Otx Genes in Brain Morphogenesis

Progress in Neurobiology 64 (2001) 69–95 www.elsevier.com/locate/pneurobio

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 SE19RT, UK c Department of Physiological Sciences, Uni6ersity of Catania, Viale A. Doria 6, 95125 Catania, Italy d 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 are the homologues of Drosophila genes coding for signalling molecules or transcription factors. Among these, the orthodenticle group includes the Drosophila orthodenticle (otd) and the vertebrate Otx1andOtx2 genes, which are mostly involved in fundamental processes of anterior neural patterning. These genes encode transcription factors that recognise specific target sequences through the DNA binding properties of the homeodomain. In Drosophila, mutations of otd cause the loss of the anteriormost head neuromere where the gene is transcribed, suggesting that it may act as a segmentation ‘gap’ gene. In mouse embryos, the expression patterns of Otx1 and Otx2 have shown a remarkable similarity with the Drosophila counterpart. This suggested that they could be part of a conserved control system operating in the brain and different from that coded by the HOX complexes controlling the hindbrain and spinal cord. To verify this hypothesis a series of mouse models have been generated in which the functions 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−/− mice are embryonically lethal, they show gastrulation impairments and fail in specifying anterior neural plate. Analysis of the Otx1−/−; Otx2+/− double mutants has shown that a minimal threshold level of the proteins they encode is required for the correct positioning of the midbrain-hindbrain boundary (MHB). In vivo otd/Otx reciprocal gene replacement experiments have provided evidence of a general functional equivalence among otd, Otx1andOtx2 in fly and mouse. Altogether these data highlight a crucial role for the Otx genes in specification, regionalization and terminal differentiation of rostral central nervous system (CNS) and lead to hypothesize that modification of their regulatory control may have influenced morphogenesis and evolution of the brain. © 2001 Elsevier Science Ltd. All rights reserved.

Contents

1. Introduction ...... 70 2. Multiple roles of the Otx1 gene in the developing and adult mouse ...... 71 2.1. Otx1 expression in early embryogenesis ...... 71 2.2. Otx1 expression during corticogenesis...... 71

Abbre6iations: ame, axial mesendoderm; AML, anterior midline; A/P, antero-posterior; AVE, anterior visceral endoderm; b-FSH, follicle-stimulating hormone; a-GSU, a-glycoprotein subunit; b-LH, luteinizing hormone; BrdU, Bromodeoxyuridine; CDGA, constitutional delay in growth and abdolescence; CNS, central nervous system; CP, cortical plate; d.p.c., days post coitum; D/V, dorso-ventral; EEG, electroencephalographic recording; EPSP, excitatory post-synaptic potentials; GABA, g-aminobutyric acid; GH, growth hormone; GnRH, gonadotropin releasing hormone; GnRHR, gonadotropin releasing hormone receptor; GRH, growth hormone releasing hormone; GRHR, growth hormone releasing hormone 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 limitans intrathalamica. * Corresponding author. Tel.: +44-20-78486536; fax: +44-20-78486550. E-mail address: [email protected] (A. Simeone).

2.3. Otx1 is required for correct brain development ...... 71 2.4. Otx1−/− mutant mice exhibit an epileptic phenotype ...... 72 2.5. Otx1 controls cortical connectivity to subcortical targets...... 73 2.6. Otx1 transiently controls GH, FSH, and LH in the pituitary ...... 73 2.7. Otx1 is necessary for correct sense organ development...... 73

3. Early requirement of Otx2 for normal gastrulation and anterior neural plate induction and maintenance...... 74 3.1. Organisers and molecules in the current scenario of the anterior brain patterning . . . 74 3.2. Speciﬁcation of anterior patterning requires Otx2 function ...... 75 3.3. The role of Otx2 in the induction and maintenance of rostral CNS identities ..... 78 3.4. AVE-restricted OTX2 function is necessary for proper expression of Lim1 and bone morphogenetic protein (BMP) antagonists in the ame ...... 79 3.5. Speciﬁc and interchangeable roles between Otx1 and Otx2...... 80

4. Brain patterning depends on a critical Otx gene dosage ...... 80 4.1. Morphogenetic origin of the IsO ...... 80 4.2. Otx genes are required for correct positioning of the isthmus ...... 81

5. otd/Otx conserved functions throughout evolution...... 85 5.1. Otx genes in the animal kingdom ...... 85 5.2. A common genetic program in insect and vertebrate head development ...... 87 5.3. Otx genes in brain evolution ...... 89

6. Conclusions ...... 90

Acknowledgements ...... 90

References ...... 91

1. Introduction specific cell populations (e.g. the anterior neural ridge and the zona limitans intrathalamica (ZLI)) and trans- The central nervous system (CNS) of vertebrates is a verse rings of neuroepithelia (e.g. the isthmic organizer very complex structure derived from a multistep process (IsO)) that possess inductive and boundary properties involving sequential molecular and morphogenetic (Marin and Puelles, 1994; Crossley et al., 1996; Houart events that pattern the epiblast first and the neural plate et al., 1998; Rubenstein et al., 1998; Ruiz i Altaba, 1998). later. During early gastrulation the concerted and se- In vertebrates, several genes controlling developmental quential action of both the anterior visceral endoderm programmes underlying brain morphogenesis have been (AVE) and the node-derived axial mesendoderm (ame, isolated and their role studied in detail. Most of them are Beddington and Robertson, 1999; Bachiller et al., 2000) the vertebrate homologues of Drosophila genes encoding drives the specification of anterior neuroectoderm that, signalling molecules or transcription factors (Lemaire later on results grossly subdivided in three main territo- and Kodjabachian, 1996; Tam and Behringer, 1997; ries (forebrain, midbrain and hindbrain, Gallera, 1971; Rubenstein et al., 1998). Among these, the orthodenticle Storey et al., 1992; Ruiz i Altaba, 1994; Shimamura and group is strictly defined by the Drosophila orthodenticle Rubenstein, 1997; Rubenstein and Beachy, 1998). The (otd) and the vertebrate Otx1 and Otx2 genes, which maintenance of this patterning is likely dependent on contain a bicoid-like homeodomain (Finkelstein and signals originating from the ame and/or within the Boncinelli, 1994; Simeone, 1998). However, other genes overlying neuroectoderm (Acampora et al., 1998b; Rhinn that might be more distantly related to otd/Otx genes et al., 1998; Shawlot et al., 1999; Camus et al., 2000). have been also identified (Muccielli et al., 1996; Szeto et Anatomical and histological studies postulate the al., 1996; Chen et al., 1997). existence of genetic fate determinants which subdivide Expression pattern analysis of Otx genes had sug- these large neural regions into progressively smaller gested that these transcription factors might play an longitudinal and transverse domains (Vaage, 1969; Alt- important role during brain morphogenesis in verte- man and Bayer, 1988; Figdor and Stern, 1993; Ruben- brates. A systematic genetic approach using transgenic stein et al., 1994). Some of the patterning events along mice is revealing that they contribute to the molecular the antero-posterior (A/P) axis require the presence of mechanisms underlying all of the major events (induc- D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 71 tion, maintenance, regionalization, corticogenesis and At mid-late gestation, high level transcription of axon connectivity) necessary to build a normal brain. Otx1 occurs only in ventricular cells, which at these stages are precursors of deep layer neurons. By the time upper layer neurons are generated, Otx1 expression 2. Multiple roles of the Otx1 gene in the developing decreases in the VZ and becomes progressively promi- and adult mouse nent in the cortical plate which consists of postmigra- tory neurons of layer 5 and 6. Otx1 is absent in later differentiated neurons of upper layers 1–4 (Frantz et 2.1. Otx1 expression in early embryogenesis al., 1994). Thus, the progressive down-regulation of Otx1inthe Otx1 starts to be expressed at early stages (2–5 ventricular cells suggests that Otx1 may confer deep- somite stage, 8.2–8.5 days post coitum (d.p.c.)) in the layer identity to young neurons. Heterochronic trans- developing mouse embryo throughout the presumptive plantation experiments have demonstrated that the forebrain and midbrain neuroepithelium (Simeone et broad differentiative potentials of the early progenitors al., 1992). From these stages onwards its expression (McConnell and Kaznowski, 1991) become progres- largely overlaps with that of Otx2, but while the ex- sively restricted over time (Frantz and McConnell, pression of the latter disappears from the dorsal telen- 1996). cephalon since 10.5 d.p.c. (Simeone et al., 1993), Otx1 Indeed, Otx1 expression is heterogeneous across the expression is maintained uniformly across the ventricu- regions of the adult cortex, suggesting that it might also lar zone (VZ) of the cortical anlage from the onset of be involved in the forming of the cortical areas. Its corticogenesis up to mid- to late gestation stages (Sime- expression in layer 5 is more prominent in the posterior one et al., 1993; Frantz et al., 1994). and lateral cortex but absent in the frontal, insular and Otx1 is also expressed at early stages in precursor orbital cortices, while in layer 6 it is more uniform structures of sense organs corresponding to the olfac- throughout the neocortex (Frantz et al., 1994). tory placode, otic and optic vesicles (Simeone et al., Mouse mutant models have been generated and 1993). Later on, Otx1 is transcribed in the olfactory analysed (Acampora et al., 1996, 1998a, 1999a,b Suda epithelium, the saccule, the cochlea and the lateral et al., 1996; Morsli et al., 1999; Weimann et al., 1999; semicircular canal of the inner ear as well as in the iris, Avanzini et al., submitted) to gain insight into the the ciliary process in the eye and the lachrymal gland different roles that Otx1 plays during brain, cortex and primordia (Simeone et al., 1993). From the birthday sense organ development. onwards, Otx1 is also expressed at a relatively low level in the anterior lobe of the pituitary gland (Acampora et 2.3. Otx1 is required for correct brain de6elopment al., 1998c). Heterozygous (Otx1+/−) mice are healthy and 2.2. Otx1 expression during corticogenesis their intercross generates homozygous mice (Otx1−/ −) in the expected mendelian ratio. However, about The cerebral cortex develops according to molecular 30% of the mutants die before the first postnatal strategies that determine the fate of precursor cells month, and appear smaller in size.Table 1 linked to specific neuronal phenotypes. Two main pro- Otx1−/− adult brains are reduced in weight and cesses have been identified so far, laminar determina- size and, at the anatomo-histological inspection, show tion, by which committed cells migrate to their reduction in thickness of the dorsal telencephalic cor- appropriate layer, and cortical areas formation, by tex, a dorsally displaced sulcus rhinalis and shrunken which cortical neurons interact to create functionally hippocampus with a divaricated dentate gyrus. The distinct regions. During corticogenesis, postmitotic neu- cortex is particularly affected at the level of the tempo- rons migrate along radial glial cells (Rakic, 1972), ral and perirhinal areas, where a 40% reduction in cell through the overlying intermediate zone (IZ), and to number is detected. Furthermore, in these same areas, the cortical plate (CP), which will later create the cortical layer organisation is less evident (Acampora et typical layered organisation of the adult cortex. The al., 1996), although the expression of layer-specific layers are generated in an inside-out pattern, in which molecular markers demonstrates that the laminar iden- cells of the deepest layers (6 and 5) are born first in the tities are preserved (Weimann et al., 1999). VZ, and those of the upper layers (4, 3, and 2) progres- The origin of the overall reduction of the Otx1−/− sively later (Rakic, 1974). brains has been investigated through experiments aimed Otx1 represents a molecular correlate of deep layer at determining possible changes of the normal number neurogenesis and its expression is confined to neurons of apoptotic or proliferating cells within the neuroep- of layers 5 and 6 in the adult cortex (Frantz et al., ithelium of the developing telencephalon. No differ- 1994). ences at apoptosis were observed by comparing 72 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 wild-type and Otx1 mutant embryos. By contrast, Bro- ity in hippocampus and cortex. Occasionally, convul- modeoxyuridine (BrdU) labelling experiments revealed sions are followed by status epilepticus and exitus. a reduction of proliferating cells (by about 25%) in the Recently, the epileptogenic mechanisms accounting dorsal telencephalic neuroepithelium of 9.75 d.p.c. for these seizures have been further investigated by Otx1−/− embryos (Acampora et al., 1998a). A defec- means of electrophysiological recordings (current clump tive proliferation of neuronal progenitors at these early intracellular recordings) made from layer 5 pyramidal stages may thus be responsible of the adult phenotype neurons in somatosensory cortical slices (Avanzini et of the Otx1 mutant mice. al., 2000). This analysis shows that g-aminobutyric acid (GABA)-mediated inhibitory post-synaptic potentials (IPSP), as compared with control pyramidal neurons, 2.4. Otx1−/− mutant mice exhibit an epileptic are more pronounced in the mutants where they seem phenotype to be involved in the synchronisation of the excitatory activity from the earliest postnatal period. On the other Otx1−/− mice exhibit both spontaneous high hand, multisynaptic excitatory post-synaptic potentials speed turning behaviour and epileptic behaviour (EPSP) are significantly more expressed in the mutants (Acampora et al., 1996). The latter consists of the than in controls, also at the end of the first postnatal combination of, (i) focal seizures characterised by au- month. These results suggest that the excessive excita- tomatisms (head bobbing and teeth chattering) and tory amino acid-mediated synaptic driving, without a electroencefalographic (EEG) recording of spikes in well-developed GABA counteraction, may lead to a hippocampus; (ii) generalised seizures characterised by hyperexcitable condition that is responsible for the convulsions and high voltage synchronised EEG activ- epileptic manifestations occurring in Otx1−/− mice.

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

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

Dorsal telencephalon Cell proliferation rate at E9.75 Reduced by 25% Full recovery Full recovery At E 13.5 and E 15.5 Reduced by 10% Full recovery Full recovery Cerebral cortex Cell number Reduced Full recovery Full recovery Layer organisation Altered Full recovery Full recovery Laminar fatea Normal N/Ab N/Ab Temporal cortex Reduced by 40% Full recovery Full recovery Perirhinal cortex Reduced by 40% Full recovery Full recovery Hippocampus ShrunkenFull recovery Full recovery Mesencephalon Size of colliculi EnlargedNormal in 30% Normal in 15% Intermediate in Intennediate in 45% 50% Identities of colliculia Normal N/Ab N/Ab Cerebellum Abnormal foliationRecovery in Recovery in 10% 50% Axonal projection from layer 5 neurons of 6isual Defective reﬁnement of exuberant projections N/Ab N/Ab cortexa Beha6iour Turning behaviour High-speed Moderate-speed Moderate-speed Epileptic seizures Present Absent Absent Pituitary gland Impaired Normal Normal Ear Lateral semicircular duct Absent Absent Absent Eye Iris Reduced Recovery in Recovery in 80% 80% Ciliary process Absent Present in 70% Present in 80% Lachrymal and Harderian glandAbsent Present in 75% Present in 34%

a Data from Weimann et al. (1999). b N/A, not analysed. D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 73

2.5. Otx1 controls cortical connecti6ity to subcortical in Otx1−/− mice, hypothalamic and pituitary cells of targets the somatotropic and gonadotropic lineages appear un- altered and that it is the ability to synthesise GH, FSH, Recent analysis of axonal projections in Otx1−/− and LH, rather than the number of cells producing mutants has shed new light on the role of Otx1 during these hormones, to be affected (Acampora et al., brain development (Weimann et al., 1999). In several 1998c). brain regions, connections usually develop by a bipha- An intriguing aspect of our observation is the fact sic mechanism in which an excess of early formed axon that transcription factors of the Ptx and Otx sub- projections is finely pruned by elimination of inappro- families recognise similar DNA target sequences (Sime- priate axon terminals. Layer 5 neurons in the visual one et al., 1993; Lamonerie et al., 1996; Szeto et al., cortex provide a good example of exuberance in con- 1996; Tremblay et al., 1998), and that Ptx1 and Ptx2 nectivity, establishing, among others (e.g. to corpus are expressed in most pituitary lineages, in particular in callosum), connections to a number of subcortical somatotrophic and gonadotrophic cells (Tremblay et targets such as the pons, superior and inferior colliculi, al., 1998). Ptx1 is the most highly expressed of these and spinal cord. During early postnatal life, they selec- genes, followed by Ptx2 and then Otx1 (Tremblay et tively eliminate connections from the inferior colliculus al., 1998). Yet, the Otx1 knock-out has a dramatic and spinal cord (Stanfield and O’Leary, 1985; Stanfield effect only during the prepuberal period. The unique et al., 1982), but the molecular mechanisms underlying activity of Otx1 during this period might reflect a this remodelling are poorly understood. specific interaction of Otx1, but not of the related Ptx Otx1 is strongly expressed in a subset of layer 5 factor(s), with a transcription co-regulator in the soma- neurons that form subcortical but not cortical connec- totrophic and gonadotrophic cells. tions (Weimann et al., 1999) and in layer 6 neurons, Taken together with previous reports, our observa- many of which forming thalamic projections. Analysis tions support the existence of complex regulatory mech- of Otx1−/− mutants reveals significant defects, spe- anisms defining combinatorial cell- and stage-specific cifically in the patterning of subcortical projections. In interactions between transcription factors belonging to fact, while callosal and thalamic projections appear the same or to different gene families for the establish- normal in the Otx1−/− mutants, there are additional ment/maintenance of pituitary function. extensive innervations of both the inferior colliculus A novel feature of this phenotype is the fact that and the spinal cord that have been erroneously main- most of the impaired functions are recovered by the tained. This phenotype suggests that Otx1 function is adult stage. Indeed, after the prepubescent stage, Otx1 required for the last step of subcortical axon develop- mutant mice begin to gradually recover from their ment, in which exuberant connections undergo exten- abnormalities, showing at 4 months of age normal sive refinement with the elimination of axon projections levels of GH, FSH and LH which are paralleled by a from inappropriate targets (Weimann et al., 1999). restored normal body weight, differentiation and size of both testis and ovary, as confirmed also by their sexual 2.6. Otx1 transiently controls GH, FSH, and LH in fertility, and by normal levels of downstream molecular the pituitary targets such as testosterone and insulin growth factor 1 (IGF1). Although we are unable to explain the mecha- Otx1 is postnatally transcribed and translated in the nism underlying this recovery, this observation might pituitary gland. Cell culture experiments indicate that represent a possible example of temporal-restricted Otx1 may activate transcription of the growth hormone competence in hormonal regulation of specific cell-lin- (GH), follicle-stimulating hormone (b-FSH), luteinizing eages by the Otx1 transcription factor. This recovery hormone (b-LH), and a-glycoprotein subunit (a-GSU) appears similar to the ‘catch-up growth’ (Boersma and genes. Analysis of Otx1 null mice (Acampora et al., Wit, 1997) described in children with delayed growth 1998c) indicates that, at the prepubescent stage, they and puberty, also called ‘constitutional delay in growth exhibit transient dwarfism and hypogonadism due to and abdolescence’, CDGA (Horner et al., 1978). low levels of pituitary GH, FSH and LH hormones which, in turn, dramatically affect downstream molecu- 2.7. Otx1 is necessary for correct sense organ lar and organ targets. Nevertheless, Otx1−/− mice de6elopment gradually recover from most of these abnormalities, showing normal levels of pituitary hormones with re- Otx1−/− mutants show inner ear and eye abnor- stored growth and gonadal function at 4 months of age. malities that are consistent with Otx1 expression pat- Expression patterns of related hypothalamic genes such tern (Acampora et al., 1996). as the growth hormone releasing hormone (GRH), Otx1 is expressed in the lateral canal and ampulla as gonadotropin releasing hormone (GnRH), and their well as in a part of the utricle, in the saccule and pituitary receptors (GRHR and GnRHR) suggest that, cochlea. Interestingly, Otx2 is co-expressed with Otx1 74 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 in the saccule and cochlea but not in the components of These results suggested the possibility that head and the pars superior. Lack of Otx1 results in the absence trunk inducing ability of the organiser might be associ- of the lateral semicircular canal and lateral ampulla, in ated to different cell populations coexisting in an early abnormal utriculosaccular and cochleosaccular ducts organiser. In mouse, at late streak stage the node is and in a poorly defined hook (the proximal part) of the located at the rostral end of the primitive streak and is cochlea (Acampora et al., 1996; Morsli et al., 1999). able to induce a secondary axis. Importantly, the sec- Defects in the shape of the saccule and cochlea are ondary axis lacks any anterior neural tissues (Bedding- variable in Otx1−/− mice and are much more severe ton, 1994) suggesting a functional parallelism with the in Otx1−/−; Otx2+/− background. In Otx1−/− amphibian late organiser. Inducing properties of an and Otx1−/−; Otx2+/− mutants the lateral crista early mouse node have been tested in transplantation is absent and the maculae of the utricle and saccule are experiments, and also in this case it fails to induce partially fused (Morsli et al., 1999). anterior identity (Tam et al., 1997), thus indicating that, In the eye and annexed structures Otx1 transcripts unlike the amphibian organiser, the mouse node is are restricted to the iris, ciliary process and ectodermal unable to induce rostral neuroectoderm, independently cells migrating from the eyelid and included in the of its age. mesenchymal component of the lachrymal glands. The mouse node gives rise to the ame that at mid-late These ectodermal cells are believed to induce differenti- streak stage underlies the anterior developing neu- ation of the mesenchymal cells into a glandular ex- roectoderm in the midline, and comprises mesoderm ocrine cell-type. In Otx1−/− mice the ciliary process (prechordal plate and notochord) and the definitive gut is absent, the iris is thinner and the lachrymal and endoderm. These node-derived tissues are similar to Harderian glands do not develop, failing the differenti- those originated by the amphibian organiser and ex- ation to a glandular cell-type. Interestingly, the ectoder- press similar genes (Beddington, 1981; Lawson et al., mal cells embedded within the mesenchymal 1991). Moreover, there is clear evidence that the murine components are not identified in Otx1−/− mice, thus node and its derivatives are able both to emanate indicating that failure in development of the glands is a neuralising signals (Ruiz i Altaba, 1993, 1994; Bedding- consequence of the impaired migration of the ectoder- ton and Robertson, 1999) with patterning properties of mal cells from the eyelid to the mesenchyme of the different A/P value (Foley et al., 1997; Shimamura and lachrymal primordium that in turn is not induced to Rubenstein, 1997) and to regulate the expression of the differentiate into the exocrine glandular phenotype region-specific neural genes En1andOtx2 in the neu- (Acampora et al., 1996). roectoderm (Ang and Rossant, 1993; Ang et al., 1994). Interestingly, recent findings have ascribed to the AVE, which surrounds the epiblast cells at the pre-early 3. Early requirement of Otx2 for normal gastrulation streak stage, a crucial role in specifying anterior neural and anterior neural plate induction and maintenance patterning in mouse (reviewed in Beddington and Robertson, 1998, 1999). Expression analysis and cell 3.1. Organisers and molecules in the current scenario of lineage studies remarkably contributed to define the the anterior brain patterning origin and the molecular identity of the AVE. AVE precursor cells are located at the distal tip of Fate and patterning of tissues depend on the activity the mouse pre-streak embryo and can be visualised by of organiser cells emanating signals to a responding the expression of the Hex gene (Thomas et al., 1998). tissue which undergoes morphogenetic changes result- Few hours later, descendants of this group of cells will ing in a specific differentiated fate (Spemann and Man- be found only in the perspective anterior region, giving gold, 1924; Waddington, 1932; Gurdon, 1987). rise to the first (molecular) asymmetry along the pre- Indeed, in amphibians, the dorsal lip of the blasto- sumptive A/P axis of the embryo. pore induces a new, ectopic secondary axis when trans- This A/P asymmetry is confirmed by the analysis of planted on the ventral side of a host embryo. Because the expression patterns of a number of genes such as of this ability, the dorsal lip of the blastopore has been Hex, Hesx1, goosecoid (gsc), cerberus-like (cer-l), called the organiser (Spemann and Mangold, 1924). Lim1, Otx2 and nodal (Simeone et al., 1993; Thomas Further experiments in amphibian and chick embryos and Beddington, 1996; Thomas et al., 1998; Belo et al., have suggested that the age of the organiser tissue 1997; Varlet et al., 1997; Dattani et al., 1998). influences the extension of the induced neural plate as Cell ablation and tissue recombination experiments well as its regional identity. An organiser deriving from have indicated the functional relevance of AVE in an early gastrula induces anterior as well as posterior inducing anterior patterning. Indeed, it has been shown neural tissue, whereas a late organiser induces only that, (i) the removal of a patch of AVE cells expressing posterior tissue (Gallera, 1971; Nieuwkoop et al., 1985; the Hesx1 gene prevents the subsequent expression of Storey et al., 1992). this gene in the rostral neuroectoderm which becomes D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 75 reduced and abnormally patterned (Thomas and Bed- anterior patterning initiated by the AVE, which per se dington, 1996); (ii) recombination of chick epiblast with might be necessary, but not sufficient, to induce the rabbit pre-streak AVE induces the expression of fore- anterior neural plate. brain markers, while chick hypoblast is unable to elicit Early patterning of the CNS primordium is also the same response, thus suggesting that chick hypoblast controlled by additional mechanisms involving planar and murine AVE might not share the property of signals acting through the neuroectodermal plane (Do- specifying anterior neural patterning (Kno¨tgen et al., niach, 1993; Ruiz i Altaba, 1993, 1994). Furthermore, it 1999); (iii) chimera and transplantation experiments as has been shown that in zebrafish a small group of well as the analysis of mouse mutants have indicated ectodermal cells located in the anteriormost head region that a number of genes, including Lim1, Otx2 and is required for the patterning and survival of the ante- nodal are required in the AVE for proper specification rior brain (Houart et al., 1998; Ruiz i Altaba, 1998). of the anterior neuroectoderm (Acampora et al., 1995; Finally, heterotopic transplantation studies have in- Shawlot and Behringer, 1995; Ang et al., 1996; Varlet et dicated that it is only from the synergistic interaction of al., 1997), and in the ame for maintenance and refine- anterior germinal layers (AVE and anterior epiblast) ment of correct identities (Acampora et al., 1998b; and a fragment of the posterior epiblast (early node) of Rhinn et al., 1998; Shawlot et al., 1999); (iv) embryos early-streak embryos that it is possible to induce a full homozygous for a null mutation in Cripto show expres- neural axis including anterior neural gene expression sion, even if abnormal, of anterior neural markers in (Tam and Steiner, 1999). Similar experiments involving the absence of node-derived mesendoderm (Ding et al., removal and ectopic transplantation of anterior midline 1998). This gene encodes a membrane-associated tissue (AML, which includes both ame and ventral protein containing epidermal growth factor-like motifs neuroectoderm) from late-streak embryos, have shown and is expressed shortly before the onset of gastrulation that AML is required for maintenance and regionaliza- in the proximal region of the epiblast where the primi- tion of the forebrain (Camus et al., 2000). tive streak forms (Dono et al., 1993; Ding et al., 1998; Among the genes required in the early specification Minchiotti et al., 2000). Cripto−/− embryos lack a of the anterior neural plate, Otx2 plays a remarkable primitive streak and do not anteriorize the AVE, thus role in both induction and maintenance of the rostral failing to convert the proximo-distal into A/P axis neural patterning (Simeone, 1998; Acampora and Sime- (Ding et al., 1998). Nevertheless, the AVE markers are one, 1999). These and other potential roles are now correctly expressed, although in a distal position and subject of intense study that takes advantage from the epiblast does express rostral neural markers such as genetically modified mouse models and embryological Otx2andEn until 8–8.5 d.p.c., thus indicating that in approaches. the absence of a node and derived tissues the epiblast is able to acquire the identity of anterior neuroectoderm. 3.2. Specification of anterior patterning requires Otx2 Together with the ectopic transpantations of the function node (Beddington, 1994; Tam et al., 1997), these results point to the possibility that in the mouse the AVE In mouse, Otx2 is already transcribed before the contains genetic information required to instruct the onset of gastrulation in the epiblast and in the visceral patterning of the rostral neuroectoderm and, hence, endoderm (VE), and at the end of gastrulation in the there might be two physically separated organising ame and rostral neural plate (Simeone et al., 1992, centres, AVE and node, responsible for the induction of 1993; Ang et al., 1994). During brain regionalization, head and trunk structures, respectively. Otx2 shows expression domains largely overlapping This idea has been re-considered in the light of new with those of Otx1, with a posterior border coincident mouse models recently generated. In particular, with the mesencephalic side of the isthmic constriction Chordin−/−; Noggin−/− (Chd−/−; Nog−/−) (Simeone et al., 1992; Millet et al., 1996). double mutants have provided evidence that they are Therefore, during gastrulation Otx2 is transcribed crucial in early forebrain development. These genes are and translated in the cells that are believed to emanate expressed in the mouse node and its derivatives but not signals in early specification and patterning of the neu- in the AVE, and single null mutation of either of them ral plate (AVE and ame) as well as in those responding does not affect anterior neural patterning. However, to these instructing signals (epiblast and anterior neu- Chd/Nog double mutants display severe defects in the roectoderm) (reviewed in Simeone, 1998; Acampora development of prosencephalon (Bachiller et al., 2000). and Simeone, 1999, Fig. 1A). The first indication that Analysis at the early stages of the mutant embryos has Otx2 is responsive to inductive interactions comes from demonstrated that the AVE is correctly established, explant-recombination experiments in gastrulating thus suggesting that either the ame cells have their own mouse embryos showing that a positive signal from head-inducing properties which are affected in these ame of headfold stage embryos is able to maintain mutants, or are required for the early maintenance of Otx2 expression in the anterior ectoderm of early 76 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95

Fig. 1. D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 77 streak embryos, and that a negative signal from the ments of extension and convergence during gastrulation posterior mesendoderm, mimicked by exogenous (for trunk and tail reduction) and/or with an Otx2-re- retinoic acid (RA), represses Otx2 expression in the quirement in the specification of anteriormost head anterior ectoderm of late streak embryos (Ang et al., structures (for the ectopic cement gland). Moreover, by 1994). Similar interactions have been also demonstrated using a dexamethasone-inducible OTX 2 protein it has in Xenopus (Blitz and Cho, 1995). been shown that the Xenopus Otx2 activity is regulated The possibility that RA might contribute to the early by regionally restricted factor(s), and that the cement distinction between fore-midbrain and hindbrain by gland-specific gene XCG may represent a direct target controlling Otx2 expression is supported by the finding of the Otx2 gene product (Gammill and Sive, 1997). that administration of exogenous RA at mid-late streak In mouse, Otx2 null embryos die early in embryogen- stage represses Otx2 expression in both the ame and the esis, lack the rostral neuroectoderm fated to become posterior neural plate (Ang et al., 1994; Simeone et al., forebrain, midbrain and rostral hindbrain, and show 1995; Avantaggiato et al., 1996). This repression corre- heavy abnormalities in their body plan (Fig. 1E) lates with the appearance of microcephalic embryos (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., that show early anteriorization of Hoxb1 expression, 1996). Heterozygous Otx2+/− embryos into an ap- hindbrain expansion (Sive and Cheng, 1991; Conlon propriate genetic background show defects of the head and Rossant, 1992; Marshall et al., 1992), loss of such as serious brain abnormalities and craniofacial forebrain molecular and morphological landmarks, and malformations, which are reminiscent of otocephalic gain of midbrain molecular markers in the rostralmost phenotypes (Matsuo et al., 1995). neuroectoderm (Simeone et al., 1995; Avantaggiato et The analysis of Otx2 null embryos revealed that at al., 1996). Moreover, Otx2 responsiveness to RA appli- late streak stage, the rostral neuroectoderm is not spe- cation is a common feature in different species includ- cified and the primitive streak as well as the node and ing Xenopus and chick (Bally-Cuif et al., 1995; Pannese the ame are severely impaired. Therefore, one possibil- et al., 1995). Nevertheless, the question of whether ity is that the resulting headless phenotype might be endogenous RA plays a physiological role in rostral due to the abnormal development of node-derived ame CNS demarcation by contributing to the establishment which lacks head organiser activity. A second possibil- of the posterior border of Otx2 expression, still remains ity is that neural inducing properties of the AVE are open. severely impaired or abolished. In embryos replacing The evidence that Otx2 may play a remarkable role Otx2 with the lacZ reporter gene, the first abnormality in rostral CNS specification derives from in vivo genetic is already detected at the early streak stage (Acampora manipulation experiments performed in mouse and et al., 1995). Indeed, at this stage, lacZ transcription Xenopus which, to some extent, complement each other. and staining are abolished in the epiblast while remain- In Xenopus, microinjection of synthetic Otx2 RNA ing high in the VE of Otx2−/− embryos (Fig. 1D); results into an abnormal reduction in size of tail and the gsc expression is undetectable or confined to the trunk structures, and in the appearance of a second proximal region of the mutant embryos (Acampora et cement gland (Blitz and Cho, 1995; Pannese et al., al., 1995); and the presumptive AVE does not anteri- 1995). These phenotypes have been interpreted either orize, thus remaining confined to the distal region of with a possible Otx2-mediated interference with move- the embryo (Fig. 1D).

Fig. 1. Schematic representation and morphology of Otx2−/− and hOtx12/hOtx12 mutant embryos as compared with wild type at different stages. 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 Otx2 null 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) (arrows in A) that are directed to the epiblast and are required for specification of anterior patterning, while at late gastrula-headfold stage, Otx2is required to maintain fore-midbrain regional identities (see also F–H) even though it cannot be assessed in which tissue (ame or neural plate) it plays 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 of the normal Otx2 allele is required in the VE to rescue signal(s) necessary for its transcription in the epiblast, for specification of anterior patterning and proper gastrulation. These findings suggest that Otx2 is an important component of VE organizing properties. The arrow in (E) points to the 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.), hOtx1 transcripts are detected correctly in AVE and epiblast cells while, in contrast, the hOTX 1 protein is restricted to the VE. This model confirms the OTX 2 gene product requirement in the AVE for specification of the early anterior neural plate and demonstrates that hOTX 1 and OTX 2 proteins are functionally equivalent in the VE. Moreover, the differential post-transcriptional control of hOtx1 transcripts makes it possible to distinguish between specification and maintenance properties of Otx2. In fact, while the specification of the anterior neural plate and normal gastrulation are rescued by AVE-restricted hOTX 1 protein (F), the maintenance of anterior patterning is not recovered for the absence of any OTX protein in the epiblast-derived cells (anterior neural plate and ame) (G). This finding leads to the attractive hypothesis that Otx2-mediated maintenance 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, primitive streak; Te, telencephalon; and VE, visceral endoderm. 78 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95

Therefore, these results indicate that headless pheno- al., 1998b; Rhinn et al., 1998; Acampora et al., unpub- type and abnormal organisation of the primitive streak lished results). Chimeric embryos containing Otx2−/− may be determined very early at the pre-early-streak epiblast and wild-type VE rescue an early Otx2−/− stage by an impairment of AVE properties. neural plate but subsequently fail to develop a brain, Moreover, as revealed by the mesodermal early marker suggesting that Otx2 is firstly required in the AVE for Brachyury (T), epiblast cells do not migrate posteriorly induction of rostral neural plate and, then, in the at the site of the primitive streak formation but remain epiblast-derived tissues for specification of forebrain and for an abnormally longer period in a ring close to midbrain regional identities. Conversely, when chimeric embryonic-extraembryonic boundary. Only later, and in embryos consist of an Otx2−/− VE and an Otx2+/+ a small number of Otx2−/− embryos, the presumptive epiblast, none of the phenotypic features of Otx2−/− AVE cells appear anteriorized. However, although ab- embryos are rescued (Rhinn et al., 1998). This latter normal, a primitive streak forms in all the Otx2−/− result also argues, as previously suggested by Otx2−/− embryos and mesodermal cells appear to be concentrated mice (Acampora et al., 1995), that impaired ame of at the posterior third of the embryo (Acampora et al., Otx2−/− embryos is a consequence of Otx2 require- 1995, 1998b; Ang et al., 1996). ment at earlier stages in the VE. These findings, therefore, favour a relationship be- Mice replacing Otx2 with Otx1 were originally gener- tween primitive streak formation and anterior location ated in order to assess whether the two proteins shared of the AVE and suggest that Otx2, normally expressed functional equivalence or, alternatively, displayed unique in both epiblast cells and AVE, might be required in these properties specified by their limited amino acid diver- two cell types in order to mediate proper positioning of gence. Murine OTX 1 and OTX 2 gene products, in fact, the AVE and normal formation of the primitive streak. share extensive sequence similarities even though in However, data deduced from the analysis of chimeric OTX1, downstream of the homeodomain, these regions embryos and further mouse models indicate that early of homology to OTX2 are separated by stretches of abnormalities in both AVE and primitive streak of additional amino acids including repetitions of alanine and histidine residues (Simeone et al., 1993). Otx2−/− embryos should be ascribed to Otx2 require- Interestingly, homozygous mutant embryos replacing ment in the VE (Acampora et al., 1998b; Rhinn et al., Otx2 with the human Otx1(hOtx1) cDNA (hOtx12/ 1998; see below). hOtx12) recover anterior neural plate induction and Interestingly, an intriguing feature of Otx2−/− normal gastrulation but show a headless phenotype from embryos is that lack of anterior neuroectoderm is paral- 9 d.p.c. onwards (Fig. 1F–H). A combined analysis of leled by failure of epiblast cells to express the lacZ both hOtx1 RNA and protein distribution during early reporter gene. gastrulation has revealed that while the hOtx1 mRNA Thus, since Otx2 is already transcribed from the is detected in the VE and epiblast, the hOTX1 protein earliest stages (unfertilised egg in Xenopus and at least is revealed only in VE (Fig. 1F). Nevertheless, this morula in mouse), this observation suggests that mainte- VE-restricted translation of the hOtx1 mRNA is suffi- nance of Otx2 transcription in the epiblast cells requires cient to recover early anterior neural plate but fails to at least one normal allele expressed in the AVE while maintain fore-midbrain identities (Fig. 1G), and, conse- Otx2 transcription in the latter is independent of the quently, hOtx12/hOtx12 embryos display a headless presence of a normal allele (Simeone, 1998; Acampora phenotype with a normal body plan (Fig. 1H) (Acam- and Simeone, 1999). pora et al., 1998b). These findings also support the existence of Otx2-me- In fact, at early-streak stage, hOtx12/hOtx12 embryos diated signal(s) emitted from the AVE and directed to the recover anteriorization of the AVE, normal primitive epiblast cells. Nevertheless, it is still unclear whether the streak and proper expression of Lim1, cer-l, Hesx1, gsc same signal operates to mediate both specification of and T. At late gastrula stage, the anterior patterning of anterior patterning and Otx2 transcription in epiblast the rostral neural plate is correctly defined by the cells or these two events are independently controlled. expression of fore-, mid- and hindbrain markers such as Six3, Pax2, Gbx2 and Hoxb1 and the ame properly 3.3. The role of Otx2 in the induction and maintenance expresses Lim1, Noggin, cer-l and Hesx1. These findings of rostral CNS identities support the idea, as indicated by chimeric studies (Rhinn et al., 1998) that Otx2 is required in the AVE for In order to understand the role of Otx2 in the initiating the specification of the anterior patterning and specification of the anterior patterning, it is of particular demarcation of forebrain and midbrain territories relevance to define its functional contribution to the (Acampora et al., 1998b). different tissues where it is expressed during gastrulation. Further analysis of hOtx12/hOtx12 embryos reveals A direct evidence proving a role for Otx2 in the AVE that at the early somite stage the forebrain markers has been recently provided by generating murine BF1 and the hOtx12 mRNA are not detected in the chimeric embryos and new mouse models (Acampora et rostral neuroectoderm where mid-hindbrain markers D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 79 such as Wnt1, En1, Fgf8, Gbx2 and Pax2 appear to be A recent in vitro study supports the possibility of a co-expressed. direct protein–protein interaction between OTX 2 C- These observations lead to argue that the OTX2 gene terminus and Lim1 homeodomain (Nakano et al., product is required from the headfold stage onwards to 2000). maintain the anterior patterning previously established by the AVE. In this respect, the phenotype observed at 3.4. AVE-restricted OTX2 function is necessary for the early somite stage appears to be the consequence of proper expression of Lim1 and bone morphogenetic an A/P repatterning process involving the entire ante- protein (BMP) antagonists in the ame rior neural plate (fore-midbrain) which, in the absence of any OTX gene product, adopts a more posterior fate In hOtx12/hOtx12 (Acampora et al., 1998b) and (hindbrain) (Acampora et al., 1998b; Acampora and chimera experiments (Rhinn et al., 1998), a tight corre- Simeone, 1999). Therefore, the hOtx12/hOtx12 mouse lation exists between correct specification of anterior model allows to uncouple two distinct phases both patterning and the recovery of normal gastrulation. requiring Otx2 early induction of anterior neural pat- Moreover, in Otx2−/− embryos failure of anterior terning that is under the control of AVE and its subse- specification always coincides with a distal location of quent maintenance that is likely mediated by the AVE and abnormal gastrulation. In particular, in epiblast-derived cells (the ame and neuroectoderm). these embryos ame is no longer identifiable, and expres- However, since Otx2 is normally transcribed and sion of Nog, Chd and Lim1 is strongly abnormal or translated in both ame and rostral neuroectoderm, absent (Acampora et al., 1998b; Rhinn et al., 1998). In 2 2 while the hOtx12 mRNA is transcribed but not trans- hOtx1 /hOtx1 , general morphology as well as expres- lated either in the ame or in the rostral neuroectoderm, sion of these genes in the node and ame appears to be it cannot be deduced from this model whether Otx2is normal, and additionally, cer-l expression in the definitive endoderm emerging from the node also appears to required in one or both of these tissues to mediate be unaffected. maintenance of the anterior identity and whether hOtx1 As previously mentioned, recent studies have shown is functionally equivalent to Otx2 also in these tissues. that (i) anterior midline tissue including ame and ven- A cell-autonomous role of Otx2 in the neuroectoderm tral neuroectoderm is required for maintenance and has emerged from the analysis of chimeras with a regionalization of forebrain (Camus et al., 2000); (ii) moderate contribution of Otx2−/− cells (Rhinn et BMP antagonists such as Nog and Chd are required in al., 1999). It has been shown that genes such as Wnt1, a temporally coherent manner in the node and ame for Hesx1andR-cadherin are selectively, not expressed in forebrain development (Bachiller et al., 2000); and (iii) Otx2−/− cells whereas other genes such as En2 and Lim1 is required in the ame for stabilising anterior Six3 are uniformly expressed in neuroectodermal cells patterning (Shawlot et al., 1999). On this basis, it can of both genotypes. be hypothesised that anterior specification by AVE-re- How Otx2 is integrated in the network of other stricted OTX function might act through ame-restricted genetic functions which have been shown to be funda- non-cell autonomous properties of Chd, Nog and Lim1. mental for anterior neural patterning is still an open This hypothesis does not exclude the existence of a question. The overall phenotype of Lim1−/− and direct inducing signal for anterior patterning emanating 2 2 hOtx1 /hOtx1 embryos, for example, show impressive from the AVE. Rather it indicates that the VE is also phenotypic similarities. Recently, the stage and the cell important for normal development of the primitive type in which Lim1 is required for head specification streak (Beddington and Robertson, 1999). In this re- have been approached by chimera and tissue recombi- spect, it is noteworthy that chimera experiments per- nation experiments (Shawlot et al., 1999). These experi- formed to assess the role of HNF3b have indicated that ments have revealed that Lim1 is required in the AVE this gene is necessary in the VE to promote proper for inducing anterior neural patterning and, subse- primitive streak elongation (Dufort et al., 1998). quently, in the ame to refine and maintain the anterior Since, contrary to OTX2, hOTX1 protein is not identity. Therefore, since maintenance of anterior pat- detected in the ame, it cannot be excluded a priori that tern requires Otx2 in the ame or in the anterior neu- OTX2 also plays a role in these cells. Noteworthy is roectoderm or in both tissues, it can be speculated that also the fact that in the absence of any OTX protein, Lim1 might contribute to mediating the release of ame hOtx12/hOtx12 embryos exhibited anterior patterning signal(s) instructing maintenance of anterior character at least until 7.75–8 d.p.c., while they subsequently fail and Otx2 might confer to neuroectoderm the compe- in proper positioning of the IsO at the MHB. Chd−/ tence to respond to this signal(s) emitted from the −; Nog−/− embryos lacked anterior brain structures mesendoderm. This possibility is also compatible with a and retained a relatively normal midbrain (Bachiller et cell-autonomous role within the neuroectoderm and al., 2000). Together, these findings suggest that while does not exclude an additional role also in the ame. early forebrain maintenance and/or induction is under 80 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 the control of Chd/Nog signalling in the ame, OTX 2 is only in the VE, where it was sufficient to rescue VE-re- required for late maintenance by specifying midbrain stricted Otx2 functions. territory and allowing the correct positioning of the In sum, these mouse models support an extended IsO. functional equivalence between OTX1 and OTX2 proteins and provide evidence that Otx1−/−; 3.5. Specific and interchangeable roles between Otx1 Otx2−/− contrasting phenotypes stem from differ- and Otx2 ences in expression patterns rather than in amino acid sequences. Mutant phenotypes, expression patterns (see above) The clearest exception to the overall Otx functional and amino acid sequence comparison support two pos- equivalence is provided by the lateral semicircular canal sibilities, (i) the functional properties of Otx1 and Otx2 of the inner ear, that in hOtx21/hOtx21 mice is never largely overlap and differences in their temporal and restored (Morsli et al., 1999) as well as in mice in which spatial transcriptional control are responsible for the Otx1 is replaced with the Drosophila otd gene (Acam- highly divergent phenotypes of Otx1−/− and pora et al., 1998a). These findings, discussed below in Otx2−/− mice; or (ii) OTX1 and OTX2 gene prod- evolutionary terms, suggest that the ability to specify ucts display unique functional properties specified by the lateral semicircular canal of the inner ear may be, their limited amino acid divergence. In order to distin- indeed, an Otx1-specific property (Acampora and guish between these two possibilities, mice models that Simeone, 1999). replace Otx1 with the human Otx2(hOtx2) cDNA and However, it is noteworthy that these studies do not vice versa were generated. address the question of whether hOTX1 might be func- Homozygous mice in which Otx1 is replaced with the tionally equivalent to OTX2 in the embryonic neu- human Otx2 cDNA (hOtx21/hOtx21), despite a re- roectoderm and ame, due to the lack of hOTX1 protein duced expression of the transgenic alleles, no longer in these tissues. Genetic replacement studies carried out show epilepsy and corticogenic abnormalities caused by by Suda et al. (1999) provide some evidence that OTX1 the absence of Otx1. This rescue is due to a restored might be able to partially rescue the headless phenotype normal cell proliferation activity in the dorsal neuroep- that was observed in hOtx12/hOtx12 mutant animals. ithelium, as revealed by BrdU labelling experiments at However, it is unclear whether the amount of OTX1 9.5 d.p.c. (Acampora et al., 1999a). This is particularly was similar to that of OTX2 in these experiments (Suda relevant when considering the different expression pat- et al., 1999). This is an important aspect since a reduc- terns of Otx1 and Otx2 genes in the dorsal telen- tion of OTX gene products below a critical threshold is cephalon from the 9.5 d.p.c. stage onwards. Indeed, at always reflected in a mis-specification of fore- and this stage, while Otx1 is expressed throughout the midbrain regions (Acampora et al., 1997, 1998b; Suda entire dorsal telencephalon, Otx2 is expressed only in et al., 1997; Broccoli et al., 1999; Millet et al., 1999; the mediodorsal area and basal neuroepithelium, Simeone, 2000). In this context, it has been recently whereas it disappears completely from the mediodorsal discovered that different roles attributed to two Hox area by 11 d.p.c. Therefore, the rescue observed in genes belonging to the same paralogous group are the hOtx21/hOtx21 mice suggests that Otx1andOtx2 have result of quantitative modulations in gene expression interchangeable roles in the cortex. To complement the rather than of qualitative properties depending on information derived by the loss-of-function and gene protein sequence (Duboule, 2000; Greer et al., 2000). replacement experiments, it would be particularly inter- Thus, even if OTX1 and OTX2 do share a certain esting to see also the effects of an OTX over-dosage on degree of functional equivalence in the neuroectoderm, the brain and, specifically, on cortical development. the limited partial recovery of the phenotype might be hOtx21/hOtx21 mice also show a significant improve- ascribed either to a reduced level of OTX1 protein or to ment of mesencephalon, eye and lachrymal gland de- an OTX2-specific function. Further investigation is fects. Some of the Otx1−/− inner ear abnormalities needed to address this important evolutionary aspect. are also rescued in the regions where the Otx genes are normally co-expressed, but not the absence of the lateral semicircular canal. As previously reported in detail, 4. Brain patterning depends on a critical Otx gene homozygous mutant mice in which Otx2 has been dosage replaced with the human Otx1(hOtx1) cDNA (hOtx12/ hOtx12) recover the anterior neural plate and proper 4.1. Morphogenetic origin of the IsO gastrulation but fail to maintain fore-midbrain identities, displaying a headless phenotype from 9 d.p.c. The initial patterning of the primitive neuroepithe- onwards (Acampora et al., 1998b). A deeper analysis lium is established during early gastrulation, when sig- has revealed that despite RNA distribution in both the nals originating from both the underlying mesoderm VE and epiblast, the hOTX1 protein was synthesised and the VE or present within the ectoderm itself, D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 81 roughly divide the future CNS into few main subdivi- However, En compound mutant mice lack the entire sions (Rubenstein and Shimamura, 1997; Beddington region resembling the Wnt1 mutant phenotype (W. and Robertson, 1998). At the end of this process, Wurst and A.L. Joyner, unpublished results) and En2 different regions have acquired their own identity ex- can rescue the En1 mutant brain defects when it is pressing a unique set of transcription factors. Later on, expressed in place of En1, revealing overlapping func- potent signalling centres are generated at the boundary tions between the two genes (Hanks et al., 1995). A between adjacent territories, which express diffusible similar brain deletion has been found also in mice molecules that refine and polarise the neighbouring carrying both Pax2 and Pax5 null alleles (Schwarz et neural tissue (Meinhardt, 1983; Rubenstein et al., al., 1997; Urbanek et al., 1997) and in the zebrafish noi 1998). The best studied centre is located at the MHB in mutants that harbour an induced mutation in the Pax2 the isthmic region. Over the last years, elegant tissue orthologue (Brand et al., 1996; Lun and Brand, 1998). transplantation experiments in chick have addressed its Likewise, Fgf8 mutant mice and zebrafish display an role in neural patterning (Martinez et al., 1991, 1995; early brain deletion due to degeneration of the mid- Marin and Puelles, 1994). In fact, when isthmic tissue is brain and isthmic tissue (Meyers et al., 1998; Reifers et grafted to ectopic sites in the brain, as the caudal al., 1998). In each mutant outlined so far, it has been prosomeres or some rhombomeres, it can not only reported that the expression of the other genes de- maintain its own identity, but can also induce the scribed is correctly initiated but no longer maintained. surrounding tissue to change its programmed fate giv- This is probably the cause that leads to the described ing rise to midbrain and cerebellar structures. Due to loss of the brain structures (McMahon et al., 1992; this potent non cell-autonomous function, the MHB Reifers et al., 1998; V. Blanquet and W. Wurst, unpub- signalling centre has been re-named IsO. However, lished results). These important findings strongly sug- within the forebrain, the territory rostral to the ZLI, gest a close functional connection among these genes, the boundary between prosomeres 2 and 3, is not where the inactivation of a gene is able to feedback competent to respond to midbrain inducing signals negatively on the transcription of all the other genes, released by the IsO (Martinez et al., 1991). In the last leading to the degeneration of brain structures. The years, different proteins specifically expressed in the genetic maintenance loop among these genes is the isthmic region and required for its development have molecular origin of the IsO and subsequent midbrain– been characterised. They include the transcription fac- hindbrain specification and morphological shaping tors EN1, EN2, PAX2, PAX5 and two secreted (Fig. 3). This hypothesis is also supported by the strik- molecules, WNT1 and FGF8 (Rowitch and McMahon, ing phenotypes obtained in gain of function experi- 1995; Joyner, 1996). Although some of these factors ments. When, in fact, acrylic beads soaked with like EN2 and PAX5 appear to be general markers of recombinant FGF8b protein are implanted in different the region, the dynamic expression profiles of the others areas of the caudal forebrain, the nearby regions are (EN1, PAX2, WNT1 and FGF8) parallel the progres- induced to change their fate into midbrain, cerebellar sive refinement of specification taking place within the and isthmic structures (Crossley et al., 1996; Irving and midbrain-hindbrain domain. In fact, if their expression Mason, 1999; Martinez et al., 1999). These results are domains are initially broad encompassing the presump- mediated by the de-novo induction of Wnt1, Pax2, tive midbrain and rostral hindbrain (in 1–5 somite old En2, En1 in the site of bead implantation. The same embryo), later on they become progressively restricted phenotype is obtained by inducing En gene expression to narrow transverse rings that encircle the neural tube ectopically in the caudal prosomeres either in zebrafish in the isthmic area (Bally-Cuif and Wassef, 1995). In (Ristoratore et al., 1999) or chick (Shamim et al., 1999). particular, Wnt1 ring of expression is almost com- Thus, single genes can trigger the activation of the pletely overlapped and localised at the caudal end of entire genetic loop formed by Wnt, Fgf, Pax and En the prospective midbrain rostral-adjacent to the Fgf8 genes to initiate the neural fate re-specification. positive ring that lies on the isthmic-cerebellum anlage (Hidalgo-Sanchez et al., 1999b, Fig. 2). The functional 4.2. Otx genes are required for correct positioning of role of all these genes (En, Pax, Fgf8 and Wnt1) has the isthmus been tested in great detail in different animal models, clearly showing their requirement for the normal devel- An important question to be answered is how all opment of this region. In fact, mice homozygous for a these genes are induced in this particular region of the Wnt1 null allele completely lack all the midbrain and developing neural tissue. Why not more anteriorly in isthmic derivatives (McMahon and Bradley, 1990; the forebrain or more caudally in the spinal cord? Thomas and Capecchi, 1990). Mutations in En1 cause a Grafting or tissue ablation experiments in chick have similar but milder phenotype (Wurst et al., 1994), shown that Fgf8 expressing tissue with properties of the whereas, mutations in En2 only lead to subtle defects in IsO is generated when midbrain and rhombomere 1 cerebellum cyto-organisation (Joyner et al., 1991). tissue are juxtaposed but not when midbrain contacts 82 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95

Fig. 2. A schematic gene expression network in mice with different Otx2, Gbx2orFgf8 gene dosage that display a re-positioning of the MHB along 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 their expression domains cause a more anterior relocation of the MHB just near to the ZLI area. Moreover, the absence of OTX gene products in the neuroepithelium in hOtx12/hOtx12 mouse embryos cause the gene expression deregulation of Gbx2, Fgf8, and Wnt1 (violet) along all the neural plate. On the contrary, in gain of function experiments, the Otx2 ectopic expression in the metencephalon (met) leads to a repression of Fgf8and Gbx2 activity in the same region, that positions the MHB more caudally (En1Otx2/+). In Gbx2 mutant embryos (Gbx2−/−), Otx2andWnt1 expression 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 metencephalic region preceded by an ectopic rostral expression of Fgf8. The same results are obtained by ectopically expressing the Fgf8b spliced formed in the mesencephalon. Note that the nomenclature of the genotypes has not standardized and they are reported as cited in the original papers. D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 83

main towards the future diencephalic regions (Fig. 2) Therefore, a level above a critical threshold of Otx gene products is required to repress Fgf8 and, therefore, to maintain its correct transcript localisation at early somite stage. This molecular event triggers, few hours later, a rostral repositioning of En1 and Wnt1 expression domains followed by a more general anterior shift of the hindbrain specific markers and by failure of activation of the mesencephalic molecular determinants. Morphological alterations start to be scored at 10.5 d.p.c., when the isthmic region is repositioned in the caudal diencephalon, leading to replacement of the mesencephalic vesicle with a metencephalic-fated tissue (Acampora et al., 1997). Similar results are obtained when analysing the compound heterozygous embryos, Otx1+/−; Otx2+/− in the CBA/C57 genetic background, where anterior expansion of rhombomere 1 combined with loss of mesencephalic tissue have been also described (Suda et al., 1997). These findings argue in favour of a negative feedback loop between Otx2 and Fgf8, that sharpens the two neighbouring boundaries of their complementary expression domains (Hidalgo-Sanchez et al., 1999b; Fig. 3). This hypothesis is strengthened by a different report (Martinez et al., Fig. 3. Genetic hierarchy determining MHB formation. In the induc- 1999) that describes a strong repression of Otx2 expres- tion phase (7.5–8.5 d.p.c.), Fgf8 expression is induced where the sion in the chick mesencephalon around the place Otx2andGbx2 expression domains face each other. Recently, several where a FGF8 secreting source is implanted. Moreover, reports have been shown that Fgf8 is activated when mesencephalic and metencephalic fated tissues are placed in contact. This secreted the ectopic expression of the Fgf8b isoform under the molecule is able to activate several transciption factors like PAX2, control of the Wnt1 regulatory regions, inhibits the PAX5, EN1, EN2 and others that are necessary for the future endogenous Otx2 expression in the mesencephalic an- specification and patterning of the region. All these molecules seem to lage and tranforms this area in hindbrain-fated tissue maintain each other into an auto-regulatory loop. Moreover, FGF8 (Liu et al., 1999; Fig. 2). An even stronger re-location can re-induce Gbx2 expression, and inhibit Otx2 activity. of the IsO in the neuroepithelium is present in the homozygous hOtx12/hOtx12 embryos described previ- any other rh or neural tissue (Hidalgo-Sanchez et al., ously (Acampora et al., 1998b). In this mouse model, 1999a; Irving and Mason, 1999). The genetic identity of where no OTX protein is present in epiblast derivatives, the tissue rostral and adjacent to the Fgf8 expression embryos normally undergo specification of the anterior domain is depending on Otx1andOtx2 transcription neural plate which, however, fails to maintain its iden- factors (Simeone et al., 1993; Simeone, 1998). As previ- tity and acquires a more posterior fate, yielding a ously mentioned, Otx2 embryos fail to specify −/− headless phenotype at late embryogenesis as assessed by forebrain, midbrain, and anterior hindbrain. The last the rostral mislocalisation at the rostral tip of the region is specified by the IsO and never expresses Otx2 neuroectoderm of all the genes normally present in the (Acampora et al. 1995; Matsuo et al., 1995; Ang et al., IsO and rostral hindbrain, like Wnt1, Fgf8, Pax2, En1 1996). This finding argues in favour of an involvement and Gbx2, (Acampora et al., 1998b; Fig. 2). This of the Otx genes in the early IsO generation. Moreover, finding provides a strong evidence for an Otx gene a more direct involvement of the Otx function in the dosage requirement in anterior neural fate determina- anterior neuroepithelium specification is provided by tion and following maintenance throughout a contin- the phenotype analysis of Otx compound mutants car- ued repression on the posteriorizing genetic rying only one Otx2 functional allele. In fact, while determinants (Simeone, 1998). Is the action played by Otx1−/−; Otx2+/− embryos do not display Otx genes only necessary or even sufficient to induce mesendoderm and gastrulation defects, they show anterior fate in the neural plate? Gain of function molecular and morphological transformation of the experiments have been carried out, in the last years, to caudal prosomeres 1–2 and mesencephalon into a strik- answer this question. As already mentioned, over-ex- ingly enlarged metencephalon (Acampora et al., 1997). pression of Otx2 RNA in Xenopus results in an abnor- This brain re-patterning is the primary consequence of mal expansion of the head coupled with a strong an abnormal enlargement of the Fgf8 expression do- reduction of the tail and trunk and with ectopic forma- 84 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 tion of the most anterior structure of the Xenopus is caudal immediately to the midbrain, and co-lo- head, that is, the cement gland (Blitz and Cho, 1995; calises with Fgf8 and Pax2 expression domains. At Pannese et al., 1995). this stage, Gbx2 expression is also detected in two To get a more specific and controlled Otx2 ectopic longitudinal columns in the hindbrain and spinal cord expression inside the neuroepithelium a knock-in ap- (Bulfone et al., 1993; Wassarmann et al., 1997; proach has been devised for targeting the Otx2cod- Shamim and Mason, 1998; Hidalgo-Sanchez et al., ing region into the En1 genomic region (Broccoli et 1999b). Its pivotal role in determining the rostral al., 1999). Therefore, the exogenous Otx2 allele be- hindbrain neural identity has been assessed by gene comes expressed ectopically in the rostral meten- targeting inactivation experiments (Wassarmann et al., cephalon where En1 expression domain is normally 1997). In fact, Gbx2 null mutants die perinatally, lack found, thus encompassing the isthmic region (Wassef the cerebellum, the locus coeruleus and the IV and V and Joyner, 1997). The mice carrying this newly-engi- motor nuclei. These defects can be traced back to an neered Otx2 allele (En1Otx2/+) display a striking alter- early stage of neuroectoderm development (at least 8 ation in the ratio between mesencephalic and d.p.c.) and are likely due to a failed specification of cerebellar tissue. In fact, while the rectum is enlarged rhombomeres 1–3 and isthmic area that are replaced up to one-third, affecting in particular the inferior by a small undetermined region (Wassarmann et al., colliculi, the cerebellum loses almost completely its 1997; Fig. 2). Moreover, the midbrain is enlarged, medial structure, called the vermis. Because of this extending caudally over its normal limit. This event is brain rearrangement, the mice develop a strong ataxia preceded by an abnormal caudal extension of the during the early postnatal life, due to the compro- Otx2 expression domain, indicating Gbx2 as one of mised cerebellar functions (Broccoli et al., 1999). This the factors that confines the Otx2 acitivity in the anterior neural plate. However, other unidentified fac- malformation, already detectable in 10.5 d.p.c. em- tor(s) prevent Otx2 expression from expanding further bryos, is the final morphological outcome of a back- caudally. To test the hypothesis that Gbx2 is involved ward expansion of mesencephalic tissue at the in inhibiting Otx2 expression, Gbx2 was expressed in expenses of the cerebellum. This repatterning process the midbrain at early somite stages under the control is driven by the knocked-in Otx2 allele that activates of the Wnt1 enhancer (Millet et al., 1999). Consistent anteriorly expressed genes like En1, Wnt1and with the initial assumption, the Otx2 caudal limit was EphrinA5 in its ectopic expression domain, whereas shifted rostrally between the diencephalon and the hindbrain molecular determinants such as Fgf8and mesencephalon, leading to an enlargement of the pre- Gbx2 result repressed parallely. As a consequence, the sumptive anterior hindbrain, and a rostral reposition- IsO is shifted slightly caudally (Broccoli et al., 1999; ing of the gene expression network that generates the Fig. 2). How far from its normal position can the IsO IsO (Fgf8, Wnt1, En1 and Pax2; Fig. 2). Unfortu- be shifted and up to what level of the A/P axis is nately, due to the Gbx2 transient ectopic expression, neural tissue competent to respond to an Otx2 ec- the transgenic embryos recover a normal proportion topic expression are the questions to be answered in between the mesencephalon and the metencephalon by the near future. While Otx2 seems to play a key role 12.5 d.p.c. (Millet et al., 1999). In conclusion, this in determining the anterior fate of the neural plate, large body of results strongly suggests that the recip- another homeobox gene, named Gbx2, has been indi- rocal repression between Otx2 and Gbx2 is the first viduated as the major molecular determinant in con- molecular event leading to the inductive process which ferring the metencephalic identity (Bouillet et al. 1995; will specify the midbrain and hindbrain (Fig. 3). In Chapman and Rathjen, 1995; von Bubnoff et al. 1995; particular, this mutual repression is instrumental to Wassarmann et al., 1997). Gbx2 is expressed at very three subsequent events occurring between 7.5 and 8.5 early stages of neuraxis development starting at 7.5 d.p.c. embryos, (i) a sharpening and positioning of the d.p.c., where it is expressed in all three germ layers of Otx2 and Gbx2 expression borders; (ii) the establish- the gastrulating embryo in a region that extends ros- ment of the mesencephalic and metencephalic identity, trally from the posterior end of the embryo into the and (iii) the induction of the molecular cascade under- prospective hindbrain. Interestingly, its anterior ex- lying the IsO genesis and its maintenance. pression boundary moves forward as the Otx2 expres- Finally, the generation of conditional mouse mu- sion domain retracts progressively into the perspective tants for Otx2andGbx2 as well as the isolation of fore-midbrain territories (Conlon, 1995). This mutu- their regulatory regions will shed more light in the ally excluding expression of these genes suggests a near future on the mechanisms by which reasonably repression mechanism, either direct or indirect, be- few transcription factors talk to each others to lay tween OTX 2 and GBX2 gene products. By 9.5 d.p.c., down the determination program leading to the final Gbx2 RNA is confined to a sharp transverse ring that complex pattern of the CNS. D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 85

5. otd/Otx conserved functions throughout evolution bilateral symmetry such as planarians (Stornaiuolo et al., 1998; Umesono et al., 1999). In the planarian 5.1. Otx genes in the animal kingdom Dugesia tigrina, Otx expression has been found in regeneration blastemas after transverse sectioning, with Genes related to Drosophila otd and vertebrate Otx an asymmetric pattern of transcripts more abundant in have been isolated from a wide range of organisms head regenerating tissues (Stornaiuolo et al., 1998). The (Fig. 4). Most of them, up to protochordates have only expression of the Otx genes in these early metazoans one member, with few exceptions where duplication reveals some features in common with chordate Otx seems to have occurred in independent lineages (Li et genes. Although not directly correlated with a defined al., 1996; Umesono et al., 1999). anterior structure, the ancient function seems to deal An Otx related gene is present already in Cnidarians, with patterning body axis and making tissues com- primitive metazoans with a defined body plan and petent to respond to anteriorizing signals (Smith et al., radial symmetry. In these organisms, the Otx function 1999), at least in budding and regeneration processes is associated to cell movements involved in the forma- which involve cell movements. Two Otx-related genes tion of new axes rather than in the formation of the have been identified only on the basis of homeodomain head (Smith et al., 1999). Rising in the evolutionary sequence homology in the nematode C. elegans (Galliot scale, Otx has been found in animals with primitive et 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 asterisks points to the two possible positions of the first Otx duplication from a single ancestral gene. The presence of only one Otx-like gene in protochordates suggests that the Otx gene duplications observed in both Tribolium castaneum and in Dugesia japonica are likely independent duplication events occurred in these organisms. 86 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 to Otx has been discovered by means of the sequencing project of the whole genome of this worm (Ruvkun and Hobert, 1998). However, no expression data are yet available about this gene. The first animal with a clearly head-associated Otx expression belongs to annelids (the leech Helobdella triserialis; Bruce and Shankland, 1998). This study is relevant particularly in evolutionary terms, since it supports the idea of the origin of bilaterians from radial ancestors (Brusca and Brusca, 1990). The passage from radials to bilaterians might have occurred through specification and subsequent expansion of a trunk precursor cell-population from one side of the radial ancestor. The leech Otx expression, which is organised radially around its mouth may represent a reminiscence of the Otx expression in a radial ancestor, thus suggesting that, if not recruited to specify trunk structures, relega- tion of head genes to head domain occurred in bilaterians as a consequence of their evolutionary origin. Otx expression in insects will be discussed in more detail in a separate section. The adult pentaradial symmetry shown by sea urchin seems to contradict the general head-associated Otx expression rule. It is held generally that the radial symmetry in echinoderms is a shared derived character Fig. 5. Schematic comparison of expression domains of Otx-related (synapomorphy) because of their embryonic and larval genes (grey) during embryogenesis in some representative protochor- bilateral symmetry (Lowe and Wray, 1997). Thus, the dates (Ascidia and Amphioxus) and vertebrates (Lamprey and mouse). Both lampreys Otx-related genes are shown because of their highly divergent and non-head specific Otx expression highly divergent expression patterns. Abbreviations — D, dien- in sea urchin can be justified as a consequence of the cephalon; Ep, epiphysis; Ey, eye; F, forebrain; FE, frontal eye; HB, highly modified body plan of this phylum. hindbrain; IO, infundibular organ; ll, lower lip; M, midbrain; MHB, Comparative studies have demonstrated the existence mid-hindbrain boundary; NC, nerve cord; Oe, olfactory epithelium; of Otx-related genes in all chordates (Simeone et al., Os, optic stalk; RV, rhomboencephalic vesicle; SC, spinal cord; SV, sensory vesicle; T, telencephalon; ul, upper lip; and VG, visceral 1992, 1993; Frantz et al., 1994; Li et al., 1994; Mori et ganglion. al., 1994; Bally-Cuif et al., 1995; Blitz and Cho, 1995; Mercier et al., 1995; Pannese et al., 1995; Kablar et al., evoluted sensory vescicle and cerebral vescicle of 1996) including urochordates (Wada et al., 1996), Ascidians and Amphioxus, respectively (Fig. 5) cephalochordates (Williams and Holland, 1996), and (Williams and Holland, 1998). Some authors have also agnathan vertebrates (Ueki et al., 1998), where they are extended this homology further backwards in evolu- expressed in the rostralmost CNS, independently of the tion. As mentioned previously, it has been proposed complexity acquired by this area during evolution. The that a primitive Otx function could be associated only critical evolutionary position of lampreys will be dis- with cell movement rather than with anterior patterning cussed below, mainly with regard to Otx gene (Smith et al., 1999). Similarly, functional data in frog duplication. (Blitz and Cho, 1995; Pannese et al., 1995) and mouse In urochordates and cephalochordates, only one Otx mutants (Acampora et al., 1995; Matsuo et al., 1995; gene has been identified so far that may be related to Ang et al., 1996) suggest a possible involvement of Otx2 (Wada et al., 1996; Williams and Holland, 1998). Otx2 in controlling the cell movements occurring dur- Indeed, in addition to similarities in amino acid se- ing gastrulation. Recently, an early role of the ascidian quence and expression, they are both expressed during Otx2-related gene Hroth has been suggested in the gastrulation in endoderm cells which suggests that their inhibition of notochord and muscle cell fate or in the oldest and primary role might be to mediate signals interference with notochord cell movements (Wada and required to specify anterior neuroectoderm. Restriction Saiga, 1999). Conservation of the expression domains of an early widespread expression of Otx2-like genes to among Otx2-like orthologues from low chordates the anterior CNS is a remarkable feature of all verte- (Wada et al., 1996; Williams and Holland, 1996; Ueki brates, which can be already observed in Ascidians and et al., 1998), to vertebrates (Acampora and Simeone, Amphioxus. This led to hypothesise the homology be- 1999 and references therein) is consistent with the low tween fore-midbrain territory of vertebrates to the less- rate of divergence of their alignable sequences. D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 87

The duplication event generating the Otx1 branch Even more convincing was the similar expression of from the ancestor Otx2-like gene in gnathostome verte- short gastrulation/chordin and decapentaplegic/Bmp4 brates cannot be dated precisely and will be discussed pairs of homologous genes, which are antagonistic key below. However, comparative sequence analysis indi- molecules of the D/V patterning (Piccolo et al., 1997) in cates clearly that Otx1-like genes evolve more rapidly, both Drosophila and vertebrates. As far as their expres- as also shown by a further duplication event occurred sion is inverted with respect to the D/V axis in in both Xenopus (Kablar et al., 1996) and Zebrafish Drosophila and Xenopus, injection experiments have (Mori et al., 1994) and by a ratio of sequence diver- shown that these molecules are functionally equivalent gence higher than Otx2-like genes (Williams and Hol- (De Robertis and Sasai, 1996), thus supporting the land, 1998). existence of a homologous mechanism of D/V pattern- These data can be reinforced by the profound ing in a common ancestor of both Drosophila and changes in the expression domains of different verte- vertebrates and reinforcing the ancient hypothesis of an brate Otx1-like genes (Simeone et al., 1993; Mori et al., inversion of the D/V axis during evolution (De Rober- 1994) which underlie rapid evolution of regulatory se- tis and Sasai, 1996; Arendt and Nu¨bler-Jung, 1999). quences as well. Finally, Gerhart (2000) has recently reviewed a number of hypotheses alternative to the inversion of the D/V axis, mainly based on the analysis of intermediate posi- 5.2. A common genetic program in insect and tions in evolution which imply intermediate anatomies, 6ertebrate head de6elopment as in hemichordates. Along the A/P axis, in both Drosophila and verte- Cloning of several master genes conserved through- brates, the conserved families of HOM/HOX and otd/ out evolution and their comparative expression analysis Otx genes play a fundamental role in the regional has been used largely as a means to identify ho- specification of the neuroectoderm destined to form mologous body regions and molecular mechanisms nerve cord/posterior brain and anterior brain, respec- among animals of different phyla (Abouheif et al., tively. This gross similarity can be refined further when 1997). looking at the precise coincidence of gene-expression The most sensational example of such a functional domains with the boundary of metameric units or conservation is represented by the Pax-6/eyeless gene, neuromeres (Reichert and Simeone, 1999) and, as for which in both Drosophila and vertebrates controls eye the HOX genes, also according to the phenomenon of development (Callaerts et al., 1997). the ‘spatial colinearity’ (Lumsden and Krumlauf, 1996). Until recently, it was assumed widely that the insect Over the last 10 years, functional experiments have and vertebrate CNS had evolved independently substanciated the equivalence suggested by the insect/ (Garstang, 1928; Lacalli, 1994). This was due to their vertebrate comparative expression data. Most of these position on opposite body sides of the dorso-ventral studies carried out in Drosophila have shown that mam- (D/V) axis. One theory, the so-called ‘auricularia hy- malian HOX genes could either partially rescue pheno- pothesis’, postulated the homology between the outer types due to mutations of their fly orthologues or elicit ectoderm of the insect embryo and the chordate CNS, responses similar to those elicited by their endogenous based on both anatomical studies made in echinoderms counterparts when transiently overexpressed (Malicki et (auricularia larvae) and urochordates, and on compara- al., 1990, 1993; Zhao et al., 1993; Bachiller et al., 1994). tive expression of the HOX genes. To further sustain the idea that some of the molecu- Starting from an ancient hypothesis of about two lar mechanisms underlying brain development were centuries ago postulated by Geoffroy Saint-Hilaire (De conserved and comparable, a final, more challenging, Robertis and Sasai, 1996), which has been object of proof remained to be verified. Could the Drosophila otd controversial debate (Arendt and Nu¨bler-Jung, 1994; gene substitute for Otx genes in the mouse? That is, can Lacalli, 1995; Peterson, 1995), an increasing body of a gene working in a structure as simple as the insect evidence has now accumulated which supports a com- CNS substitute for homologous genetic functions in an mon evolutionary origin of insect and vertebrate CNS. enormously more complex organ such as the brain of The first clues have been provided when it was shown mammals? that the overall arrangement of neuroblasts in three The embryonic Drosophila brain is composed of two longitudinal columns on either side of the midline in supraesophageal ganglia, each subdivided into three both insect and vertebrate CNS (Doe and Goodman, neuromeres. The anterior ganglion is subdivided into 1985; Chitnis et al., 1995) was paralleled by conserved protocerebral, deutocerebral, and tritocerebral neu- topographical expression (although with inverted D/V romeres. The gap gene otd is expressed mainly in the polarity) of pairs of homologous genes (NK-2/NK-2.2; anteriormost (protocerebral) neuromere, which is ind/Gsh and Msh/Msx; Arendt and Nu¨bler-Jung, 1996; deleted almost entirely in otd null embryos (Finkelstein D’Alessio and Frasch, 1996; Weiss et al., 1998). and Perrimon, 1990; Cohen and Ju¨rgens, 1991; Hirth et 88 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 al., 1995; Younossi-Hartenstein et al., 1997). This phe- In otd 1/otd 1 adult mice, brain size as well as the notype is due to the failure of expression of the otd thickness and cell number of the temporal and perirhi- downstream gene lethal of scute, one of the proneural nal cortices, both reduced in Otx1−/− mice, are very genes regulating the three-column arrangement of neu- similar to wild-type. The rescue is likely to be ascribed roblasts mentioned above. As a consequence, in the at least in part, to a restored normal proliferating absence of otd, proneural genes are not activated and a activity of the dorsal telencephalic neuroepithelium at gap-like deletion of the rostral brain neuromere (proto- 9.75 d.p.c. (Acampora et al., 1998a). As described in a cerebrum) results in late mutant embryos (Hirth et al., previous section, Otx1 and Otx2 co-operate in brain 1995). Other defects are also observed in the ventral morphogenesis and a minimal threshold (corresponding nerve cord and in non-neural structures. Flies that are to either two copies of Otx2 or to one copy of Otx1 homozygotes for ocelliless (oc), a different otd allele, are and one copy of Otx2) of their gene products is re- viable and lack the ocelli (light sensing organs) and quired for correct brain regionalization (Acampora et associated sensory bristles of the vertex (Finkelstein et al., 1997). When otd is expressed in place of both copies al., 1990). Moreover, in cephalic development, different of Otx1 and in the presence of one functional copy of levels of OTD protein are required for the formation of Otx2(otd 1/−; Otx2+/− or otd 1/otd 1; Otx2+/−), specific subdomains of the adult head (Royet and brain patterning abnormalities of the Otx1−/−; Finkelstein, 1995). Despite the overall morphological Otx2+/− mutants are rescued partially in a otd dose- differences, however, expression pattern and mutant dependent manner, both at morphological and molecu- phenotypes of Drosophila otd and mouse Otx genes can lar level (Acampora et al., 1998a). The extent of this be paralleled (see previous sections) easily. Neverthe- rescue decreases progressively along the A/P axis, being less, OTD and OTX proteins are highly conserved only the posterior mesencephalon still severely affected with in the homeodomain, which represents roughly one- respect to a normal telencephalon and to ameliorated tenth of the whole OTD protein and about one-sixth diencephalic and anterior mesencephalic structures. All and one-fifth of OTX 1 and OTX 2 proteins, respec- of these data indicate strongly that regionalization of tively (Simeone et al., 1993). the brain requires levels of OTX proteins that increase Outside the homeodomain, homology is restricted to along the A/P axis and are particularly critical at the a few very short sequences. Drosophila OTD protein MHB (Acampora et al., 1999b). also lacks the so-called ‘‘OTX tail’’ (Freud et al., 1997), A partial rescue is also observed in some sensory and a conserved motif of about 20 amino acids which is sensory-associated structures such as iris, ciliary process present in single copy in echinoderm, Ascidian and and Harderian glands. Amphioxus Otx-related genes, whilst is tandemly dupli- On the contrary, the lateral semicircular canal of the cated in all vertebrate OTX proteins at the COOH inner ear (last to be established in evolution) (Fritzsch terminus (Williams and Holland, 1998). et al., 1986; Torres and Giraldez, 1998) is never re- The limited sequence homology shared by OTD and 1 1 OTX proteins underlines the importance that the home- stored in otd /otd mice, suggesting that, either it re- odomain might have in triggering the CNS develop- quires higher levels of protein or that specification of mental programmes in both insects and vertebrates. this structure is dependent upon an Otx1-newly estab- Outside the homeodomain, the proteins could either lished function. This seems indeed to be the case since, 1 1 have diverged, to allow mammals to acquire new and as previously mentioned, in hOtx2 /hOtx2 mice, Otx2 specific functions or might be more homologous in is able to rescue some ear defects in the regions where terms of tertiary structure than what the primary se- both Otx genes are transcribed but not in the lateral quence alignment indicate. To gain insight into the semicircular canal, where only Otx1 is normally ex- possibility that otd and Otx genes might share con- pressed (Acampora et al., 1999a; Morsli et al., 1999). served genetic functions during CNS development, we The absence of the lateral canal in the inner ear of undertook experiments in which a Drosophila otd Otx1−/− mice might represent a back-evolutionary cDNA replaces both mouse Otx1andOtx2 genes. mutation that can indicate the presumptive date of When a full-coding Drosophila otd cDNA is intro- duplication of the Otx genes. This event should have duced into a disrupted Otx1 locus by homologous occurred after that the lineage leading to cephalochor- recombination many abnormalities of the Otx1−/− dates had split from that leading to vertebrates mice are rescued, regardless of a lower level of OTD (Williams and Holland, 1998) and probably after the (about 30%) as compared with the endogenous OTX 1 latest passage leading from Agnatha to Gnathostomes. level (Acampora et al., 1998a). Homozygous knock-in In fact, in lampreys, extant agnathan vertebrates, only otd mice (otd 1/otd 1) show no significant perinatal death two semicircular ducts (anterior and posterior) are (with respect to a 30% death of Otx1−/− newborns) present in the inner ear, and despite two Otx-like genes and, most importantly, they have neither the abnormal have been identified, none of them is clearly related to behaviour nor EEG characteristics observed in the Otx1. However, it cannot be excluded that the Otx Otx1 null mutants. genes evolved by duplication in a common ancestor of D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 89 lampreys and gnathostomes (Fig. 4) and that the un- latory genes may be conserved across the two phyla and classified lamprey Otx represents a still evolutionary indicates that new functions have been acquired by the unstable version of an Otx1-like gene (Ueki et al., vertebrate genes during evolution. 1998). More recently, we have generated a mouse model in 5.3. Otx genes in brain e6olution which it is the Otx2 gene to be substituted by the Drosophila otd cDNA (otd2), using the same targeting One of the most interesting observations raised by strategy as in the replacement with lacZ (Acampora et our mouse models is the existence of a differential al., 1995) and with hOtx1 cDNA (Acampora et al., post-transcriptional control of the hOtx1/otd and Otx2 1998b). Also in this case, the otd mRNA is detected in mRNAs between VE and epiblast cells. both AVE and epiblast, whereas the protein only in the In heterozygous hOtx12/Otx 2 and otd 2/Otx 2 em- VE. The OTD protein, exactly as the hOTX1 protein bryos, Otx2 mRNA and protein always colocalize, does, is able to take over all of the Otx2 functions in while the hOtx1 and otd mRNAs are translated only in the AVE, thus recovering both gastrulation defects and the VE, thus suggesting that the knocked-in mRNAs absence of an early anterior neural plate due to the lack detected in epiblast cells are post-transcriptionally regu- of Otx2. Later on, as in the hOtx12/hOtx12 mutants, lated by an Otx2-independent cis-acting control however, otd 2/otd 2 embryos fail to maintain the anteri- (Acampora etal., 1998b). ormost identities of the brain and become headless. In Otx2+/− embryos, the same Otx2 region that is These results, as far as limited to the AVE, provide a replaced by the hOtx1 and otd cDNA is substituted further proof of functional equivalence, shared by otd/ with the lacZ gene (Acampora et al., 1995). In these Otx genes, via the activation of the same basic genetic embryos the lacZ mRNA is correctly detected in both pathway(s). AVE and epiblast while the b-gal staining is, again, Similar conclusions have been drawn by complemen- heavily reduced in the epiblast at early-mid streak stage tary experiments in Drosophila (Leuzinger et al., 1998; (Acampora et al., 1995). Nagao et al., 1998). Heat-shock induced expression of Therefore, three different mouse models replacing the hOTX1 and hOTX2 proteins in the fly is able to rescue same Otx2 genomic region with three different genes the CNS defects of otd null mutant embryos (Leuzinger (lacZ, hOtx1 and otd) are suggestive of a different et al., 1998) as well as cephalic defects of the ocelliless post-transcriptional control between AVE and epiblast mutations, both at the morphological and molecular cells. One or more Otx2 regions deleted in these models level (Nagao et al., 1998), whereas in a wild-type back- (introns, untranslated or coding sequences) should be ground, OTX overexpression leads to induction of ec- responsible for this differential control. topic neural structures (Leuzinger et al., 1998). Loss of mRNA translation in the epiblast might be As previously mentioned, even though these cross- also influenced by abnormal molecular event(s) affect- phylum rescues highlight the fundamental role of the ing RNA stability, processing and transport of the homeodomain because it is the only recognisable region chimeric knocked-in transcripts. Whatever the impair- conserved between OTD and OTX proteins, they do ments are, since in the AVE the mRNA is correctly not exclude that functional equivalence may be ex- translated, the absence of protein should be considered tended to the overall structure of OTD/OTX proteins. a peculiar event occurring in epiblast cells and their The fact that OTD and OTX proteins are able to derivatives. drive cephalic development through the activation of The finding that Otx2 escapes this post-transcrip- genetic pathways conserved between the two taxa, rein- tional control suggests that this is necessary for the forces the idea that insect and chordate CNS are indeed maintenance of fore-midbrain territory specified by the homologous structures originated from a common an- AVE (Acampora et al., 1998b) and might have evolu- cestor and controlled by a common basic genetic pro- tionary implications. Indeed, the architectural compo- gram (Sharman and Brand, 1998; Acampora and nents of the vertebrate brain (telencephalon, Simeone, 1999; Reichert and Simeone, 1999). diencephalon and mesencephalon) are less clear in pro- Interestingly, substitution of a different key gene tochordates and a vertebrate-type brain, which is com- involved in brain patterning processes with its posed of a midbrain and six prosomers, firstly appears Drosophila orthologue has led to very similar conclu- in Petromyzontoidea. It may be hypothesized that the sions (Hanks et al., 1998). In fact, Drosophila engrailed specification of this prosomeric brain might have re- was able to substitute for mouse Engrailed 1(En1) quired the presence of a sufficient level of OTX2 functions in mid- and hind-brain regions but not in protein in the early neuroectoderm cells and this event limb development. Therefore, despite a higher degree of might have been acquired by modifying post-transcrip- homology between EN proteins with respect to OTD/ tional control of Otx2 transcripts rather than its coding OTX proteins, this rescue reinforces the idea that some sequence which would have retained common func- of the biochemical properties of highly conserved regu- tional properties among the otd-related genes through- 90 D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 out evolution. The low ratio of Otx2 sequence diver- mammalian brain, for example by increasing the extent gence among the species fits with this hypothesis. of neuroectodermal territory recruited to form the Therefore, although this conservation in expression brain. This event might involve an improvement of pattern and coding sequence might favour a remarkable proliferative activity of early neuronal progenitors role and functional equivalence of otd/Otx genes, it is (Acampora et al., 1998a, 1999a) and/or the positioning unclear why the brain vescicles of protochordates have of the MHB (Acampora et al., 1997, 1998b; Suda et al., been so deeply and suddenly modified in a prosomeric 1997). Additional property(ies) may be also conferred territory that has been maintained in its basic topogra- to the duplicated gene by altering its coding sequence. phy until mammals (Rubenstein et al., 1998). Thus, the limited amino acidic divergence between A rather obvious answer to this question is that new OTX1 and OTX2 might underlie modifications of their genetic functions have been gained and recruited in functional properties, as shown by the non-rescued developmental pathways. Indeed, it might be that con- absence of the lateral semicircular canal of the inner ear served genes such as the Otx acquired different roles in mice replacing Otx1 with Otx2 (Acampora et al., even while retaining an evolutionary functional equiva- 1996, 1999a; Morsli et al., 1999). lence. Based on this hypothesis, it is expected that drastic evolutionary events should act on the regulatory control (transcription and translation) of Otx-related 6. Conclusions genes rather than on their coding sequences. Interest- ingly, the findings that, (i) the Drosophila otd rescues in In the last decade an impressive amount of knowl- mouse most of the Otx1−/− impairments (Acampora edge has contributed to shed some light on the com- et al., 1998a); (ii) the human Otx1 and Otx2 rescue plexity of molecules and mechanisms involved in the most of the otd defects in flies (Leuzinger et al., 1998; development of the vertebrate brain. In this context Nagao et al., 1998; Sharman and Brand, 1998); (iii) otd/Otx genes play a cardinal role. Previous mouse Otx1 rescues Otx2 requirements in VE (Acampora et models have indicated that Otx1andOtx2 are required al., 1998b); (iv) Otx2 rescues most of the Otx1−/− for normal brain and sense organ development and, defects (Acampora et al., 1999a); (v) the Drosophila otd, together with the Drosophila orthodenticle (otd) gene similarly to Otx1, rescues Otx2 requirements in the VE they share a high degree of recriprocal functional equiv- (Acampora et al., unpublished results), indicate that alence. As far as the Otx genes are concerned, their Otx1, Otx2 and otd genes show a high degree of interchangeability indicates that the differential tran- functional equivalence in the tissues and body regions scriptional control between Otx1 and Otx2 is the major where they are properly expressed. Therefore, these molecular reason responsible for generating Otx1−/− data support the notion that otd/Otx functions have and Otx2−/− divergent phenotypes. Nevertheless, been established in a common ancestor of fly and one of the most interesting observations raised by mouse and retained throughout evolution, while copy mouse models replacing Otx2 with hOtx1orotd is the number and regulatory control of their expression have existence of a differential post-transcriptional and/or been modified and re-adapted by evolutionary events translational control between the VE and epiblast. In- that have led to the specification of the increasingly deed, hOtx1orotd mRNA were detected in VE, complex vertebrate brain (Sharman and Brand, 1998; epiblast and its derivatives while hOTX1 or OTD Simeone, 1998; Acampora and Simeone, 1999; Hirth proteins in the VE only. This event results in the failure and Reichert, 1999; Reichert and Simeone, 1999). Gene of maintenance of fore-midbrain identities in the mu- duplication may allow the duplicated gene to acquire tant embryos. new specific function(s) either retaining or losing ances- In our opinion one of the most interesting perspec- tral properties, and similarly, modification of the regu- tives will be the understanding of the molecular mecha- latory control of gene expression may establish new nism(s) underlying this phenomenon, and its potential expression patterns which might alter pre-existing cell- relevance in the morphogenetic changes leading to the fates by generating new specialised cellular functions. evolution of the mammalian brain. A likely consequence of both increased genomic complexity and modification of regulatory control of gene expression may result in an greater number of molecu- Acknowledgements lar interactions. This may contribute to modifying relevant morphogenetic processes that, in turn, can confer We thank A. Secondulfo for manuscript preparation. a change in shape and size of the body plan as well as This work was supported by the the EC BIOTECH in the generation of cell-types with new developmental programme, the MRC Grant programme (c potentials. On this basis, Otx gene duplication and G9900955), the Italian Association for Cancer Research subsequent or contemporary modification of regulatory (A.I.R.C.) and the ‘MURST-CNR Biotechnology Pro- control might have contributed to the evolution of the gramme Legge 95/95’. D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95 91

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