSH, follicle-stim-ulating hormone; a-GSU, a-glycoprotein subunit; b-LH, luteinizing hormone; BrdU, Bromodeoxyuridine; CDGA, constitutional delay in growthand abdolescence; CNS, central nervous system; CP, cortical plate; d.p.c., days post coitum; D/V, dorso-ventral; EEG, electroencephalographicrecording; EPSP, excitatory post-synaptic potentials; GABA, g-aminobutyric acid; GH, growth hormone; GnRH, gonadotropin releasinghormone; GnRHR, gonadotropin releasing hormone receptor; GRH, growth hormone releasing hormone; GRHR, growth hormone releasinghormone receptor; IGF1, insulin growth factor 1; IPSP, inhibitory post-synaptic potentials; IsO, isthmic organizer; IZ, intermediate zone; MHB,midbrain-hindbrain boundary; otd, Drosophila orthodenticle ; RA, retinoic acid; VE, visceral endoderm; VZ, ventricular zone; ZLI, zona limitansintrathalamica.

* Corresponding author. Tel.: +44-20-78486536; fax: +44-20-78486550.E-mail address: [email protected] (A. Simeone).

0301-0082/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.

PII: S0301-0082(00)00042-3

D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–9570

2.3. Otx1 is required for correct brain development . . . . . . . . . . . . . . . . . . . . . . . 712.4. Otx1−/− mutant mice exhibit an epileptic phenotype . . . . . . . . . . . . . . . . . . 722.5. Otx1 controls cortical connectivity to subcortical targets. . . . . . . . . . . . . . . . . . 732.6. Otx1 transiently controls GH, FSH, and LH in the pituitary . . . . . . . . . . . . . . . 732.7. Otx1 is necessary for correct sense organ development . . . . . . . . . . . . . . . . . . . 73

3. Early requirement of Otx2 for normal gastrulation and anterior neural plate induction andmaintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743.1. Organisers and molecules in the current scenario of the anterior brain patterning . . . 743.2. Specification of anterior patterning requires Otx2 function . . . . . . . . . . . . . . . . 753.3. The role of Otx2 in the induction and maintenance of rostral CNS identities . . . . . 783.4. AVE-restricted OTX2 function is necessary for proper expression of Lim1 and bone

morphogenetic protein (BMP) antagonists in the ame . . . . . . . . . . . . . . 793.5. Specific and interchangeable roles between Otx1 and Otx2 . . . . . . . . . . . . . . . . 80

4. Brain patterning depends on a critical Otx gene dosage . . . . . . . . . . . . . . . . . . . . . 804.1. Morphogenetic origin of the IsO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804.2. Otx genes are required for correct positioning of the isthmus . . . . . . . . . . . . . . . 81

5. otd/Otx conserved functions throughout evolution. . . . . . . . . . . . . . . . . . . . . . . . . 855.1. Otx genes in the animal kingdom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855.2. A common genetic program in insect and vertebrate head development . . . . . . . . . 875.3. Otx genes in brain evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

1. Introduction

The central nervous system (CNS) of vertebrates is avery complex structure derived from a multistep processinvolving sequential molecular and morphogeneticevents that pattern the epiblast first and the neural platelater. During early gastrulation the concerted and se-quential action of both the anterior visceral endoderm(AVE) and the node-derived axial mesendoderm (ame,Beddington and Robertson, 1999; Bachiller et al., 2000)drives the specification of anterior neuroectoderm that,later on results grossly subdivided in three main territo-ries (forebrain, midbrain and hindbrain, Gallera, 1971;Storey et al., 1992; Ruiz i Altaba, 1994; Shimamura andRubenstein, 1997; Rubenstein and Beachy, 1998). Themaintenance of this patterning is likely dependent onsignals originating from the ame and/or within theoverlying neuroectoderm (Acampora et al., 1998b; Rhinnet al., 1998; Shawlot et al., 1999; Camus et al., 2000).

Anatomical and histological studies postulate theexistence of genetic fate determinants which subdividethese large neural regions into progressively smallerlongitudinal and transverse domains (Vaage, 1969; Alt-man and Bayer, 1988; Figdor and Stern, 1993; Ruben-stein et al., 1994). Some of the patterning events alongthe antero-posterior (A/P) axis require the presence of

specific cell populations (e.g. the anterior neural ridgeand the zona limitans intrathalamica (ZLI)) and trans-verse rings of neuroepithelia (e.g. the isthmic organizer(IsO)) that possess inductive and boundary properties(Marin and Puelles, 1994; Crossley et al., 1996; Houartet al., 1998; Rubenstein et al., 1998; Ruiz i Altaba, 1998).

In vertebrates, several genes controlling developmentalprogrammes underlying brain morphogenesis have beenisolated and their role studied in detail. Most of them arethe vertebrate homologues of Drosophila genes encodingsignalling molecules or transcription factors (Lemaireand Kodjabachian, 1996; Tam and Behringer, 1997;Rubenstein et al., 1998). Among these, the orthodenticlegroup is strictly defined by the Drosophila orthodenticle(otd) and the vertebrate Otx1 and Otx2 genes, whichcontain a bicoid-like homeodomain (Finkelstein andBoncinelli, 1994; Simeone, 1998). However, other genesthat might be more distantly related to otd/Otx geneshave been also identified (Muccielli et al., 1996; Szeto etal., 1996; Chen et al., 1997).

Expression pattern analysis of Otx genes had sug-gested that these transcription factors might play animportant role during brain morphogenesis in verte-brates. A systematic genetic approach using transgenicmice is revealing that they contribute to the molecularmechanisms underlying all of the major events (induc-

D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 71

tion, maintenance, regionalization, corticogenesis andaxon connectivity) necessary to build a normal brain.

2. Multiple roles of the Otx1 gene in the developingand adult mouse

2.1. Otx1 expression in early embryogenesis

Otx1 starts to be expressed at early stages (2–5somite stage, 8.2–8.5 days post coitum (d.p.c.)) in thedeveloping mouse embryo throughout the presumptiveforebrain and midbrain neuroepithelium (Simeone etal., 1992). From these stages onwards its expressionlargely overlaps with that of Otx2, but while the ex-pression of the latter disappears from the dorsal telen-cephalon since 10.5 d.p.c. (Simeone et al., 1993), Otx1expression is maintained uniformly across the ventricu-lar zone (VZ) of the cortical anlage from the onset ofcorticogenesis up to mid- to late gestation stages (Sime-one et al., 1993; Frantz et al., 1994).

Otx1 is also expressed at early stages in precursorstructures of sense organs corresponding to the olfac-tory placode, otic and optic vesicles (Simeone et al.,1993). Later on, Otx1 is transcribed in the olfactoryepithelium, the saccule, the cochlea and the lateralsemicircular canal of the inner ear as well as in the iris,the ciliary process in the eye and the lachrymal glandprimordia (Simeone et al., 1993). From the birthdayonwards, Otx1 is also expressed at a relatively low levelin the anterior lobe of the pituitary gland (Acampora etal., 1998c).

2.2. Otx1 expression during corticogenesis

The cerebral cortex develops according to molecularstrategies that determine the fate of precursor cellslinked to specific neuronal phenotypes. Two main pro-cesses have been identified so far, laminar determina-tion, by which committed cells migrate to theirappropriate layer, and cortical areas formation, bywhich cortical neurons interact to create functionallydistinct regions. During corticogenesis, postmitotic neu-rons migrate along radial glial cells (Rakic, 1972),through the overlying intermediate zone (IZ), and tothe cortical plate (CP), which will later create thetypical layered organisation of the adult cortex. Thelayers are generated in an inside-out pattern, in whichcells of the deepest layers (6 and 5) are born first in theVZ, and those of the upper layers (4, 3, and 2) progres-sively later (Rakic, 1974).

Otx1 represents a molecular correlate of deep layerneurogenesis and its expression is confined to neuronsof layers 5 and 6 in the adult cortex (Frantz et al.,1994).

At mid-late gestation, high level transcription ofOtx1 occurs only in ventricular cells, which at thesestages are precursors of deep layer neurons. By the timeupper layer neurons are generated, Otx1 expressiondecreases in the VZ and becomes progressively promi-nent in the cortical plate which consists of postmigra-tory neurons of layer 5 and 6. Otx1 is absent in laterdifferentiated neurons of upper layers 1–4 (Frantz etal., 1994).

Thus, the progressive down-regulation of Otx1 in theventricular cells suggests that Otx1 may confer deep-layer identity to young neurons. Heterochronic trans-plantation experiments have demonstrated that thebroad differentiative potentials of the early progenitors(McConnell and Kaznowski, 1991) become progres-sively restricted over time (Frantz and McConnell,1996).

Indeed, Otx1 expression is heterogeneous across theregions of the adult cortex, suggesting that it might alsobe involved in the forming of the cortical areas. Itsexpression in layer 5 is more prominent in the posteriorand lateral cortex but absent in the frontal, insular andorbital cortices, while in layer 6 it is more uniformthroughout the neocortex (Frantz et al., 1994).

Mouse mutant models have been generated andanalysed (Acampora et al., 1996, 1998a, 1999a,b Sudaet al., 1996; Morsli et al., 1999; Weimann et al., 1999;Avanzini et al., submitted) to gain insight into thedifferent roles that Otx1 plays during brain, cortex andsense organ development.

2.3. Otx1 is required for correct brain de6elopment

Heterozygous (Otx1+/− ) mice are healthy andtheir intercross generates homozygous mice (Otx1−/− ) in the expected mendelian ratio. However, about30% of the mutants die before the first postnatalmonth, and appear smaller in size.Table 1

Otx1−/− adult brains are reduced in weight andsize and, at the anatomo-histological inspection, showreduction in thickness of the dorsal telencephalic cor-tex, a dorsally displaced sulcus rhinalis and shrunkenhippocampus with a divaricated dentate gyrus. Thecortex is particularly affected at the level of the tempo-ral and perirhinal areas, where a 40% reduction in cellnumber is detected. Furthermore, in these same areas,cortical layer organisation is less evident (Acampora etal., 1996), although the expression of layer-specificmolecular markers demonstrates that the laminar iden-tities are preserved (Weimann et al., 1999).

The origin of the overall reduction of the Otx1−/−brains has been investigated through experiments aimedat determining possible changes of the normal numberof apoptotic or proliferating cells within the neuroep-ithelium of the developing telencephalon. No differ-ences at apoptosis were observed by comparing


wild-type and Otx1 mutant embryos. By contrast, Bro-modeoxyuridine (BrdU) labelling experiments revealeda reduction of proliferating cells (by about 25%) in thedorsal telencephalic neuroepithelium of 9.75 d.p.c.Otx1−/− embryos (Acampora et al., 1998a). A defec-tive proliferation of neuronal progenitors at these earlystages may thus be responsible of the adult phenotypeof the Otx1 mutant mice.

2.4. Otx1−/− mutant mice exhibit an epilepticphenotype

Otx1−/− mice exhibit both spontaneous highspeed turning behaviour and epileptic behaviour(Acampora et al., 1996). The latter consists of thecombination of, (i) focal seizures characterised by au-tomatisms (head bobbing and teeth chattering) andelectroencefalographic (EEG) recording of spikes inhippocampus; (ii) generalised seizures characterised byconvulsions and high voltage synchronised EEG activ-

ity in hippocampus and cortex. Occasionally, convul-sions are followed by status epilepticus and exitus.

Recently, the epileptogenic mechanisms accountingfor these seizures have been further investigated bymeans of electrophysiological recordings (current clumpintracellular recordings) made from layer 5 pyramidalneurons in somatosensory cortical slices (Avanzini etal., 2000). This analysis shows that g-aminobutyric acid(GABA)-mediated inhibitory post-synaptic potentials(IPSP), as compared with control pyramidal neurons,are more pronounced in the mutants where they seemto be involved in the synchronisation of the excitatoryactivity from the earliest postnatal period. On the otherhand, multisynaptic excitatory post-synaptic potentials(EPSP) are significantly more expressed in the mutantsthan in controls, also at the end of the first postnatalmonth. These results suggest that the excessive excita-tory amino acid-mediated synaptic driving, without awell-developed GABA counteraction, may lead to ahyperexcitable condition that is responsible for theepileptic manifestations occurring in Otx1−/− mice.

Table 1Major phenotypes observed in Otx l−/−; hOtx21/hOtx21 and otd1/otd1 mutant mice

Major phenotypes Otx1−/− hOtx21/hOtx21 otd1/otd1

Dorsal telencephalonReduced by 25%Cell proliferation rate at E9.75 Full recoveryFull recoveryReduced by 10% Full recoveryAt E 13.5 and E 15.5 Full recovery

Cerebral cortexReducedCell number Full recovery Full recovery

Full recovery Full recoveryLayer organisation AlteredN/Ab N/AbLaminar fatea Normal

Full recoveryFull recoveryTemporal cortex Reduced by 40%Reduced by 40%Perirhinal cortex Full recovery Full recovery

Hippocampus Full recoveryShrunken Full recovery

MesencephalonSize of colliculi Normal in 30%Enlarged Normal in 15%

Intermediate in Intennediate in45% 50%

Identities of colliculia N/AbN/AbNormalRecovery inAbnormal foliation Recovery in 10%Cerebellum50%

Axonal projection from layer 5 neurons of 6isual N/AbDefective refinement of exuberant projections N/Ab

cortexa

Beha6iourTurning behaviour High-speed Moderate-speed Moderate-speed

Present AbsentEpileptic seizures Absent

Impaired NormalPituitary gland Normal

EarAbsentAbsentAbsentLateral semicircular duct

EyeIris Reduced Recovery in Recovery in 80%

80%Absent Present in 70% Present in 80%Ciliary process

Lachrymal and Harderian gland Present in 75%Absent Present in 34%

a Data from Weimann et al. (1999).b N/A, not analysed.


2.5. Otx1 controls cortical connecti6ity to subcorticaltargets

Recent analysis of axonal projections in Otx1−/−mutants has shed new light on the role of Otx1 duringbrain development (Weimann et al., 1999). In severalbrain regions, connections usually develop by a bipha-sic mechanism in which an excess of early formed axonprojections is finely pruned by elimination of inappro-priate axon terminals. Layer 5 neurons in the visualcortex provide a good example of exuberance in con-nectivity, establishing, among others (e.g. to corpuscallosum), connections to a number of subcorticaltargets such as the pons, superior and inferior colliculi,and spinal cord. During early postnatal life, they selec-tively eliminate connections from the inferior colliculusand spinal cord (Stanfield and O’Leary, 1985; Stanfieldet al., 1982), but the molecular mechanisms underlyingthis remodelling are poorly understood.

Otx1 is strongly expressed in a subset of layer 5neurons that form subcortical but not cortical connec-tions (Weimann et al., 1999) and in layer 6 neurons,many of which forming thalamic projections. Analysisof Otx1−/− mutants reveals significant defects, spe-cifically in the patterning of subcortical projections. Infact, while callosal and thalamic projections appearnormal in the Otx1−/− mutants, there are additionalextensive innervations of both the inferior colliculusand the spinal cord that have been erroneously main-tained. This phenotype suggests that Otx1 function isrequired for the last step of subcortical axon develop-ment, in which exuberant connections undergo exten-sive refinement with the elimination of axon projectionsfrom inappropriate targets (Weimann et al., 1999).

2.6. Otx1 transiently controls GH, FSH, and LH inthe pituitary

Otx1 is postnatally transcribed and translated in thepituitary gland. Cell culture experiments indicate thatOtx1 may activate transcription of the growth hormone(GH), follicle-stimulating hormone (b-FSH), luteinizinghormone (b-LH), and a-glycoprotein subunit (a-GSU)genes. Analysis of Otx1 null mice (Acampora et al.,1998c) indicates that, at the prepubescent stage, theyexhibit transient dwarfism and hypogonadism due tolow levels of pituitary GH, FSH and LH hormoneswhich, in turn, dramatically affect downstream molecu-lar and organ targets. Nevertheless, Otx1−/− micegradually recover from most of these abnormalities,showing normal levels of pituitary hormones with re-stored growth and gonadal function at 4 months of age.Expression patterns of related hypothalamic genes suchas the growth hormone releasing hormone (GRH),gonadotropin releasing hormone (GnRH), and theirpituitary receptors (GRHR and GnRHR) suggest that,

in Otx1−/− mice, hypothalamic and pituitary cells ofthe somatotropic and gonadotropic lineages appear un-altered and that it is the ability to synthesise GH, FSH,and LH, rather than the number of cells producingthese hormones, to be affected (Acampora et al.,1998c).

An intriguing aspect of our observation is the factthat transcription factors of the Ptx and Otx sub-families recognise similar DNA target sequences (Sime-one et al., 1993; Lamonerie et al., 1996; Szeto et al.,1996; Tremblay et al., 1998), and that Ptx1 and Ptx2are expressed in most pituitary lineages, in particular insomatotrophic and gonadotrophic cells (Tremblay etal., 1998). Ptx1 is the most highly expressed of thesegenes, followed by Ptx2 and then Otx1 (Tremblay etal., 1998). Yet, the Otx1 knock-out has a dramaticeffect only during the prepuberal period. The uniqueactivity of Otx1 during this period might reflect aspecific interaction of Otx1, but not of the related Ptxfactor(s), with a transcription co-regulator in the soma-totrophic and gonadotrophic cells.

Taken together with previous reports, our observa-tions support the existence of complex regulatory mech-anisms defining combinatorial cell- and stage-specificinteractions between transcription factors belonging tothe same or to different gene families for the establish-ment/maintenance of pituitary function.

A novel feature of this phenotype is the fact thatmost of the impaired functions are recovered by theadult stage. Indeed, after the prepubescent stage, Otx1mutant mice begin to gradually recover from theirabnormalities, showing at 4 months of age normallevels of GH, FSH and LH which are paralleled by arestored normal body weight, differentiation and size ofboth testis and ovary, as confirmed also by their sexualfertility, and by normal levels of downstream moleculartargets such as testosterone and insulin growth factor 1(IGF1). Although we are unable to explain the mecha-nism underlying this recovery, this observation mightrepresent a possible example of temporal-restrictedcompetence in hormonal regulation of specific cell-lin-eages by the Otx1 transcription factor. This recoveryappears similar to the ‘catch-up growth’ (Boersma andWit, 1997) described in children with delayed growthand puberty, also called ‘constitutional delay in growthand abdolescence’, CDGA (Horner et al., 1978).

2.7. Otx1 is necessary for correct sense organde6elopment

Otx1−/− mutants show inner ear and eye abnor-malities that are consistent with Otx1 expression pat-tern (Acampora et al., 1996).

Otx1 is expressed in the lateral canal and ampulla aswell as in a part of the utricle, in the saccule andcochlea. Interestingly, Otx2 is co-expressed with Otx1


in the saccule and cochlea but not in the components ofthe pars superior. Lack of Otx1 results in the absenceof the lateral semicircular canal and lateral ampulla, inabnormal utriculosaccular and cochleosaccular ductsand in a poorly defined hook (the proximal part) of thecochlea (Acampora et al., 1996; Morsli et al., 1999).Defects in the shape of the saccule and cochlea arevariable in Otx1−/− mice and are much more severein Otx1−/− ; Otx2+/− background. In Otx1−/−and Otx1−/− ; Otx2+/− mutants the lateral cristais absent and the maculae of the utricle and saccule arepartially fused (Morsli et al., 1999).

In the eye and annexed structures Otx1 transcriptsare restricted to the iris, ciliary process and ectodermalcells migrating from the eyelid and included in themesenchymal component of the lachrymal glands.These ectodermal cells are believed to induce differenti-ation of the mesenchymal cells into a glandular ex-ocrine cell-type. In Otx1−/− mice the ciliary processis absent, the iris is thinner and the lachrymal andHarderian glands do not develop, failing the differenti-ation to a glandular cell-type. Interestingly, the ectoder-mal cells embedded within the mesenchymalcomponents are not identified in Otx1−/− mice, thusindicating that failure in development of the glands is aconsequence of the impaired migration of the ectoder-mal cells from the eyelid to the mesenchyme of thelachrymal primordium that in turn is not induced todifferentiate into the exocrine glandular phenotype(Acampora et al., 1996).

3. Early requirement of Otx2 for normal gastrulationand anterior neural plate induction and maintenance

3.1. Organisers and molecules in the current scenario ofthe anterior brain patterning

Fate and patterning of tissues depend on the activityof organiser cells emanating signals to a respondingtissue which undergoes morphogenetic changes result-ing in a specific differentiated fate (Spemann and Man-gold, 1924; Waddington, 1932; Gurdon, 1987).

Indeed, in amphibians, the dorsal lip of the blasto-pore induces a new, ectopic secondary axis when trans-planted on the ventral side of a host embryo. Becauseof this ability, the dorsal lip of the blastopore has beencalled the organiser (Spemann and Mangold, 1924).Further experiments in amphibian and chick embryoshave suggested that the age of the organiser tissueinfluences the extension of the induced neural plate aswell as its regional identity. An organiser deriving froman early gastrula induces anterior as well as posteriorneural tissue, whereas a late organiser induces onlyposterior tissue (Gallera, 1971; Nieuwkoop et al., 1985;Storey et al., 1992).

These results suggested the possibility that head andtrunk inducing ability of the organiser might be associ-ated to different cell populations coexisting in an earlyorganiser. In mouse, at late streak stage the node islocated at the rostral end of the primitive streak and isable to induce a secondary axis. Importantly, the sec-ondary axis lacks any anterior neural tissues (Bedding-ton, 1994) suggesting a functional parallelism with theamphibian late organiser. Inducing properties of anearly mouse node have been tested in transplantationexperiments, and also in this case it fails to induceanterior identity (Tam et al., 1997), thus indicating that,unlike the amphibian organiser, the mouse node isunable to induce rostral neuroectoderm, independentlyof its age.

The mouse node gives rise to the ame that at mid-latestreak stage underlies the anterior developing neu-roectoderm in the midline, and comprises mesoderm(prechordal plate and notochord) and the definitive gutendoderm. These node-derived tissues are similar tothose originated by the amphibian organiser and ex-press similar genes (Beddington, 1981; Lawson et al.,1991). Moreover, there is clear evidence that the murinenode and its derivatives are able both to emanateneuralising signals (Ruiz i Altaba, 1993, 1994; Bedding-ton and Robertson, 1999) with patterning properties ofdifferent A/P value (Foley et al., 1997; Shimamura andRubenstein, 1997) and to regulate the expression of theregion-specific neural genes En1 and Otx2 in the neu-roectoderm (Ang and Rossant, 1993; Ang et al., 1994).

Interestingly, recent findings have ascribed to theAVE, which surrounds the epiblast cells at the pre-earlystreak stage, a crucial role in specifying anterior neuralpatterning in mouse (reviewed in Beddington andRobertson, 1998, 1999). Expression analysis and celllineage studies remarkably contributed to define theorigin and the molecular identity of the AVE.

AVE precursor cells are located at the distal tip ofthe mouse pre-streak embryo and can be visualised bythe expression of the Hex gene (Thomas et al., 1998).Few hours later, descendants of this group of cells willbe found only in the perspective anterior region, givingrise to the first (molecular) asymmetry along the pre-sumptive A/P axis of the embryo.

This A/P asymmetry is confirmed by the analysis ofthe expression patterns of a number of genes such asHex, Hesx1, goosecoid (gsc), cerberus-like (cer-l),Lim1, Otx2 and nodal (Simeone et al., 1993; Thomasand Beddington, 1996; Thomas et al., 1998; Belo et al.,1997; Varlet et al., 1997; Dattani et al., 1998).

Cell ablation and tissue recombination experimentshave indicated the functional relevance of AVE ininducing anterior patterning. Indeed, it has been shownthat, (i) the removal of a patch of AVE cells expressingthe Hesx1 gene prevents the subsequent expression ofthis gene in the rostral neuroectoderm which becomes


reduced and abnormally patterned (Thomas and Bed-dington, 1996); (ii) recombination of chick epiblast withrabbit pre-streak AVE induces the expression of fore-brain markers, while chick hypoblast is unable to elicitthe same response, thus suggesting that chick hypoblastand murine AVE might not share the property ofspecifying anterior neural patterning (Knotgen et al.,1999); (iii) chimera and transplantation experiments aswell as the analysis of mouse mutants have indicatedthat a number of genes, including Lim1, Otx2 andnodal are required in the AVE for proper specificationof the anterior neuroectoderm (Acampora et al., 1995;Shawlot and Behringer, 1995; Ang et al., 1996; Varlet etal., 1997), and in the ame for maintenance and refine-ment of correct identities (Acampora et al., 1998b;Rhinn et al., 1998; Shawlot et al., 1999); (iv) embryoshomozygous for a null mutation in Cripto show expres-sion, even if abnormal, of anterior neural markers inthe absence of node-derived mesendoderm (Ding et al.,1998). This gene encodes a membrane-associatedprotein containing epidermal growth factor-like motifsand is expressed shortly before the onset of gastrulationin the proximal region of the epiblast where the primi-tive streak forms (Dono et al., 1993; Ding et al., 1998;Minchiotti et al., 2000). Cripto−/− embryos lack aprimitive streak and do not anteriorize the AVE, thusfailing to convert the proximo-distal into A/P axis(Ding et al., 1998). Nevertheless, the AVE markers arecorrectly expressed, although in a distal position andthe epiblast does express rostral neural markers such asOtx2 and En until 8–8.5 d.p.c., thus indicating that inthe absence of a node and derived tissues the epiblast isable to acquire the identity of anterior neuroectoderm.

Together with the ectopic transpantations of thenode (Beddington, 1994; Tam et al., 1997), these resultspoint to the possibility that in the mouse the AVEcontains genetic information required to instruct thepatterning of the rostral neuroectoderm and, hence,there might be two physically separated organisingcentres, AVE and node, responsible for the induction ofhead and trunk structures, respectively.

This idea has been re-considered in the light of newmouse models recently generated. In particular,Chordin−/− ; Noggin−/− (Chd−/− ; Nog−/− )double mutants have provided evidence that they arecrucial in early forebrain development. These genes areexpressed in the mouse node and its derivatives but notin the AVE, and single null mutation of either of themdoes not affect anterior neural patterning. However,Chd/Nog double mutants display severe defects in thedevelopment of prosencephalon (Bachiller et al., 2000).Analysis at the early stages of the mutant embryos hasdemonstrated that the AVE is correctly established,thus suggesting that either the ame cells have their ownhead-inducing properties which are affected in thesemutants, or are required for the early maintenance of

anterior patterning initiated by the AVE, which per semight be necessary, but not sufficient, to induce theanterior neural plate.

Early patterning of the CNS primordium is alsocontrolled by additional mechanisms involving planarsignals acting through the neuroectodermal plane (Do-niach, 1993; Ruiz i Altaba, 1993, 1994). Furthermore, ithas been shown that in zebrafish a small group ofectodermal cells located in the anteriormost head regionis required for the patterning and survival of the ante-rior brain (Houart et al., 1998; Ruiz i Altaba, 1998).

Finally, heterotopic transplantation studies have in-dicated that it is only from the synergistic interaction ofanterior germinal layers (AVE and anterior epiblast)and a fragment of the posterior epiblast (early node) ofearly-streak embryos that it is possible to induce a fullneural axis including anterior neural gene expression(Tam and Steiner, 1999). Similar experiments involvingremoval and ectopic transplantation of anterior midlinetissue (AML, which includes both ame and ventralneuroectoderm) from late-streak embryos, have shownthat AML is required for maintenance and regionaliza-tion of the forebrain (Camus et al., 2000).

Among the genes required in the early specificationof the anterior neural plate, Otx2 plays a remarkablerole in both induction and maintenance of the rostralneural patterning (Simeone, 1998; Acampora and Sime-one, 1999). These and other potential roles are nowsubject of intense study that takes advantage fromgenetically modified mouse models and embryologicalapproaches.

3.2. Specification of anterior patterning requires Otx2function

In mouse, Otx2 is already transcribed before theonset of gastrulation in the epiblast and in the visceralendoderm (VE), and at the end of gastrulation in theame and rostral neural plate (Simeone et al., 1992,1993; Ang et al., 1994). During brain regionalization,Otx2 shows expression domains largely overlappingwith those of Otx1, with a posterior border coincidentwith the mesencephalic side of the isthmic constriction(Simeone et al., 1992; Millet et al., 1996).

Therefore, during gastrulation Otx2 is transcribedand translated in the cells that are believed to emanatesignals in early specification and patterning of the neu-ral plate (AVE and ame) as well as in those respondingto these instructing signals (epiblast and anterior neu-roectoderm) (reviewed in Simeone, 1998; Acamporaand Simeone, 1999, Fig. 1A). The first indication thatOtx2 is responsive to inductive interactions comes fromexplant-recombination experiments in gastrulatingmouse embryos showing that a positive signal fromame of headfold stage embryos is able to maintainOtx2 expression in the anterior ectoderm of early


Fig. 1.


streak embryos, and that a negative signal from theposterior mesendoderm, mimicked by exogenousretinoic acid (RA), represses Otx2 expression in theanterior ectoderm of late streak embryos (Ang et al.,1994). Similar interactions have been also demonstratedin Xenopus (Blitz and Cho, 1995).

The possibility that RA might contribute to the earlydistinction between fore-midbrain and hindbrain bycontrolling Otx2 expression is supported by the findingthat administration of exogenous RA at mid-late streakstage represses Otx2 expression in both the ame and theposterior neural plate (Ang et al., 1994; Simeone et al.,1995; Avantaggiato et al., 1996). This repression corre-lates with the appearance of microcephalic embryosthat show early anteriorization of Hoxb1 expression,hindbrain expansion (Sive and Cheng, 1991; Conlonand Rossant, 1992; Marshall et al., 1992), loss offorebrain molecular and morphological landmarks, andgain of midbrain molecular markers in the rostralmostneuroectoderm (Simeone et al., 1995; Avantaggiato etal., 1996). Moreover, Otx2 responsiveness to RA appli-cation is a common feature in different species includ-ing Xenopus and chick (Bally-Cuif et al., 1995; Panneseet al., 1995). Nevertheless, the question of whetherendogenous RA plays a physiological role in rostralCNS demarcation by contributing to the establishmentof the posterior border of Otx2 expression, still remainsopen.

The evidence that Otx2 may play a remarkable rolein rostral CNS specification derives from in vivo geneticmanipulation experiments performed in mouse andXenopus which, to some extent, complement each other.In Xenopus, microinjection of synthetic Otx2 RNAresults into an abnormal reduction in size of tail andtrunk structures, and in the appearance of a secondcement gland (Blitz and Cho, 1995; Pannese et al.,1995). These phenotypes have been interpreted eitherwith a possible Otx2-mediated interference with move-

ments of extension and convergence during gastrulation(for trunk and tail reduction) and/or with an Otx2-re-quirement in the specification of anteriormost headstructures (for the ectopic cement gland). Moreover, byusing a dexamethasone-inducible OTX 2 protein it hasbeen shown that the Xenopus Otx2 activity is regulatedby regionally restricted factor(s), and that the cementgland-specific gene XCG may represent a direct targetof the Otx2 gene product (Gammill and Sive, 1997).

In mouse, Otx2 null embryos die early in embryogen-esis, lack the rostral neuroectoderm fated to becomeforebrain, midbrain and rostral hindbrain, and showheavy abnormalities in their body plan (Fig. 1E)(Acampora et al., 1995; Matsuo et al., 1995; Ang et al.,1996). Heterozygous Otx2+/− embryos into an ap-propriate genetic background show defects of the headsuch as serious brain abnormalities and craniofacialmalformations, which are reminiscent of otocephalicphenotypes (Matsuo et al., 1995).

The analysis of Otx2 null embryos revealed that atlate streak stage, the rostral neuroectoderm is not spe-cified and the primitive streak as well as the node andthe ame are severely impaired. Therefore, one possibil-ity is that the resulting headless phenotype might bedue to the abnormal development of node-derived amewhich lacks head organiser activity. A second possibil-ity is that neural inducing properties of the AVE areseverely impaired or abolished. In embryos replacingOtx2 with the lacZ reporter gene, the first abnormalityis already detected at the early streak stage (Acamporaet al., 1995). Indeed, at this stage, lacZ transcriptionand staining are abolished in the epiblast while remain-ing high in the VE of Otx2−/− embryos (Fig. 1D);the gsc expression is undetectable or confined to theproximal region of the mutant embryos (Acampora etal., 1995); and the presumptive AVE does not anteri-orize, thus remaining confined to the distal region ofthe embryo (Fig. 1D).

Fig. 1. Schematic representation and morphology of Otx2−/− and hOtx12/hOtx12 mutant embryos as compared with wild type at differentstages. In wild-type embryos at early streak stage (A) (6.5 d.p.c.) Otx2 mRNA and protein colocalize in VE and epiblast cells. Findings from Otx2null embryos (D, E) suggest that in wild-type embryos (A–C) at the early-streak stage Otx2 is required in the VE to mediate signal(s) (arrowsin A) that are directed to the epiblast and are required for specification of anterior patterning, while at late gastrula-headfold stage, Otx2 isrequired to maintain fore-midbrain regional identities (see also F–H) even though it cannot be assessed in which tissue (ame or neural plate) itplays this role. In null embryos (D, E), where Otx2 is replaced with the lacZ gene, the first impairment is seen at the pre-early streak stage (D)when lacZ transcription is lost in the epiblast and confined at the distal tip of the embryo. This observation indicates that at least one copy ofthe normal Otx2 allele is required in the VE to rescue signal(s) necessary for its transcription in the epiblast, for specification of anterior patterningand proper gastrulation. These findings suggest that Otx2 is an important component of VE organizing properties. The arrow in (E) points tothe rostral limit of the neuroectoderm in an Otx2−/− embryo. In hOtx12/hOtx12 embryos (F–H), at early streak stage (F) (6.5 d.p.c.), hOtx1transcripts are detected correctly in AVE and epiblast cells while, in contrast, the hOTX 1 protein is restricted to the VE. This model confirmsthe OTX 2 gene product requirement in the AVE for specification of the early anterior neural plate and demonstrates that hOTX 1 and OTX 2proteins are functionally equivalent in the VE. Moreover, the differential post-transcriptional control of hOtx1 transcripts makes it possible todistinguish between specification and maintenance properties of Otx2. In fact, while the specification of the anterior neural plate and normalgastrulation are rescued by AVE-restricted hOTX 1 protein (F), the maintenance of anterior patterning is not recovered for the absence of anyOTX protein in the epiblast-derived cells (anterior neural plate and ame) (G). This finding leads to the attractive hypothesis that Otx2-mediatedmaintenance properties (Otx2 translation in epiblast cells) might have been acquired during evolution to specify the vertebrate head. Abbreviations— AVE, anterior visceral endoderm; epi, epiblast; Hb, hindbrain; is, isthmus; Ms, mesencephalon; np, neural plate; Ov, otic vescicle; ps, primitivestreak; Te, telencephalon; and VE, visceral endoderm.


Therefore, these results indicate that headless pheno-type and abnormal organisation of the primitive streakmay be determined very early at the pre-early-streakstage by an impairment of AVE properties.

Moreover, as revealed by the mesodermal early markerBrachyury (T), epiblast cells do not migrate posteriorlyat the site of the primitive streak formation but remainfor an abnormally longer period in a ring close toembryonic-extraembryonic boundary. Only later, and ina small number of Otx2−/− embryos, the presumptiveAVE cells appear anteriorized. However, although ab-normal, a primitive streak forms in all the Otx2−/−embryos and mesodermal cells appear to be concentratedat the posterior third of the embryo (Acampora et al.,1995, 1998b; Ang et al., 1996).

These findings, therefore, favour a relationship be-tween primitive streak formation and anterior locationof the AVE and suggest that Otx2, normally expressedin both epiblast cells and AVE, might be required in thesetwo cell types in order to mediate proper positioning ofthe AVE and normal formation of the primitive streak.However, data deduced from the analysis of chimericembryos and further mouse models indicate that earlyabnormalities in both AVE and primitive streak ofOtx2−/− embryos should be ascribed to Otx2 require-ment in the VE (Acampora et al., 1998b; Rhinn et al.,1998; see below).

Interestingly, an intriguing feature of Otx2−/−embryos is that lack of anterior neuroectoderm is paral-leled by failure of epiblast cells to express the lacZreporter gene.

Thus, since Otx2 is already transcribed from theearliest stages (unfertilised egg in Xenopus and at leastmorula in mouse), this observation suggests that mainte-nance of Otx2 transcription in the epiblast cells requiresat least one normal allele expressed in the AVE whileOtx2 transcription in the latter is independent of thepresence of a normal allele (Simeone, 1998; Acamporaand Simeone, 1999).

These findings also support the existence of Otx2-me-diated signal(s) emitted from the AVE and directed to theepiblast cells. Nevertheless, it is still unclear whether thesame signal operates to mediate both specification ofanterior patterning and Otx2 transcription in epiblastcells or these two events are independently controlled.

3.3. The role of Otx2 in the induction and maintenanceof rostral CNS identities

In order to understand the role of Otx2 in thespecification of the anterior patterning, it is of particularrelevance to define its functional contribution to thedifferent tissues where it is expressed during gastrulation.

A direct evidence proving a role for Otx2 in the AVEhas been recently provided by generating murinechimeric embryos and new mouse models (Acampora et

al., 1998b; Rhinn et al., 1998; Acampora et al., unpub-lished results). Chimeric embryos containing Otx2−/−epiblast and wild-type VE rescue an early Otx2−/−neural plate but subsequently fail to develop a brain,suggesting that Otx2 is firstly required in the AVE forinduction of rostral neural plate and, then, in theepiblast-derived tissues for specification of forebrain andmidbrain regional identities. Conversely, when chimericembryos consist of an Otx2−/− VE and an Otx2+/+epiblast, none of the phenotypic features of Otx2−/−embryos are rescued (Rhinn et al., 1998). This latterresult also argues, as previously suggested by Otx2−/−mice (Acampora et al., 1995), that impaired ame ofOtx2−/− embryos is a consequence of Otx2 require-ment at earlier stages in the VE.

Mice replacing Otx2 with Otx1 were originally gener-ated in order to assess whether the two proteins sharedfunctional equivalence or, alternatively, displayed uniqueproperties specified by their limited amino acid diver-gence. Murine OTX 1 and OTX 2 gene products, in fact,share extensive sequence similarities even though inOTX1, downstream of the homeodomain, these regionsof homology to OTX2 are separated by stretches ofadditional amino acids including repetitions of alanineand histidine residues (Simeone et al., 1993).

Interestingly, homozygous mutant embryos replacingOtx2 with the human Otx1 (hOtx1) cDNA (hOtx12/hOtx12) recover anterior neural plate induction andnormal gastrulation but show a headless phenotype from9 d.p.c. onwards (Fig. 1F–H). A combined analysis ofboth hOtx1 RNA and protein distribution during earlygastrulation has revealed that while the hOtx1 mRNAis detected in the VE and epiblast, the hOTX1 proteinis revealed only in VE (Fig. 1F). Nevertheless, thisVE-restricted translation of the hOtx1 mRNA is suffi-cient to recover early anterior neural plate but fails tomaintain fore-midbrain identities (Fig. 1G), and, conse-quently, hOtx12/hOtx12 embryos display a headlessphenotype with a normal body plan (Fig. 1H) (Acam-pora et al., 1998b).

In fact, at early-streak stage, hOtx12/hOtx12 embryosrecover anteriorization of the AVE, normal primitivestreak and proper expression of Lim1, cer-l, Hesx1, gscand T. At late gastrula stage, the anterior patterning ofthe rostral neural plate is correctly defined by theexpression of fore-, mid- and hindbrain markers such asSix3, Pax2, Gbx2 and Hoxb1 and the ame properlyexpresses Lim1, Noggin, cer-l and Hesx1. These findingssupport the idea, as indicated by chimeric studies (Rhinnet al., 1998) that Otx2 is required in the AVE forinitiating the specification of the anterior patterning anddemarcation of forebrain and midbrain territories(Acampora et al., 1998b).

Further analysis of hOtx12/hOtx12 embryos revealsthat at the early somite stage the forebrain markersBF1 and the hOtx12 mRNA are not detected in therostral neuroectoderm where mid-hindbrain markers


such as Wnt1, En1, Fgf8, Gbx2 and Pax2 appear to beco-expressed.

These observations lead to argue that the OTX2 geneproduct is required from the headfold stage onwards tomaintain the anterior patterning previously establishedby the AVE. In this respect, the phenotype observed atthe early somite stage appears to be the consequence ofan A/P repatterning process involving the entire ante-rior neural plate (fore-midbrain) which, in the absenceof any OTX gene product, adopts a more posterior fate(hindbrain) (Acampora et al., 1998b; Acampora andSimeone, 1999). Therefore, the hOtx12/hOtx12 mousemodel allows to uncouple two distinct phases bothrequiring Otx2 early induction of anterior neural pat-terning that is under the control of AVE and its subse-quent maintenance that is likely mediated byepiblast-derived cells (the ame and neuroectoderm).

However, since Otx2 is normally transcribed andtranslated in both ame and rostral neuroectoderm,while the hOtx12 mRNA is transcribed but not trans-lated either in the ame or in the rostral neuroectoderm,it cannot be deduced from this model whether Otx2 isrequired in one or both of these tissues to mediatemaintenance of the anterior identity and whether hOtx1is functionally equivalent to Otx2 also in these tissues.A cell-autonomous role of Otx2 in the neuroectodermhas emerged from the analysis of chimeras with amoderate contribution of Otx2−/− cells (Rhinn etal., 1999). It has been shown that genes such as Wnt1,Hesx1 and R-cadherin are selectively, not expressed inOtx2−/− cells whereas other genes such as En2 andSix3 are uniformly expressed in neuroectodermal cellsof both genotypes.

How Otx2 is integrated in the network of othergenetic functions which have been shown to be funda-mental for anterior neural patterning is still an openquestion. The overall phenotype of Lim1−/− andhOtx12/hOtx12 embryos, for example, show impressivephenotypic similarities. Recently, the stage and the celltype in which Lim1 is required for head specificationhave been approached by chimera and tissue recombi-nation experiments (Shawlot et al., 1999). These experi-ments have revealed that Lim1 is required in the AVEfor inducing anterior neural patterning and, subse-quently, in the ame to refine and maintain the anterioridentity. Therefore, since maintenance of anterior pat-tern requires Otx2 in the ame or in the anterior neu-roectoderm or in both tissues, it can be speculated thatLim1 might contribute to mediating the release of amesignal(s) instructing maintenance of anterior characterand Otx2 might confer to neuroectoderm the compe-tence to respond to this signal(s) emitted from themesendoderm. This possibility is also compatible with acell-autonomous role within the neuroectoderm anddoes not exclude an additional role also in the ame.

A recent in vitro study supports the possibility of adirect protein–protein interaction between OTX 2 C-terminus and Lim1 homeodomain (Nakano et al.,2000).

3.4. AVE-restricted OTX2 function is necessary forproper expression of Lim1 and bone morphogeneticprotein (BMP) antagonists in the ame

In hOtx12/hOtx12 (Acampora et al., 1998b) andchimera experiments (Rhinn et al., 1998), a tight corre-lation exists between correct specification of anteriorpatterning and the recovery of normal gastrulation.Moreover, in Otx2−/− embryos failure of anteriorspecification always coincides with a distal location ofthe AVE and abnormal gastrulation. In particular, inthese embryos ame is no longer identifiable, and expres-sion of Nog, Chd and Lim1 is strongly abnormal orabsent (Acampora et al., 1998b; Rhinn et al., 1998). InhOtx12/hOtx12, general morphology as well as expres-sion of these genes in the node and ame appears to benormal, and additionally, cer-l expression in the defini-tive endoderm emerging from the node also appears tobe unaffected.

As previously mentioned, recent studies have shownthat (i) anterior midline tissue including ame and ven-tral neuroectoderm is required for maintenance andregionalization of forebrain (Camus et al., 2000); (ii)BMP antagonists such as Nog and Chd are required ina temporally coherent manner in the node and ame forforebrain development (Bachiller et al., 2000); and (iii)Lim1 is required in the ame for stabilising anteriorpatterning (Shawlot et al., 1999). On this basis, it canbe hypothesised that anterior specification by AVE-re-stricted OTX function might act through ame-restrictednon-cell autonomous properties of Chd, Nog and Lim1.This hypothesis does not exclude the existence of adirect inducing signal for anterior patterning emanatingfrom the AVE. Rather it indicates that the VE is alsoimportant for normal development of the primitivestreak (Beddington and Robertson, 1999). In this re-spect, it is noteworthy that chimera experiments per-formed to assess the role of HNF3b have indicated thatthis gene is necessary in the VE to promote properprimitive streak elongation (Dufort et al., 1998).

Since, contrary to OTX2, hOTX1 protein is notdetected in the ame, it cannot be excluded a priori thatOTX2 also plays a role in these cells. Noteworthy isalso the fact that in the absence of any OTX protein,hOtx12/hOtx12 embryos exhibited anterior patterningat least until 7.75–8 d.p.c., while they subsequently failin proper positioning of the IsO at the MHB. Chd−/− ; Nog−/− embryos lacked anterior brain structuresand retained a relatively normal midbrain (Bachiller etal., 2000). Together, these findings suggest that whileearly forebrain maintenance and/or induction is under


the control of Chd/Nog signalling in the ame, OTX 2 isrequired for late maintenance by specifying midbrainterritory and allowing the correct positioning of theIsO.

3.5. Specific and interchangeable roles between Otx1and Otx2

Mutant phenotypes, expression patterns (see above)and amino acid sequence comparison support two pos-sibilities, (i) the functional properties of Otx1 and Otx2largely overlap and differences in their temporal andspatial transcriptional control are responsible for thehighly divergent phenotypes of Otx1−/− andOtx2−/− mice; or (ii) OTX1 and OTX2 gene prod-ucts display unique functional properties specified bytheir limited amino acid divergence. In order to distin-guish between these two possibilities, mice models thatreplace Otx1 with the human Otx2 (hOtx2) cDNA andvice versa were generated.

Homozygous mice in which Otx1 is replaced with thehuman Otx2 cDNA (hOtx21/hOtx21), despite a re-duced expression of the transgenic alleles, no longershow epilepsy and corticogenic abnormalities caused bythe absence of Otx1. This rescue is due to a restorednormal cell proliferation activity in the dorsal neuroep-ithelium, as revealed by BrdU labelling experiments at9.5 d.p.c. (Acampora et al., 1999a). This is particularlyrelevant when considering the different expression pat-terns of Otx1 and Otx2 genes in the dorsal telen-cephalon from the 9.5 d.p.c. stage onwards. Indeed, atthis stage, while Otx1 is expressed throughout theentire dorsal telencephalon, Otx2 is expressed only inthe mediodorsal area and basal neuroepithelium,whereas it disappears completely from the mediodorsalarea by 11 d.p.c. Therefore, the rescue observed inhOtx21/hOtx21 mice suggests that Otx1 and Otx2 haveinterchangeable roles in the cortex. To complement theinformation derived by the loss-of-function and genereplacement experiments, it would be particularly inter-esting to see also the effects of an OTX over-dosage onthe brain and, specifically, on cortical development.

hOtx21/hOtx21 mice also show a significant improve-ment of mesencephalon, eye and lachrymal gland de-fects. Some of the Otx1−/− inner ear abnormalitiesare also rescued in the regions where the Otx genes arenormally co-expressed, but not the absence of the lat-eral semicircular canal. As previously reported in detail,homozygous mutant mice in which Otx2 has beenreplaced with the human Otx1 (hOtx1) cDNA (hOtx12/hOtx12) recover the anterior neural plate and propergastrulation but fail to maintain fore-midbrain identi-ties, displaying a headless phenotype from 9 d.p.c.onwards (Acampora et al., 1998b). A deeper analysishas revealed that despite RNA distribution in both theVE and epiblast, the hOTX1 protein was synthesised

only in the VE, where it was sufficient to rescue VE-re-stricted Otx2 functions.

In sum, these mouse models support an extendedfunctional equivalence between OTX1 and OTX2proteins and provide evidence that Otx1−/− ;Otx2−/− contrasting phenotypes stem from differ-ences in expression patterns rather than in amino acidsequences.

The clearest exception to the overall Otx functionalequivalence is provided by the lateral semicircular canalof the inner ear, that in hOtx21/hOtx21 mice is neverrestored (Morsli et al., 1999) as well as in mice in whichOtx1 is replaced with the Drosophila otd gene (Acam-pora et al., 1998a). These findings, discussed below inevolutionary terms, suggest that the ability to specifythe lateral semicircular canal of the inner ear may be,indeed, an Otx1-specific property (Acampora andSimeone, 1999).

However, it is noteworthy that these studies do notaddress the question of whether hOTX1 might be func-tionally equivalent to OTX2 in the embryonic neu-roectoderm and ame, due to the lack of hOTX1 proteinin these tissues. Genetic replacement studies carried outby Suda et al. (1999) provide some evidence that OTX1might be able to partially rescue the headless phenotypethat was observed in hOtx12/hOtx12 mutant animals.However, it is unclear whether the amount of OTX1was similar to that of OTX2 in these experiments (Sudaet al., 1999). This is an important aspect since a reduc-tion of OTX gene products below a critical threshold isalways reflected in a mis-specification of fore- andmidbrain regions (Acampora et al., 1997, 1998b; Sudaet al., 1997; Broccoli et al., 1999; Millet et al., 1999;Simeone, 2000). In this context, it has been recentlydiscovered that different roles attributed to two Hoxgenes belonging to the same paralogous group are theresult of quantitative modulations in gene expressionrather than of qualitative properties depending onprotein sequence (Duboule, 2000; Greer et al., 2000).Thus, even if OTX1 and OTX2 do share a certaindegree of functional equivalence in the neuroectoderm,the limited partial recovery of the phenotype might beascribed either to a reduced level of OTX1 protein or toan OTX2-specific function. Further investigation isneeded to address this important evolutionary aspect.

4. Brain patterning depends on a critical Otx genedosage

4.1. Morphogenetic origin of the IsO

The initial patterning of the primitive neuroepithe-lium is established during early gastrulation, when sig-nals originating from both the underlying mesodermand the VE or present within the ectoderm itself,


roughly divide the future CNS into few main subdivi-sions (Rubenstein and Shimamura, 1997; Beddingtonand Robertson, 1998). At the end of this process,different regions have acquired their own identity ex-pressing a unique set of transcription factors. Later on,potent signalling centres are generated at the boundarybetween adjacent territories, which express diffusiblemolecules that refine and polarise the neighbouringneural tissue (Meinhardt, 1983; Rubenstein et al.,1998). The best studied centre is located at the MHB inthe isthmic region. Over the last years, elegant tissuetransplantation experiments in chick have addressed itsrole in neural patterning (Martinez et al., 1991, 1995;Marin and Puelles, 1994). In fact, when isthmic tissue isgrafted to ectopic sites in the brain, as the caudalprosomeres or some rhombomeres, it can not onlymaintain its own identity, but can also induce thesurrounding tissue to change its programmed fate giv-ing rise to midbrain and cerebellar structures. Due tothis potent non cell-autonomous function, the MHBsignalling centre has been re-named IsO. However,within the forebrain, the territory rostral to the ZLI,the boundary between prosomeres 2 and 3, is notcompetent to respond to midbrain inducing signalsreleased by the IsO (Martinez et al., 1991). In the lastyears, different proteins specifically expressed in theisthmic region and required for its development havebeen characterised. They include the transcription fac-tors EN1, EN2, PAX2, PAX5 and two secretedmolecules, WNT1 and FGF8 (Rowitch and McMahon,1995; Joyner, 1996). Although some of these factorslike EN2 and PAX5 appear to be general markers ofthe region, the dynamic expression profiles of the others(EN1, PAX2, WNT1 and FGF8) parallel the progres-sive refinement of specification taking place within themidbrain-hindbrain domain. In fact, if their expressiondomains are initially broad encompassing the presump-tive midbrain and rostral hindbrain (in 1–5 somite oldembryo), later on they become progressively restrictedto narrow transverse rings that encircle the neural tubein the isthmic area (Bally-Cuif and Wassef, 1995). Inparticular, Wnt1 ring of expression is almost com-pletely overlapped and localised at the caudal end ofthe prospective midbrain rostral-adjacent to the Fgf8positive ring that lies on the isthmic-cerebellum anlage(Hidalgo-Sanchez et al., 1999b, Fig. 2). The functionalrole of all these genes (En, Pax, Fgf8 and Wnt1) hasbeen tested in great detail in different animal models,clearly showing their requirement for the normal devel-opment of this region. In fact, mice homozygous for aWnt1 null allele completely lack all the midbrain andisthmic derivatives (McMahon and Bradley, 1990;Thomas and Capecchi, 1990). Mutations in En1 cause asimilar but milder phenotype (Wurst et al., 1994),whereas, mutations in En2 only lead to subtle defects incerebellum cyto-organisation (Joyner et al., 1991).

However, En compound mutant mice lack the entireregion resembling the Wnt1 mutant phenotype (W.Wurst and A.L. Joyner, unpublished results) and En2can rescue the En1 mutant brain defects when it isexpressed in place of En1, revealing overlapping func-tions between the two genes (Hanks et al., 1995). Asimilar brain deletion has been found also in micecarrying both Pax2 and Pax5 null alleles (Schwarz etal., 1997; Urbanek et al., 1997) and in the zebrafish noimutants that harbour an induced mutation in the Pax2orthologue (Brand et al., 1996; Lun and Brand, 1998).Likewise, Fgf8 mutant mice and zebrafish display anearly brain deletion due to degeneration of the mid-brain and isthmic tissue (Meyers et al., 1998; Reifers etal., 1998). In each mutant outlined so far, it has beenreported that the expression of the other genes de-scribed is correctly initiated but no longer maintained.This is probably the cause that leads to the describedloss of the brain structures (McMahon et al., 1992;Reifers et al., 1998; V. Blanquet and W. Wurst, unpub-lished results). These important findings strongly sug-gest a close functional connection among these genes,where the inactivation of a gene is able to feedbacknegatively on the transcription of all the other genes,leading to the degeneration of brain structures. Thegenetic maintenance loop among these genes is themolecular origin of the IsO and subsequent midbrain–hindbrain specification and morphological shaping(Fig. 3). This hypothesis is also supported by the strik-ing phenotypes obtained in gain of function experi-ments. When, in fact, acrylic beads soaked withrecombinant FGF8b protein are implanted in differentareas of the caudal forebrain, the nearby regions areinduced to change their fate into midbrain, cerebellarand isthmic structures (Crossley et al., 1996; Irving andMason, 1999; Martinez et al., 1999). These results aremediated by the de-novo induction of Wnt1, Pax2,En2, En1 in the site of bead implantation. The samephenotype is obtained by inducing En gene expressionectopically in the caudal prosomeres either in zebrafish(Ristoratore et al., 1999) or chick (Shamim et al., 1999).Thus, single genes can trigger the activation of theentire genetic loop formed by Wnt, Fgf, Pax and Engenes to initiate the neural fate re-specification.

4.2. Otx genes are required for correct positioning ofthe isthmus

An important question to be answered is how allthese genes are induced in this particular region of thedeveloping neural tissue. Why not more anteriorly inthe forebrain or more caudally in the spinal cord?Grafting or tissue ablation experiments in chick haveshown that Fgf8 expressing tissue with properties of theIsO is generated when midbrain and rhombomere 1tissue are juxtaposed but not when midbrain contacts


Fig. 2. A schematic gene expression network in mice with different Otx2, Gbx2 or Fgf8 gene dosage that display a re-positioning of the MHBalong the neural tube axis. In embryos with a decreasing Otx gene dosage (Otx1−/− ; Otx2+/− ), the Gbx2 (orange) and Fgf8 (grey)expression is abnormally spread into the rostral regions of the mesencephalon and 1st, 2nd, and 3rd prosomeres (p). The rostral shift of theirexpression domains cause a more anterior relocation of the MHB just near to the ZLI area. Moreover, the absence of OTX gene products in theneuroepithelium in hOtx12/hOtx12 mouse embryos cause the gene expression deregulation of Gbx2, Fgf8, and Wnt1 (violet) along all the neuralplate. On the contrary, in gain of function experiments, the Otx2 ectopic expression in the metencephalon (met) leads to a repression of Fgf8 andGbx2 activity in the same region, that positions the MHB more caudally (En1Otx2/+). In Gbx2 mutant embryos (Gbx2−/− ), Otx2 and Wnt1expression domains are enlarged caudally including rhombomere (rh) 3, and the mesencephalic tissue (mes) is expanded into the cerebellar anlage.Ectopic expression of Gbx2, under the control of the Wnt1 regulatory regions (Wnt1-Gbx2), leads to a transient expansion of the metencephalicregion preceded by an ectopic rostral expression of Fgf8. The same results are obtained by ectopically expressing the Fgf8b spliced formed in themesencephalon. Note that the nomenclature of the genotypes has not standardized and they are reported as cited in the original papers.


Fig. 3. Genetic hierarchy determining MHB formation. In the induc-tion phase (7.5–8.5 d.p.c.), Fgf8 expression is induced where theOtx2 and Gbx2 expression domains face each other. Recently, severalreports have been shown that Fgf8 is activated when mesencephalicand metencephalic fated tissues are placed in contact. This secretedmolecule is able to activate several transciption factors like PAX2,PAX5, EN1, EN2 and others that are necessary for the futurespecification and patterning of the region. All these molecules seem tomaintain each other into an auto-regulatory loop. Moreover, FGF8can re-induce Gbx2 expression, and inhibit Otx2 activity.

main towards the future diencephalic regions (Fig. 2)Therefore, a level above a critical threshold of Otx geneproducts is required to repress Fgf8 and, therefore, tomaintain its correct transcript localisation at earlysomite stage. This molecular event triggers, few hourslater, a rostral repositioning of En1 and Wnt1 expres-sion domains followed by a more general anterior shiftof the hindbrain specific markers and by failure ofactivation of the mesencephalic molecular determi-nants. Morphological alterations start to be scored at10.5 d.p.c., when the isthmic region is repositioned inthe caudal diencephalon, leading to replacement of themesencephalic vesicle with a metencephalic-fated tissue(Acampora et al., 1997). Similar results are obtainedwhen analysing the compound heterozygous embryos,Otx1+/− ; Otx2+/− in the CBA/C57 genetic back-ground, where anterior expansion of rhombomere 1combined with loss of mesencephalic tissue have beenalso described (Suda et al., 1997). These findings arguein favour of a negative feedback loop between Otx2and Fgf8, that sharpens the two neighbouringboundaries of their complementary expression domains(Hidalgo-Sanchez et al., 1999b; Fig. 3). This hypothesisis strengthened by a different report (Martinez et al.,1999) that describes a strong repression of Otx2 expres-sion in the chick mesencephalon around the placewhere a FGF8 secreting source is implanted. Moreover,the ectopic expression of the Fgf8b isoform under thecontrol of the Wnt1 regulatory regions, inhibits theendogenous Otx2 expression in the mesencephalic an-lage and tranforms this area in hindbrain-fated tissue(Liu et al., 1999; Fig. 2). An even stronger re-locationof the IsO in the neuroepithelium is present in thehomozygous hOtx12/hOtx12 embryos described previ-ously (Acampora et al., 1998b). In this mouse model,where no OTX protein is present in epiblast derivatives,embryos normally undergo specification of the anteriorneural plate which, however, fails to maintain its iden-tity and acquires a more posterior fate, yielding aheadless phenotype at late embryogenesis as assessed bythe rostral mislocalisation at the rostral tip of theneuroectoderm of all the genes normally present in theIsO and rostral hindbrain, like Wnt1, Fgf8, Pax2, En1and Gbx2, (Acampora et al., 1998b; Fig. 2). Thisfinding provides a strong evidence for an Otx genedosage requirement in anterior neural fate determina-tion and following maintenance throughout a contin-ued repression on the posteriorizing geneticdeterminants (Simeone, 1998). Is the action played byOtx genes only necessary or even sufficient to induceanterior fate in the neural plate? Gain of functionexperiments have been carried out, in the last years, toanswer this question. As already mentioned, over-ex-pression of Otx2 RNA in Xenopus results in an abnor-mal expansion of the head coupled with a strongreduction of the tail and trunk and with ectopic forma-

any other rh or neural tissue (Hidalgo-Sanchez et al.,1999a; Irving and Mason, 1999). The genetic identity ofthe tissue rostral and adjacent to the Fgf8 expressiondomain is depending on Otx1 and Otx2 transcriptionfactors (Simeone et al., 1993; Simeone, 1998). As previ-ously mentioned, Otx2−/− embryos fail to specifyforebrain, midbrain, and anterior hindbrain. The lastregion is specified by the IsO and never expresses Otx2(Acampora et al. 1995; Matsuo et al., 1995; Ang et al.,1996). This finding argues in favour of an involvementof the Otx genes in the early IsO generation. Moreover,a more direct involvement of the Otx function in theanterior neuroepithelium specification is provided bythe phenotype analysis of Otx compound mutants car-rying only one Otx2 functional allele. In fact, whileOtx1−/− ; Otx2+/− embryos do not displaymesendoderm and gastrulation defects, they showmolecular and morphological transformation of thecaudal prosomeres 1–2 and mesencephalon into a strik-ingly enlarged metencephalon (Acampora et al., 1997).This brain re-patterning is the primary consequence ofan abnormal enlargement of the Fgf8 expression do-


tion of the most anterior structure of the Xenopushead, that is, the cement gland (Blitz and Cho, 1995;Pannese et al., 1995).

To get a more specific and controlled Otx2 ectopicexpression inside the neuroepithelium a knock-in ap-proach has been devised for targeting the Otx2 cod-ing region into the En1 genomic region (Broccoli etal., 1999). Therefore, the exogenous Otx2 allele be-comes expressed ectopically in the rostral meten-cephalon where En1 expression domain is normallyfound, thus encompassing the isthmic region (Wassefand Joyner, 1997). The mice carrying this newly-engi-neered Otx2 allele (En1Otx2/+) display a striking alter-ation in the ratio between mesencephalic andcerebellar tissue. In fact, while the rectum is enlargedup to one-third, affecting in particular the inferiorcolliculi, the cerebellum loses almost completely itsmedial structure, called the vermis. Because of thisbrain rearrangement, the mice develop a strong ataxiaduring the early postnatal life, due to the compro-mised cerebellar functions (Broccoli et al., 1999). Thismalformation, already detectable in 10.5 d.p.c. em-bryos, is the final morphological outcome of a back-ward expansion of mesencephalic tissue at theexpenses of the cerebellum. This repatterning processis driven by the knocked-in Otx2 allele that activatesanteriorly expressed genes like En1, Wnt1 andEphrinA5 in its ectopic expression domain, whereashindbrain molecular determinants such as Fgf8 andGbx2 result repressed parallely. As a consequence, theIsO is shifted slightly caudally (Broccoli et al., 1999;Fig. 2). How far from its normal position can the IsObe shifted and up to what level of the A/P axis isneural tissue competent to respond to an Otx2 ec-topic expression are the questions to be answered inthe near future. While Otx2 seems to play a key rolein determining the anterior fate of the neural plate,another homeobox gene, named Gbx2, has been indi-viduated as the major molecular determinant in con-ferring the metencephalic identity (Bouillet et al. 1995;Chapman and Rathjen, 1995; von Bubnoff et al. 1995;Wassarmann et al., 1997). Gbx2 is expressed at veryearly stages of neuraxis development starting at 7.5d.p.c., where it is expressed in all three germ layers ofthe gastrulating embryo in a region that extends ros-trally from the posterior end of the embryo into theprospective hindbrain. Interestingly, its anterior ex-pression boundary moves forward as the Otx2 expres-sion domain retracts progressively into the perspectivefore-midbrain territories (Conlon, 1995). This mutu-ally excluding expression of these genes suggests arepression mechanism, either direct or indirect, be-tween OTX 2 and GBX2 gene products. By 9.5 d.p.c.,Gbx2 RNA is confined to a sharp transverse ring that

is caudal immediately to the midbrain, and co-lo-calises with Fgf8 and Pax2 expression domains. Atthis stage, Gbx2 expression is also detected in twolongitudinal columns in the hindbrain and spinal cord(Bulfone et al., 1993; Wassarmann et al., 1997;Shamim and Mason, 1998; Hidalgo-Sanchez et al.,1999b). Its pivotal role in determining the rostralhindbrain neural identity has been assessed by genetargeting inactivation experiments (Wassarmann et al.,1997). In fact, Gbx2 null mutants die perinatally, lackthe cerebellum, the locus coeruleus and the IV and Vmotor nuclei. These defects can be traced back to anearly stage of neuroectoderm development (at least 8d.p.c.) and are likely due to a failed specification ofrhombomeres 1–3 and isthmic area that are replacedby a small undetermined region (Wassarmann et al.,1997; Fig. 2). Moreover, the midbrain is enlarged,extending caudally over its normal limit. This event ispreceded by an abnormal caudal extension of theOtx2 expression domain, indicating Gbx2 as one ofthe factors that confines the Otx2 acitivity in theanterior neural plate. However, other unidentified fac-tor(s) prevent Otx2 expression from expanding furthercaudally. To test the hypothesis that Gbx2 is involvedin inhibiting Otx2 expression, Gbx2 was expressed inthe midbrain at early somite stages under the controlof the Wnt1 enhancer (Millet et al., 1999). Consistentwith the initial assumption, the Otx2 caudal limit wasshifted rostrally between the diencephalon and themesencephalon, leading to an enlargement of the pre-sumptive anterior hindbrain, and a rostral reposition-ing of the gene expression network that generates theIsO (Fgf8, Wnt1, En1 and Pax2; Fig. 2). Unfortu-nately, due to the Gbx2 transient ectopic expression,the transgenic embryos recover a normal proportionbetween the mesencephalon and the metencephalon by12.5 d.p.c. (Millet et al., 1999). In conclusion, thislarge body of results strongly suggests that the recip-rocal repression between Otx2 and Gbx2 is the firstmolecular event leading to the inductive process whichwill specify the midbrain and hindbrain (Fig. 3). Inparticular, this mutual repression is instrumental tothree subsequent events occurring between 7.5 and 8.5d.p.c. embryos, (i) a sharpening and positioning of theOtx2 and Gbx2 expression borders; (ii) the establish-ment of the mesencephalic and metencephalic identity,and (iii) the induction of the molecular cascade under-lying the IsO genesis and its maintenance.

Finally, the generation of conditional mouse mu-tants for Otx2 and Gbx2 as well as the isolation oftheir regulatory regions will shed more light in thenear future on the mechanisms by which reasonablyfew transcription factors talk to each others to laydown the determination program leading to the finalcomplex pattern of the CNS.


5. otd/Otx conserved functions throughout evolution

5.1. Otx genes in the animal kingdom

Genes related to Drosophila otd and vertebrate Otxhave been isolated from a wide range of organisms(Fig. 4). Most of them, up to protochordates have onlyone member, with few exceptions where duplicationseems to have occurred in independent lineages (Li etal., 1996; Umesono et al., 1999).

An Otx related gene is present already in Cnidarians,primitive metazoans with a defined body plan andradial symmetry. In these organisms, the Otx functionis associated to cell movements involved in the forma-tion of new axes rather than in the formation of thehead (Smith et al., 1999). Rising in the evolutionaryscale, Otx has been found in animals with primitive

bilateral symmetry such as planarians (Stornaiuolo etal., 1998; Umesono et al., 1999). In the planarianDugesia tigrina, Otx expression has been found in re-generation blastemas after transverse sectioning, withan asymmetric pattern of transcripts more abundant inhead regenerating tissues (Stornaiuolo et al., 1998). Theexpression of the Otx genes in these early metazoansreveals some features in common with chordate Otxgenes. Although not directly correlated with a definedanterior structure, the ancient function seems to dealwith patterning body axis and making tissues com-petent to respond to anteriorizing signals (Smith et al.,1999), at least in budding and regeneration processeswhich involve cell movements. Two Otx-related geneshave been identified only on the basis of homeodomainsequence homology in the nematode C. elegans (Galliotet al., 1999). A third gene, more confidentially related

Fig. 4. A simplified phylogenetic tree of the Metazoa indicating the major phyla and where Otx-related genes have been isolated. The asteriskspoints to the two possible positions of the first Otx duplication from a single ancestral gene. The presence of only one Otx-like gene inprotochordates suggests that the Otx gene duplications observed in both Tribolium castaneum and in Dugesia japonica are likely independentduplication events occurred in these organisms.


to Otx has been discovered by means of the sequencingproject of the whole genome of this worm (Ruvkun andHobert, 1998). However, no expression data are yetavailable about this gene.

The first animal with a clearly head-associated Otxexpression belongs to annelids (the leech Helobdellatriserialis ; Bruce and Shankland, 1998). This study isrelevant particularly in evolutionary terms, since it sup-ports the idea of the origin of bilaterians from radialancestors (Brusca and Brusca, 1990). The passage fromradials to bilaterians might have occurred through spe-cification and subsequent expansion of a trunk precur-sor cell-population from one side of the radial ancestor.The leech Otx expression, which is organised radiallyaround its mouth may represent a reminiscence of theOtx expression in a radial ancestor, thus suggestingthat, if not recruited to specify trunk structures, relega-tion of head genes to head domain occurred in bilateri-ans as a consequence of their evolutionary origin.

Otx expression in insects will be discussed in moredetail in a separate section.

The adult pentaradial symmetry shown by sea urchinseems to contradict the general head-associated Otxexpression rule. It is held generally that the radialsymmetry in echinoderms is a shared derived character(synapomorphy) because of their embryonic and larvalbilateral symmetry (Lowe and Wray, 1997). Thus, thehighly divergent and non-head specific Otx expressionin sea urchin can be justified as a consequence of thehighly modified body plan of this phylum.

Comparative studies have demonstrated the existenceof Otx-related genes in all chordates (Simeone et al.,1992, 1993; Frantz et al., 1994; Li et al., 1994; Mori etal., 1994; Bally-Cuif et al., 1995; Blitz and Cho, 1995;Mercier et al., 1995; Pannese et al., 1995; Kablar et al.,1996) including urochordates (Wada et al., 1996),cephalochordates (Williams and Holland, 1996), andagnathan vertebrates (Ueki et al., 1998), where they areexpressed in the rostralmost CNS, independently of thecomplexity acquired by this area during evolution. Thecritical evolutionary position of lampreys will be dis-cussed below, mainly with regard to Otx geneduplication.

In urochordates and cephalochordates, only one Otxgene has been identified so far that may be related toOtx2 (Wada et al., 1996; Williams and Holland, 1998).Indeed, in addition to similarities in amino acid se-quence and expression, they are both expressed duringgastrulation in endoderm cells which suggests that theiroldest and primary role might be to mediate signalsrequired to specify anterior neuroectoderm. Restrictionof an early widespread expression of Otx2-like genes tothe anterior CNS is a remarkable feature of all verte-brates, which can be already observed in Ascidians andAmphioxus. This led to hypothesise the homology be-tween fore-midbrain territory of vertebrates to the less-

Fig. 5. Schematic comparison of expression domains of Otx-relatedgenes (grey) during embryogenesis in some representative protochor-dates (Ascidia and Amphioxus) and vertebrates (Lamprey andmouse). Both lampreys Otx-related genes are shown because of theirhighly divergent expression patterns. Abbreviations — D, dien-cephalon; Ep, epiphysis; Ey, eye; F, forebrain; FE, frontal eye; HB,hindbrain; IO, infundibular organ; ll, lower lip; M, midbrain; MHB,mid-hindbrain boundary; NC, nerve cord; Oe, olfactory epithelium;Os, optic stalk; RV, rhomboencephalic vesicle; SC, spinal cord; SV,sensory vesicle; T, telencephalon; ul, upper lip; and VG, visceralganglion.

evoluted sensory vescicle and cerebral vescicle ofAscidians and Amphioxus, respectively (Fig. 5)(Williams and Holland, 1998). Some authors have alsoextended this homology further backwards in evolu-tion. As mentioned previously, it has been proposedthat a primitive Otx function could be associated onlywith cell movement rather than with anterior patterning(Smith et al., 1999). Similarly, functional data in frog(Blitz and Cho, 1995; Pannese et al., 1995) and mousemutants (Acampora et al., 1995; Matsuo et al., 1995;Ang et al., 1996) suggest a possible involvement ofOtx2 in controlling the cell movements occurring dur-ing gastrulation. Recently, an early role of the ascidianOtx2-related gene Hroth has been suggested in theinhibition of notochord and muscle cell fate or in theinterference with notochord cell movements (Wada andSaiga, 1999). Conservation of the expression domainsamong Otx2-like orthologues from low chordates(Wada et al., 1996; Williams and Holland, 1996; Uekiet al., 1998), to vertebrates (Acampora and Simeone,1999 and references therein) is consistent with the lowrate of divergence of their alignable sequences.


The duplication event generating the Otx1 branchfrom the ancestor Otx2-like gene in gnathostome verte-brates cannot be dated precisely and will be discussedbelow. However, comparative sequence analysis indi-cates clearly that Otx1-like genes evolve more rapidly,as also shown by a further duplication event occurredin both Xenopus (Kablar et al., 1996) and Zebrafish(Mori et al., 1994) and by a ratio of sequence diver-gence higher than Otx2-like genes (Williams and Hol-land, 1998).

These data can be reinforced by the profoundchanges in the expression domains of different verte-brate Otx1-like genes (Simeone et al., 1993; Mori et al.,1994) which underlie rapid evolution of regulatory se-quences as well.

5.2. A common genetic program in insect and6ertebrate head de6elopment

Cloning of several master genes conserved through-out evolution and their comparative expression analysishas been used largely as a means to identify ho-mologous body regions and molecular mechanismsamong animals of different phyla (Abouheif et al.,1997).

The most sensational example of such a functionalconservation is represented by the Pax-6/eyeless gene,which in both Drosophila and vertebrates controls eyedevelopment (Callaerts et al., 1997).

Until recently, it was assumed widely that the insectand vertebrate CNS had evolved independently(Garstang, 1928; Lacalli, 1994). This was due to theirposition on opposite body sides of the dorso-ventral(D/V) axis. One theory, the so-called ‘auricularia hy-pothesis’, postulated the homology between the outerectoderm of the insect embryo and the chordate CNS,based on both anatomical studies made in echinoderms(auricularia larvae) and urochordates, and on compara-tive expression of the HOX genes.

Starting from an ancient hypothesis of about twocenturies ago postulated by Geoffroy Saint-Hilaire (DeRobertis and Sasai, 1996), which has been object ofcontroversial debate (Arendt and Nubler-Jung, 1994;Lacalli, 1995; Peterson, 1995), an increasing body ofevidence has now accumulated which supports a com-mon evolutionary origin of insect and vertebrate CNS.The first clues have been provided when it was shownthat the overall arrangement of neuroblasts in threelongitudinal columns on either side of the midline inboth insect and vertebrate CNS (Doe and Goodman,1985; Chitnis et al., 1995) was paralleled by conservedtopographical expression (although with inverted D/Vpolarity) of pairs of homologous genes (NK-2/NK-2.2;ind/Gsh and Msh/Msx ; Arendt and Nubler-Jung, 1996;D’Alessio and Frasch, 1996; Weiss et al., 1998).

Even more convincing was the similar expression ofshort gastrulation/chordin and decapentaplegic/Bmp4pairs of homologous genes, which are antagonistic keymolecules of the D/V patterning (Piccolo et al., 1997) inboth Drosophila and vertebrates. As far as their expres-sion is inverted with respect to the D/V axis inDrosophila and Xenopus, injection experiments haveshown that these molecules are functionally equivalent(De Robertis and Sasai, 1996), thus supporting theexistence of a homologous mechanism of D/V pattern-ing in a common ancestor of both Drosophila andvertebrates and reinforcing the ancient hypothesis of aninversion of the D/V axis during evolution (De Rober-tis and Sasai, 1996; Arendt and Nubler-Jung, 1999).Finally, Gerhart (2000) has recently reviewed a numberof hypotheses alternative to the inversion of the D/Vaxis, mainly based on the analysis of intermediate posi-tions in evolution which imply intermediate anatomies,as in hemichordates.

Along the A/P axis, in both Drosophila and verte-brates, the conserved families of HOM/HOX and otd/Otx genes play a fundamental role in the regionalspecification of the neuroectoderm destined to formnerve cord/posterior brain and anterior brain, respec-tively. This gross similarity can be refined further whenlooking at the precise coincidence of gene-expressiondomains with the boundary of metameric units orneuromeres (Reichert and Simeone, 1999) and, as forthe HOX genes, also according to the phenomenon ofthe ‘spatial colinearity’ (Lumsden and Krumlauf, 1996).

Over the last 10 years, functional experiments havesubstanciated the equivalence suggested by the insect/vertebrate comparative expression data. Most of thesestudies carried out in Drosophila have shown that mam-malian HOX genes could either partially rescue pheno-types due to mutations of their fly orthologues or elicitresponses similar to those elicited by their endogenouscounterparts when transiently overexpressed (Malicki etal., 1990, 1993; Zhao et al., 1993; Bachiller et al., 1994).

To further sustain the idea that some of the molecu-lar mechanisms underlying brain development wereconserved and comparable, a final, more challenging,proof remained to be verified. Could the Drosophila otdgene substitute for Otx genes in the mouse? That is, cana gene working in a structure as simple as the insectCNS substitute for homologous genetic functions in anenormously more complex organ such as the brain ofmammals?

The embryonic Drosophila brain is composed of twosupraesophageal ganglia, each subdivided into threeneuromeres. The anterior ganglion is subdivided intoprotocerebral, deutocerebral, and tritocerebral neu-romeres. The gap gene otd is expressed mainly in theanteriormost (protocerebral) neuromere, which isdeleted almost entirely in otd null embryos (Finkelsteinand Perrimon, 1990; Cohen and Jurgens, 1991; Hirth et


al., 1995; Younossi-Hartenstein et al., 1997). This phe-notype is due to the failure of expression of the otddownstream gene lethal of scute, one of the proneuralgenes regulating the three-column arrangement of neu-roblasts mentioned above. As a consequence, in theabsence of otd, proneural genes are not activated and agap-like deletion of the rostral brain neuromere (proto-cerebrum) results in late mutant embryos (Hirth et al.,1995). Other defects are also observed in the ventralnerve cord and in non-neural structures. Flies that arehomozygotes for ocelliless (oc), a different otd allele, areviable and lack the ocelli (light sensing organs) andassociated sensory bristles of the vertex (Finkelstein etal., 1990). Moreover, in cephalic development, differentlevels of OTD protein are required for the formation ofspecific subdomains of the adult head (Royet andFinkelstein, 1995). Despite the overall morphologicaldifferences, however, expression pattern and mutantphenotypes of Drosophila otd and mouse Otx genes canbe paralleled (see previous sections) easily. Neverthe-less, OTD and OTX proteins are highly conserved onlyin the homeodomain, which represents roughly one-tenth of the whole OTD protein and about one-sixthand one-fifth of OTX 1 and OTX 2 proteins, respec-tively (Simeone et al., 1993).

Outside the homeodomain, homology is restricted toa few very short sequences. Drosophila OTD proteinalso lacks the so-called ‘‘OTX tail’’ (Freud et al., 1997),a conserved motif of about 20 amino acids which ispresent in single copy in echinoderm, Ascidian andAmphioxus Otx-related genes, whilst is tandemly dupli-cated in all vertebrate OTX proteins at the COOHterminus (Williams and Holland, 1998).

The limited sequence homology shared by OTD andOTX proteins underlines the importance that the home-odomain might have in triggering the CNS develop-mental programmes in both insects and vertebrates.Outside the homeodomain, the proteins could eitherhave diverged, to allow mammals to acquire new andspecific functions or might be more homologous interms of tertiary structure than what the primary se-quence alignment indicate. To gain insight into thepossibility that otd and Otx genes might share con-served genetic functions during CNS development, weundertook experiments in which a Drosophila otdcDNA replaces both mouse Otx1 and Otx2 genes.

When a full-coding Drosophila otd cDNA is intro-duced into a disrupted Otx1 locus by homologousrecombination many abnormalities of the Otx1−/−mice are rescued, regardless of a lower level of OTD(about 30%) as compared with the endogenous OTX 1level (Acampora et al., 1998a). Homozygous knock-inotd mice (otd1/otd1) show no significant perinatal death(with respect to a 30% death of Otx1−/− newborns)and, most importantly, they have neither the abnormalbehaviour nor EEG characteristics observed in theOtx1 null mutants.

In otd1/otd1 adult mice, brain size as well as thethickness and cell number of the temporal and perirhi-nal cortices, both reduced in Otx1−/− mice, are verysimilar to wild-type. The rescue is likely to be ascribedat least in part, to a restored normal proliferatingactivity of the dorsal telencephalic neuroepithelium at9.75 d.p.c. (Acampora et al., 1998a). As described in aprevious section, Otx1 and Otx2 co-operate in brainmorphogenesis and a minimal threshold (correspondingto either two copies of Otx2 or to one copy of Otx1and one copy of Otx2) of their gene products is re-quired for correct brain regionalization (Acampora etal., 1997). When otd is expressed in place of both copiesof Otx1 and in the presence of one functional copy ofOtx2 (otd1/− ; Otx2+/− or otd1/otd1; Otx2+/− ),brain patterning abnormalities of the Otx1−/− ;Otx2+/− mutants are rescued partially in a otd dose-dependent manner, both at morphological and molecu-lar level (Acampora et al., 1998a). The extent of thisrescue decreases progressively along the A/P axis, beingthe posterior mesencephalon still severely affected withrespect to a normal telencephalon and to ameliorateddiencephalic and anterior mesencephalic structures. Allof these data indicate strongly that regionalization ofthe brain requires levels of OTX proteins that increasealong the A/P axis and are particularly critical at theMHB (Acampora et al., 1999b).

A partial rescue is also observed in some sensory andsensory-associated structures such as iris, ciliary processand Harderian glands.

On the contrary, the lateral semicircular canal of theinner ear (last to be established in evolution) (Fritzschet al., 1986; Torres and Giraldez, 1998) is never re-stored in otd1/otd1 mice, suggesting that, either it re-quires higher levels of protein or that specification ofthis structure is dependent upon an Otx1-newly estab-lished function. This seems indeed to be the case since,as previously mentioned, in hOtx21/hOtx21 mice, Otx2is able to rescue some ear defects in the regions whereboth Otx genes are transcribed but not in the lateralsemicircular canal, where only Otx1 is normally ex-pressed (Acampora et al., 1999a; Morsli et al., 1999).

The absence of the lateral canal in the inner ear ofOtx1−/− mice might represent a back-evolutionarymutation that can indicate the presumptive date ofduplication of the Otx genes. This event should haveoccurred after that the lineage leading to cephalochor-dates had split from that leading to vertebrates(Williams and Holland, 1998) and probably after thelatest passage leading from Agnatha to Gnathostomes.In fact, in lampreys, extant agnathan vertebrates, onlytwo semicircular ducts (anterior and posterior) arepresent in the inner ear, and despite two Otx-like geneshave been identified, none of them is clearly related toOtx1. However, it cannot be excluded that the Otxgenes evolved by duplication in a common ancestor of


lampreys and gnathostomes (Fig. 4) and that the un-classified lamprey Otx represents a still evolutionaryunstable version of an Otx1-like gene (Ueki et al.,1998).

More recently, we have generated a mouse model inwhich it is the Otx2 gene to be substituted by theDrosophila otd cDNA (otd2), using the same targetingstrategy as in the replacement with lacZ (Acampora etal., 1995) and with hOtx1 cDNA (Acampora et al.,1998b). Also in this case, the otd mRNA is detected inboth AVE and epiblast, whereas the protein only in theVE. The OTD protein, exactly as the hOTX1 proteindoes, is able to take over all of the Otx2 functions inthe AVE, thus recovering both gastrulation defects andabsence of an early anterior neural plate due to the lackof Otx2. Later on, as in the hOtx12/hOtx12 mutants,however, otd2/otd2 embryos fail to maintain the anteri-ormost identities of the brain and become headless.These results, as far as limited to the AVE, provide afurther proof of functional equivalence, shared by otd/Otx genes, via the activation of the same basic geneticpathway(s).

Similar conclusions have been drawn by complemen-tary experiments in Drosophila (Leuzinger et al., 1998;Nagao et al., 1998). Heat-shock induced expression ofhOTX1 and hOTX2 proteins in the fly is able to rescuethe CNS defects of otd null mutant embryos (Leuzingeret al., 1998) as well as cephalic defects of the ocellilessmutations, both at the morphological and molecularlevel (Nagao et al., 1998), whereas in a wild-type back-ground, OTX overexpression leads to induction of ec-topic neural structures (Leuzinger et al., 1998).

As previously mentioned, even though these cross-phylum rescues highlight the fundamental role of thehomeodomain because it is the only recognisable regionconserved between OTD and OTX proteins, they donot exclude that functional equivalence may be ex-tended to the overall structure of OTD/OTX proteins.

The fact that OTD and OTX proteins are able todrive cephalic development through the activation ofgenetic pathways conserved between the two taxa, rein-forces the idea that insect and chordate CNS are indeedhomologous structures originated from a common an-cestor and controlled by a common basic genetic pro-gram (Sharman and Brand, 1998; Acampora andSimeone, 1999; Reichert and Simeone, 1999).

Interestingly, substitution of a different key geneinvolved in brain patterning processes with itsDrosophila orthologue has led to very similar conclu-sions (Hanks et al., 1998). In fact, Drosophila engrailedwas able to substitute for mouse Engrailed 1 (En1)functions in mid- and hind-brain regions but not inlimb development. Therefore, despite a higher degree ofhomology between EN proteins with respect to OTD/OTX proteins, this rescue reinforces the idea that someof the biochemical properties of highly conserved regu-

latory genes may be conserved across the two phyla andindicates that new functions have been acquired by thevertebrate genes during evolution.

5.3. Otx genes in brain e6olution

One of the most interesting observations raised byour mouse models is the existence of a differentialpost-transcriptional control of the hOtx1/otd and Otx2mRNAs between VE and epiblast cells.

In heterozygous hOtx12/Otx2 and otd2/Otx2 em-bryos, Otx2 mRNA and protein always colocalize,while the hOtx1 and otd mRNAs are translated only inthe VE, thus suggesting that the knocked-in mRNAsdetected in epiblast cells are post-transcriptionally regu-lated by an Otx2-independent cis-acting control(Acampora etal., 1998b).

In Otx2+/− embryos, the same Otx2 region that isreplaced by the hOtx1 and otd cDNA is substitutedwith the lacZ gene (Acampora et al., 1995). In theseembryos the lacZ mRNA is correctly detected in bothAVE and epiblast while the b-gal staining is, again,heavily reduced in the epiblast at early-mid streak stage(Acampora et al., 1995).

Therefore, three different mouse models replacing thesame Otx2 genomic region with three different genes(lacZ, hOtx1 and otd) are suggestive of a differentpost-transcriptional control between AVE and epiblastcells. One or more Otx2 regions deleted in these models(introns, untranslated or coding sequences) should beresponsible for this differential control.

Loss of mRNA translation in the epiblast might bealso influenced by abnormal molecular event(s) affect-ing RNA stability, processing and transport of thechimeric knocked-in transcripts. Whatever the impair-ments are, since in the AVE the mRNA is correctlytranslated, the absence of protein should be considereda peculiar event occurring in epiblast cells and theirderivatives.

The finding that Otx2 escapes this post-transcrip-tional control suggests that this is necessary for themaintenance of fore-midbrain territory specified by theAVE (Acampora et al., 1998b) and might have evolu-tionary implications. Indeed, the architectural compo-nents of the vertebrate brain (telencephalon,diencephalon and mesencephalon) are less clear in pro-tochordates and a vertebrate-type brain, which is com-posed of a midbrain and six prosomers, firstly appearsin Petromyzontoidea. It may be hypothesized that thespecification of this prosomeric brain might have re-quired the presence of a sufficient level of OTX2protein in the early neuroectoderm cells and this eventmight have been acquired by modifying post-transcrip-tional control of Otx2 transcripts rather than its codingsequence which would have retained common func-tional properties among the otd-related genes through-


out evolution. The low ratio of Otx2 sequence diver-gence among the species fits with this hypothesis.

Therefore, although this conservation in expressionpattern and coding sequence might favour a remarkablerole and functional equivalence of otd/Otx genes, it isunclear why the brain vescicles of protochordates havebeen so deeply and suddenly modified in a prosomericterritory that has been maintained in its basic topogra-phy until mammals (Rubenstein et al., 1998).

A rather obvious answer to this question is that newgenetic functions have been gained and recruited indevelopmental pathways. Indeed, it might be that con-served genes such as the Otx acquired different roleseven while retaining an evolutionary functional equiva-lence. Based on this hypothesis, it is expected thatdrastic evolutionary events should act on the regulatorycontrol (transcription and translation) of Otx-relatedgenes rather than on their coding sequences. Interest-ingly, the findings that, (i) the Drosophila otd rescues inmouse most of the Otx1−/− impairments (Acamporaet al., 1998a); (ii) the human Otx1 and Otx2 rescuemost of the otd defects in flies (Leuzinger et al., 1998;Nagao et al., 1998; Sharman and Brand, 1998); (iii)Otx1 rescues Otx2 requirements in VE (Acampora etal., 1998b); (iv) Otx2 rescues most of the Otx1−/−defects (Acampora et al., 1999a); (v) the Drosophila otd,similarly to Otx1, rescues Otx2 requirements in the VE(Acampora et al., unpublished results), indicate thatOtx1, Otx2 and otd genes show a high degree offunctional equivalence in the tissues and body regionswhere they are properly expressed. Therefore, thesedata support the notion that otd/Otx functions havebeen established in a common ancestor of fly andmouse and retained throughout evolution, while copynumber and regulatory control of their expression havebeen modified and re-adapted by evolutionary eventsthat have led to the specification of the increasinglycomplex vertebrate brain (Sharman and Brand, 1998;Simeone, 1998; Acampora and Simeone, 1999; Hirthand Reichert, 1999; Reichert and Simeone, 1999). Geneduplication may allow the duplicated gene to acquirenew specific function(s) either retaining or losing ances-tral properties, and similarly, modification of the regu-latory control of gene expression may establish newexpression patterns which might alter pre-existing cell-fates by generating new specialised cellular functions.

A likely consequence of both increased genomic com-plexity and modification of regulatory control of geneexpression may result in an greater number of molecu-lar interactions. This may contribute to modifying rele-vant morphogenetic processes that, in turn, can confera change in shape and size of the body plan as well asin the generation of cell-types with new developmentalpotentials. On this basis, Otx gene duplication andsubsequent or contemporary modification of regulatorycontrol might have contributed to the evolution of the

mammalian brain, for example by increasing the extentof neuroectodermal territory recruited to form thebrain. This event might involve an improvement ofproliferative activity of early neuronal progenitors(Acampora et al., 1998a, 1999a) and/or the positioningof the MHB (Acampora et al., 1997, 1998b; Suda et al.,1997). Additional property(ies) may be also conferredto the duplicated gene by altering its coding sequence.Thus, the limited amino acidic divergence betweenOTX1 and OTX2 might underlie modifications of theirfunctional properties, as shown by the non-rescuedabsence of the lateral semicircular canal of the inner earin mice replacing Otx1 with Otx2 (Acampora et al.,1996, 1999a; Morsli et al., 1999).

6. Conclusions

In the last decade an impressive amount of knowl-edge has contributed to shed some light on the com-plexity of molecules and mechanisms involved in thedevelopment of the vertebrate brain. In this contextotd/Otx genes play a cardinal role. Previous mousemodels have indicated that Otx1 and Otx2 are requiredfor normal brain and sense organ development and,together with the Drosophila orthodenticle (otd) genethey share a high degree of recriprocal functional equiv-alence. As far as the Otx genes are concerned, theirinterchangeability indicates that the differential tran-scriptional control between Otx1 and Otx2 is the majormolecular reason responsible for generating Otx1−/−and Otx2−/− divergent phenotypes. Nevertheless,one of the most interesting observations raised bymouse models replacing Otx2 with hOtx1 or otd is theexistence of a differential post-transcriptional and/ortranslational control between the VE and epiblast. In-deed, hOtx1 or otd mRNA were detected in VE,epiblast and its derivatives while hOTX1 or OTDproteins in the VE only. This event results in the failureof maintenance of fore-midbrain identities in the mu-tant embryos.

In our opinion one of the most interesting perspec-tives will be the understanding of the molecular mecha-nism(s) underlying this phenomenon, and its potentialrelevance in the morphogenetic changes leading to theevolution of the mammalian brain.

Acknowledgements

We thank A. Secondulfo for manuscript preparation.This work was supported by the the EC BIOTECHprogramme, the MRC Grant programme (cG9900955), the Italian Association for Cancer Research(A.I.R.C.) and the ‘MURST-CNR Biotechnology Pro-gramme Legge 95/95’.


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Otx genes in brain morphogenesis - · PDF fileMarconi 12, 80125Naples, Italy b ... Otx1 transiently controls GH, FSH, and LH in the pituitary ... (Acampora et al., 1998c). 2.2. Otx1expression