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Progress in Neurobiology 64 (2001) 69 – 95
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Otx genes in brain morphogenesis
Dario Acampora a,b, Massimo Gulisano c, Vania Broccoli d, Antonio Simeone a,b,*
b
a
International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples, Italy
MRC Centre for De6elopmental Neurobiology, 4th Floor, King’s College London, Guy’s Campus, New Hunts House, London Bridge,
London SE1 9RT, UK
c
Department of Physiological Sciences, Uni6ersity of Catania, Viale A. Doria 6, 95125 Catania, 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 Otx1 and Otx2 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, Otx1 and Otx2 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 . . . . . . . . . . . . . . . . . . . . . . . . .
2. Multiple roles of the Otx1 gene in the developing and
2.1. Otx1 expression in early embryogenesis . . . . .
2.2. Otx1 expression during corticogenesis. . . . . . .
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adult mouse
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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).
0301-0082/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0301-0082(00)00042-3
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
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2.3.
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2.7.
Otx1 is required for correct brain development . . . . . . . . .
Otx1−/− mutant mice exhibit an epileptic phenotype . . . .
Otx1 controls cortical connectivity to subcortical targets. . . .
Otx1 transiently controls GH, FSH, and LH in the pituitary .
Otx1 is necessary for correct sense organ development . . . . .
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3. Early requirement of Otx2 for normal gastrulation and anterior neural plate induction and
maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Organisers and molecules in the current scenario of the anterior brain patterning . . .
3.2. Specification of anterior patterning requires Otx2 function . . . . . . . . . . . . . . . .
3.3. The role of Otx2 in the induction and maintenance of rostral CNS identities . . . . .
3.4. AVE-restricted OTX2 function is necessary for proper expression of Lim1 and bone
morphogenetic protein (BMP) antagonists in the ame . . . . . . . . . . . . . .
3.5. Specific and interchangeable roles between Otx1 and Otx2 . . . . . . . . . . . . . . . .
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4. Brain patterning depends on a critical Otx gene dosage . . . . . . . . . . . . . . . . . . . . .
4.1. Morphogenetic origin of the IsO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Otx genes are required for correct positioning of the isthmus . . . . . . . . . . . . . . .
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5. otd/Otx conserved functions throughout evolution. . . . .
5.1. Otx genes in the animal kingdom . . . . . . . . . . .
5.2. A common genetic program in insect and vertebrate
5.3. Otx genes in brain evolution . . . . . . . . . . . . . .
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6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
The central nervous system (CNS) of vertebrates is a
very complex structure derived from a multistep process
involving sequential molecular and morphogenetic
events that pattern the epiblast first and the neural plate
later. During early gastrulation the concerted and sequential action of both the anterior visceral endoderm
(AVE) and the node-derived axial mesendoderm (ame,
Beddington and Robertson, 1999; Bachiller et al., 2000)
drives the specification of anterior neuroectoderm that,
later on results grossly subdivided in three main territories (forebrain, midbrain and hindbrain, Gallera, 1971;
Storey et al., 1992; Ruiz i Altaba, 1994; Shimamura and
Rubenstein, 1997; Rubenstein and Beachy, 1998). The
maintenance of this patterning is likely dependent on
signals originating from the ame and/or within the
overlying neuroectoderm (Acampora et al., 1998b; Rhinn
et al., 1998; Shawlot et al., 1999; Camus et al., 2000).
Anatomical and histological studies postulate the
existence of genetic fate determinants which subdivide
these large neural regions into progressively smaller
longitudinal and transverse domains (Vaage, 1969; Altman and Bayer, 1988; Figdor and Stern, 1993; Rubenstein et al., 1994). Some of the patterning events along
the antero-posterior (A/P) axis require the presence of
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head development
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specific cell populations (e.g. the anterior neural ridge
and the zona limitans intrathalamica (ZLI)) and transverse rings of neuroepithelia (e.g. the isthmic organizer
(IsO)) that possess inductive and boundary properties
(Marin and Puelles, 1994; Crossley et al., 1996; Houart
et al., 1998; Rubenstein et al., 1998; Ruiz i Altaba, 1998).
In vertebrates, several genes controlling developmental
programmes underlying brain morphogenesis have been
isolated and their role studied in detail. Most of them are
the vertebrate homologues of Drosophila genes encoding
signalling molecules or transcription factors (Lemaire
and Kodjabachian, 1996; Tam and Behringer, 1997;
Rubenstein et al., 1998). Among these, the orthodenticle
group is strictly defined by the Drosophila orthodenticle
(otd) and the vertebrate Otx1 and Otx2 genes, which
contain a bicoid-like homeodomain (Finkelstein and
Boncinelli, 1994; Simeone, 1998). However, other genes
that might be more distantly related to otd/Otx genes
have been also identified (Muccielli et al., 1996; Szeto et
al., 1996; Chen et al., 1997).
Expression pattern analysis of Otx genes had suggested that these transcription factors might play an
important role during brain morphogenesis in vertebrates. A systematic genetic approach using transgenic
mice is revealing that they contribute to the molecular
mechanisms underlying all of the major events (induc-
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
tion, maintenance, regionalization, corticogenesis and
axon connectivity) necessary to build a normal brain.
2. Multiple roles of the Otx1 gene in the developing
and adult mouse
2.1. Otx1 expression in early embryogenesis
Otx1 starts to be expressed at early stages (2–5
somite stage, 8.2 –8.5 days post coitum (d.p.c.)) in the
developing mouse embryo throughout the presumptive
forebrain and midbrain neuroepithelium (Simeone et
al., 1992). From these stages onwards its expression
largely overlaps with that of Otx2, but while the expression of the latter disappears from the dorsal telencephalon since 10.5 d.p.c. (Simeone et al., 1993), Otx1
expression is maintained uniformly across the ventricular zone (VZ) of the cortical anlage from the onset of
corticogenesis up to mid- to late gestation stages (Simeone et al., 1993; Frantz et al., 1994).
Otx1 is also expressed at early stages in precursor
structures of sense organs corresponding to the olfactory placode, otic and optic vesicles (Simeone et al.,
1993). Later on, Otx1 is transcribed in the olfactory
epithelium, the saccule, the cochlea and the lateral
semicircular canal of the inner ear as well as in the iris,
the ciliary process in the eye and the lachrymal gland
primordia (Simeone et al., 1993). From the birthday
onwards, Otx1 is also expressed at a relatively low level
in the anterior lobe of the pituitary gland (Acampora et
al., 1998c).
2.2. Otx1 expression during corticogenesis
The cerebral cortex develops according to molecular
strategies that determine the fate of precursor cells
linked to specific neuronal phenotypes. Two main processes have been identified so far, laminar determination, by which committed cells migrate to their
appropriate layer, and cortical areas formation, by
which cortical neurons interact to create functionally
distinct regions. During corticogenesis, postmitotic neurons migrate along radial glial cells (Rakic, 1972),
through the overlying intermediate zone (IZ), and to
the cortical plate (CP), which will later create the
typical layered organisation of the adult cortex. The
layers are generated in an inside-out pattern, in which
cells of the deepest layers (6 and 5) are born first in the
VZ, and those of the upper layers (4, 3, and 2) progressively later (Rakic, 1974).
Otx1 represents a molecular correlate of deep layer
neurogenesis and its expression is confined to neurons
of layers 5 and 6 in the adult cortex (Frantz et al.,
1994).
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At mid-late gestation, high level transcription of
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
decreases in the VZ and becomes progressively prominent in the cortical plate which consists of postmigratory neurons of layer 5 and 6. Otx1 is absent in later
differentiated neurons of upper layers 1–4 (Frantz et
al., 1994).
Thus, the progressive down-regulation of Otx1 in the
ventricular cells suggests that Otx1 may confer deeplayer identity to young neurons. Heterochronic transplantation experiments have demonstrated that the
broad differentiative potentials of the early progenitors
(McConnell and Kaznowski, 1991) become progressively restricted over time (Frantz and McConnell,
1996).
Indeed, Otx1 expression is heterogeneous across the
regions of the adult cortex, suggesting that it might also
be involved in the forming of the cortical areas. Its
expression in layer 5 is more prominent in the posterior
and lateral cortex but absent in the frontal, insular and
orbital cortices, while in layer 6 it is more uniform
throughout the neocortex (Frantz et al., 1994).
Mouse mutant models have been generated and
analysed (Acampora et al., 1996, 1998a, 1999a,b Suda
et al., 1996; Morsli et al., 1999; Weimann et al., 1999;
Avanzini et al., submitted) to gain insight into the
different roles that Otx1 plays during brain, cortex and
sense organ development.
2.3. Otx1 is required for correct brain de6elopment
Heterozygous (Otx1 + /−) mice are healthy and
their intercross generates homozygous mice (Otx1−/
− ) in the expected mendelian ratio. However, about
30% of the mutants die before the first postnatal
month, and appear smaller in size.Table 1
Otx1− /− adult brains are reduced in weight and
size and, at the anatomo-histological inspection, show
reduction in thickness of the dorsal telencephalic cortex, a dorsally displaced sulcus rhinalis and shrunken
hippocampus with a divaricated dentate gyrus. The
cortex is particularly affected at the level of the temporal and perirhinal areas, where a 40% reduction in cell
number is detected. Furthermore, in these same areas,
cortical layer organisation is less evident (Acampora et
al., 1996), although the expression of layer-specific
molecular markers demonstrates that the laminar identities are preserved (Weimann et al., 1999).
The origin of the overall reduction of the Otx1−/−
brains has been investigated through experiments aimed
at determining possible changes of the normal number
of apoptotic or proliferating cells within the neuroepithelium of the developing telencephalon. No differences at apoptosis were observed by comparing
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
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wild-type and Otx1 mutant embryos. By contrast, Bromodeoxyuridine (BrdU) labelling experiments revealed
a reduction of proliferating cells (by about 25%) in the
dorsal telencephalic neuroepithelium of 9.75 d.p.c.
Otx1−/− embryos (Acampora et al., 1998a). A defective proliferation of neuronal progenitors at these early
stages may thus be responsible of the adult phenotype
of the Otx1 mutant mice.
2.4. Otx1 −/− mutant mice exhibit an epileptic
phenotype
Otx1−/− mice exhibit both spontaneous high
speed turning behaviour and epileptic behaviour
(Acampora et al., 1996). The latter consists of the
combination of, (i) focal seizures characterised by automatisms (head bobbing and teeth chattering) and
electroencefalographic (EEG) recording of spikes in
hippocampus; (ii) generalised seizures characterised by
convulsions and high voltage synchronised EEG activ-
ity in hippocampus and cortex. Occasionally, convulsions are followed by status epilepticus and exitus.
Recently, the epileptogenic mechanisms accounting
for these seizures have been further investigated by
means of electrophysiological recordings (current clump
intracellular recordings) made from layer 5 pyramidal
neurons in somatosensory cortical slices (Avanzini et
al., 2000). This analysis shows that g-aminobutyric acid
(GABA)-mediated inhibitory post-synaptic potentials
(IPSP), as compared with control pyramidal neurons,
are more pronounced in the mutants where they seem
to be involved in the synchronisation of the excitatory
activity from the earliest postnatal period. On the other
hand, multisynaptic excitatory post-synaptic potentials
(EPSP) are significantly more expressed in the mutants
than in controls, also at the end of the first postnatal
month. These results suggest that the excessive excitatory amino acid-mediated synaptic driving, without a
well-developed GABA counteraction, may lead to a
hyperexcitable condition that is responsible for the
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
otd 1/otd 1
Dorsal telencephalon
Cell proliferation rate at E9.75
At E 13.5 and E 15.5
Reduced by 25%
Reduced by 10%
Full recovery
Full recovery
Full recovery
Full recovery
Cerebral cortex
Cell number
Layer organisation
Laminar fatea
Temporal cortex
Perirhinal cortex
Hippocampus
Reduced
Altered
Normal
Reduced by 40%
Reduced by 40%
Shrunken
Full recovery
Full recovery
N/Ab
Full recovery
Full recovery
Full recovery
Full recovery
Full recovery
N/Ab
Full recovery
Full recovery
Full recovery
Mesencephalon
Size of colliculi
Enlarged
Identities of colliculia
Cerebellum
Normal
Abnormal foliation
Normal in 15%
Intennediate in
50%
N/Ab
Recovery in 10%
Axonal projection from layer 5 neurons of 6isual
cortex a
Defective refinement of exuberant projections
Normal in 30%
Intermediate in
45%
N/Ab
Recovery in
50%
N/Ab
Beha6iour
Turning behaviour
Epileptic seizures
High-speed
Present
Moderate-speed
Absent
Moderate-speed
Absent
Pituitary gland
Impaired
Normal
Normal
Ear
Lateral semicircular duct
Absent
Absent
Absent
Eye
Iris
Reduced
Recovery in 80%
Ciliary process
Lachrymal and Harderian gland
Absent
Absent
Recovery in
80%
Present in 70%
Present in 75%
a
b
Data from Weimann et al. (1999).
N/A, not analysed.
N/Ab
Present in 80%
Present in 34%
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
2.5. Otx1 controls cortical connecti6ity to subcortical
targets
Recent analysis of axonal projections in Otx1 − /−
mutants has shed new light on the role of Otx1 during
brain development (Weimann et al., 1999). In several
brain regions, connections usually develop by a biphasic mechanism in which an excess of early formed axon
projections is finely pruned by elimination of inappropriate axon terminals. Layer 5 neurons in the visual
cortex provide a good example of exuberance in connectivity, establishing, among others (e.g. to corpus
callosum), connections to a number of subcortical
targets such as the pons, superior and inferior colliculi,
and spinal cord. During early postnatal life, they selectively eliminate connections from the inferior colliculus
and spinal cord (Stanfield and O’Leary, 1985; Stanfield
et al., 1982), but the molecular mechanisms underlying
this remodelling are poorly understood.
Otx1 is strongly expressed in a subset of layer 5
neurons that form subcortical but not cortical connections (Weimann et al., 1999) and in layer 6 neurons,
many of which forming thalamic projections. Analysis
of Otx1−/− mutants reveals significant defects, specifically in the patterning of subcortical projections. In
fact, while callosal and thalamic projections appear
normal in the Otx1−/ − mutants, there are additional
extensive innervations of both the inferior colliculus
and the spinal cord that have been erroneously maintained. This phenotype suggests that Otx1 function is
required for the last step of subcortical axon development, in which exuberant connections undergo extensive refinement with the elimination of axon projections
from inappropriate targets (Weimann et al., 1999).
2.6. Otx1 transiently controls GH, FSH, and LH in
the pituitary
Otx1 is postnatally transcribed and translated in the
pituitary gland. Cell culture experiments indicate that
Otx1 may activate transcription of the growth hormone
(GH), follicle-stimulating hormone (b-FSH), luteinizing
hormone (b-LH), and a-glycoprotein subunit (a-GSU)
genes. Analysis of Otx1 null mice (Acampora et al.,
1998c) indicates that, at the prepubescent stage, they
exhibit transient dwarfism and hypogonadism due to
low levels of pituitary GH, FSH and LH hormones
which, in turn, dramatically affect downstream molecular and organ targets. Nevertheless, Otx1 −/ − mice
gradually recover from most of these abnormalities,
showing normal levels of pituitary hormones with restored growth and gonadal function at 4 months of age.
Expression patterns of related hypothalamic genes such
as the growth hormone releasing hormone (GRH),
gonadotropin releasing hormone (GnRH), and their
pituitary receptors (GRHR and GnRHR) suggest that,
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in Otx1− /− mice, hypothalamic and pituitary cells of
the somatotropic and gonadotropic lineages appear unaltered and that it is the ability to synthesise GH, FSH,
and LH, rather than the number of cells producing
these hormones, to be affected (Acampora et al.,
1998c).
An intriguing aspect of our observation is the fact
that transcription factors of the Ptx and Otx subfamilies recognise similar DNA target sequences (Simeone et al., 1993; Lamonerie et al., 1996; Szeto et al.,
1996; Tremblay et al., 1998), and that Ptx1 and Ptx2
are expressed in most pituitary lineages, in particular in
somatotrophic and gonadotrophic cells (Tremblay et
al., 1998). Ptx1 is the most highly expressed of these
genes, followed by Ptx2 and then Otx1 (Tremblay et
al., 1998). Yet, the Otx1 knock-out has a dramatic
effect only during the prepuberal period. The unique
activity of Otx1 during this period might reflect a
specific interaction of Otx1, but not of the related Ptx
factor(s), with a transcription co-regulator in the somatotrophic and gonadotrophic cells.
Taken together with previous reports, our observations support the existence of complex regulatory mechanisms defining combinatorial cell- and stage-specific
interactions between transcription factors belonging to
the same or to different gene families for the establishment/maintenance of pituitary function.
A novel feature of this phenotype is the fact that
most of the impaired functions are recovered by the
adult stage. Indeed, after the prepubescent stage, Otx1
mutant mice begin to gradually recover from their
abnormalities, showing at 4 months of age normal
levels of GH, FSH and LH which are paralleled by a
restored normal body weight, differentiation and size of
both testis and ovary, as confirmed also by their sexual
fertility, and by normal levels of downstream molecular
targets such as testosterone and insulin growth factor 1
(IGF1). Although we are unable to explain the mechanism underlying this recovery, this observation might
represent a possible example of temporal-restricted
competence in hormonal regulation of specific cell-lineages by the Otx1 transcription factor. This recovery
appears similar to the ‘catch-up growth’ (Boersma and
Wit, 1997) described in children with delayed growth
and puberty, also called ‘constitutional delay in growth
and abdolescence’, CDGA (Horner et al., 1978).
2.7. Otx1 is necessary for correct sense organ
de6elopment
Otx1− /− mutants show inner ear and eye abnormalities that are consistent with Otx1 expression pattern (Acampora et al., 1996).
Otx1 is expressed in the lateral canal and ampulla as
well as in a part of the utricle, in the saccule and
cochlea. Interestingly, Otx2 is co-expressed with Otx1
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D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
in the saccule and cochlea but not in the components of
the pars superior. Lack of Otx1 results in the absence
of the lateral semicircular canal and lateral ampulla, in
abnormal utriculosaccular and cochleosaccular ducts
and in a poorly defined hook (the proximal part) of the
cochlea (Acampora et al., 1996; Morsli et al., 1999).
Defects in the shape of the saccule and cochlea are
variable in Otx1 −/ − mice and are much more severe
in Otx1− /−; Otx2 +/ − background. In Otx1 − /−
and Otx1 −/−; Otx2 +/ − mutants the lateral crista
is absent and the maculae of the utricle and saccule are
partially fused (Morsli et al., 1999).
In the eye and annexed structures Otx1 transcripts
are restricted to the iris, ciliary process and ectodermal
cells migrating from the eyelid and included in the
mesenchymal component of the lachrymal glands.
These ectodermal cells are believed to induce differentiation of the mesenchymal cells into a glandular exocrine cell-type. In Otx1 −/ − mice the ciliary process
is absent, the iris is thinner and the lachrymal and
Harderian glands do not develop, failing the differentiation to a glandular cell-type. Interestingly, the ectodermal cells embedded within the mesenchymal
components are not identified in Otx1 −/ − mice, thus
indicating that failure in development of the glands is a
consequence of the impaired migration of the ectodermal cells from the eyelid to the mesenchyme of the
lachrymal primordium that in turn is not induced to
differentiate into the exocrine glandular phenotype
(Acampora et al., 1996).
3. Early requirement of Otx2 for normal gastrulation
and anterior neural plate induction and maintenance
3.1. Organisers and molecules in the current scenario of
the anterior brain patterning
Fate and patterning of tissues depend on the activity
of organiser cells emanating signals to a responding
tissue which undergoes morphogenetic changes resulting in a specific differentiated fate (Spemann and Mangold, 1924; Waddington, 1932; Gurdon, 1987).
Indeed, in amphibians, the dorsal lip of the blastopore induces a new, ectopic secondary axis when transplanted on the ventral side of a host embryo. Because
of this ability, the dorsal lip of the blastopore has been
called the organiser (Spemann and Mangold, 1924).
Further experiments in amphibian and chick embryos
have suggested that the age of the organiser tissue
influences the extension of the induced neural plate as
well as its regional identity. An organiser deriving from
an early gastrula induces anterior as well as posterior
neural tissue, whereas a late organiser induces only
posterior tissue (Gallera, 1971; Nieuwkoop et al., 1985;
Storey et al., 1992).
These results suggested the possibility that head and
trunk inducing ability of the organiser might be associated to different cell populations coexisting in an early
organiser. In mouse, at late streak stage the node is
located at the rostral end of the primitive streak and is
able to induce a secondary axis. Importantly, the secondary axis lacks any anterior neural tissues (Beddington, 1994) suggesting a functional parallelism with the
amphibian late organiser. Inducing properties of an
early mouse node have been tested in transplantation
experiments, and also in this case it fails to induce
anterior identity (Tam et al., 1997), thus indicating that,
unlike the amphibian organiser, the mouse node is
unable to induce rostral neuroectoderm, independently
of its age.
The mouse node gives rise to the ame that at mid-late
streak stage underlies the anterior developing neuroectoderm in the midline, and comprises mesoderm
(prechordal plate and notochord) and the definitive gut
endoderm. These node-derived tissues are similar to
those originated by the amphibian organiser and express similar genes (Beddington, 1981; Lawson et al.,
1991). Moreover, there is clear evidence that the murine
node and its derivatives are able both to emanate
neuralising signals (Ruiz i Altaba, 1993, 1994; Beddington and Robertson, 1999) with patterning properties of
different A/P value (Foley et al., 1997; Shimamura and
Rubenstein, 1997) and to regulate the expression of the
region-specific neural genes En1 and Otx2 in the neuroectoderm (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
streak stage, a crucial role in specifying anterior neural
patterning in mouse (reviewed in Beddington and
Robertson, 1998, 1999). Expression analysis and cell
lineage studies remarkably contributed to define the
origin and the molecular identity of the AVE.
AVE precursor cells are located at the distal tip of
the mouse pre-streak embryo and can be visualised by
the expression of the Hex gene (Thomas et al., 1998).
Few hours later, descendants of this group of cells will
be found only in the perspective anterior region, giving
rise to the first (molecular) asymmetry along the presumptive A/P axis of the embryo.
This A/P asymmetry is confirmed by the analysis of
the expression patterns of a number of genes such as
Hex, Hesx1, goosecoid (gsc), cerberus-like (cer-l),
Lim1, Otx2 and nodal (Simeone et al., 1993; Thomas
and Beddington, 1996; Thomas et al., 1998; Belo et al.,
1997; Varlet et al., 1997; Dattani et al., 1998).
Cell ablation and tissue recombination experiments
have indicated the functional relevance of AVE in
inducing anterior patterning. Indeed, it has been shown
that, (i) the removal of a patch of AVE cells expressing
the Hesx1 gene prevents the subsequent expression of
this gene in the rostral neuroectoderm which becomes
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
reduced and abnormally patterned (Thomas and Beddington, 1996); (ii) recombination of chick epiblast with
rabbit pre-streak AVE induces the expression of forebrain markers, while chick hypoblast is unable to elicit
the same response, thus suggesting that chick hypoblast
and murine AVE might not share the property of
specifying anterior neural patterning (Knötgen et al.,
1999); (iii) chimera and transplantation experiments as
well as the analysis of mouse mutants have indicated
that a number of genes, including Lim1, Otx2 and
nodal are required in the AVE for proper specification
of the anterior neuroectoderm (Acampora et al., 1995;
Shawlot and Behringer, 1995; Ang et al., 1996; Varlet et
al., 1997), and in the ame for maintenance and refinement of correct identities (Acampora et al., 1998b;
Rhinn et al., 1998; Shawlot et al., 1999); (iv) embryos
homozygous for a null mutation in Cripto show expression, even if abnormal, of anterior neural markers in
the absence of node-derived mesendoderm (Ding et al.,
1998). This gene encodes a membrane-associated
protein containing epidermal growth factor-like motifs
and is expressed shortly before the onset of gastrulation
in the proximal region of the epiblast where the primitive streak forms (Dono et al., 1993; Ding et al., 1998;
Minchiotti et al., 2000). Cripto −/ − embryos lack a
primitive streak and do not anteriorize the AVE, thus
failing to convert the proximo-distal into A/P axis
(Ding et al., 1998). Nevertheless, the AVE markers are
correctly expressed, although in a distal position and
the epiblast does express rostral neural markers such as
Otx2 and En until 8 – 8.5 d.p.c., thus indicating that in
the absence of a node and derived tissues the epiblast is
able to acquire the identity of anterior neuroectoderm.
Together with the ectopic transpantations of the
node (Beddington, 1994; Tam et al., 1997), these results
point to the possibility that in the mouse the AVE
contains genetic information required to instruct the
patterning of the rostral neuroectoderm and, hence,
there might be two physically separated organising
centres, AVE and node, responsible for the induction of
head and trunk structures, respectively.
This idea has been re-considered in the light of new
mouse models recently generated. In particular,
Chordin−/−; Noggin −/ − (Chd −/ − ; Nog − /−)
double mutants have provided evidence that they are
crucial in early forebrain development. These genes are
expressed in the mouse node and its derivatives but not
in the AVE, and single null mutation of either of them
does not affect anterior neural patterning. However,
Chd/Nog double mutants display severe defects in the
development of prosencephalon (Bachiller et al., 2000).
Analysis at the early stages of the mutant embryos has
demonstrated that the AVE is correctly established,
thus suggesting that either the ame cells have their own
head-inducing properties which are affected in these
mutants, or are required for the early maintenance of
75
anterior patterning initiated by the AVE, which per se
might be necessary, but not sufficient, to induce the
anterior neural plate.
Early patterning of the CNS primordium is also
controlled by additional mechanisms involving planar
signals acting through the neuroectodermal plane (Doniach, 1993; Ruiz i Altaba, 1993, 1994). Furthermore, it
has been shown that in zebrafish a small group of
ectodermal cells located in the anteriormost head region
is required for the patterning and survival of the anterior brain (Houart et al., 1998; Ruiz i Altaba, 1998).
Finally, heterotopic transplantation studies have indicated that it is only from the synergistic interaction of
anterior germinal layers (AVE and anterior epiblast)
and a fragment of the posterior epiblast (early node) of
early-streak embryos that it is possible to induce a full
neural axis including anterior neural gene expression
(Tam and Steiner, 1999). Similar experiments involving
removal and ectopic transplantation of anterior midline
tissue (AML, which includes both ame and ventral
neuroectoderm) from late-streak embryos, have shown
that AML is required for maintenance and regionalization of the forebrain (Camus et al., 2000).
Among the genes required in the early specification
of the anterior neural plate, Otx2 plays a remarkable
role in both induction and maintenance of the rostral
neural patterning (Simeone, 1998; Acampora and Simeone, 1999). These and other potential roles are now
subject of intense study that takes advantage from
genetically modified mouse models and embryological
approaches.
3.2. Specification of anterior patterning requires Otx2
function
In mouse, Otx2 is already transcribed before the
onset of gastrulation in the epiblast and in the visceral
endoderm (VE), and at the end of gastrulation in the
ame and rostral neural plate (Simeone et al., 1992,
1993; Ang et al., 1994). During brain regionalization,
Otx2 shows expression domains largely overlapping
with those of Otx1, with a posterior border coincident
with the mesencephalic side of the isthmic constriction
(Simeone et al., 1992; Millet et al., 1996).
Therefore, during gastrulation Otx2 is transcribed
and translated in the cells that are believed to emanate
signals in early specification and patterning of the neural plate (AVE and ame) as well as in those responding
to these instructing signals (epiblast and anterior neuroectoderm) (reviewed in Simeone, 1998; Acampora
and Simeone, 1999, Fig. 1A). The first indication that
Otx2 is responsive to inductive interactions comes from
explant-recombination experiments in gastrulating
mouse embryos showing that a positive signal from
ame of headfold stage embryos is able to maintain
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
streak embryos, and that a negative signal from the
posterior mesendoderm, mimicked by exogenous
retinoic acid (RA), represses Otx2 expression in the
anterior ectoderm of late streak embryos (Ang et al.,
1994). Similar interactions have been also demonstrated
in Xenopus (Blitz and Cho, 1995).
The possibility that RA might contribute to the early
distinction between fore-midbrain and hindbrain by
controlling Otx2 expression is supported by the finding
that administration of exogenous RA at mid-late streak
stage represses Otx2 expression in both the ame and the
posterior neural plate (Ang et al., 1994; Simeone et al.,
1995; Avantaggiato et al., 1996). This repression correlates with the appearance of microcephalic embryos
that show early anteriorization of Hoxb1 expression,
hindbrain expansion (Sive and Cheng, 1991; Conlon
and Rossant, 1992; Marshall et al., 1992), loss of
forebrain molecular and morphological landmarks, and
gain of midbrain molecular markers in the rostralmost
neuroectoderm (Simeone et al., 1995; Avantaggiato et
al., 1996). Moreover, Otx2 responsiveness to RA application is a common feature in different species including Xenopus and chick (Bally-Cuif et al., 1995; Pannese
et al., 1995). Nevertheless, the question of whether
endogenous RA plays a physiological role in rostral
CNS demarcation by contributing to the establishment
of the posterior border of Otx2 expression, still remains
open.
The evidence that Otx2 may play a remarkable role
in rostral CNS specification derives from in vivo genetic
manipulation experiments performed in mouse and
Xenopus which, to some extent, complement each other.
In Xenopus, microinjection of synthetic Otx2 RNA
results into an abnormal reduction in size of tail and
trunk structures, and in the appearance of a second
cement gland (Blitz and Cho, 1995; Pannese et al.,
1995). These phenotypes have been interpreted either
with a possible Otx2-mediated interference with move-
77
ments of extension and convergence during gastrulation
(for trunk and tail reduction) and/or with an Otx2-requirement in the specification of anteriormost head
structures (for the ectopic cement gland). Moreover, by
using a dexamethasone-inducible OTX 2 protein it has
been shown that the Xenopus Otx2 activity is regulated
by regionally restricted factor(s), and that the cement
gland-specific gene XCG may represent a direct target
of the Otx2 gene product (Gammill and Sive, 1997).
In mouse, Otx2 null embryos die early in embryogenesis, lack the rostral neuroectoderm fated to become
forebrain, midbrain and rostral hindbrain, and show
heavy abnormalities in their body plan (Fig. 1E)
(Acampora et al., 1995; Matsuo et al., 1995; Ang et al.,
1996). Heterozygous Otx2+ /− embryos into an appropriate genetic background show defects of the head
such as serious brain abnormalities and craniofacial
malformations, which are reminiscent of otocephalic
phenotypes (Matsuo et al., 1995).
The analysis of Otx2 null embryos revealed that at
late streak stage, the rostral neuroectoderm is not specified and the primitive streak as well as the node and
the ame are severely impaired. Therefore, one possibility is that the resulting headless phenotype might be
due to the abnormal development of node-derived ame
which lacks head organiser activity. A second possibility is that neural inducing properties of the AVE are
severely impaired or abolished. In embryos replacing
Otx2 with the lacZ reporter gene, the first abnormality
is already detected at the early streak stage (Acampora
et al., 1995). Indeed, at this stage, lacZ transcription
and staining are abolished in the epiblast while remaining high in the VE of Otx2− /− embryos (Fig. 1D);
the gsc expression is undetectable or confined to the
proximal region of the mutant embryos (Acampora et
al., 1995); and the presumptive AVE does not anteriorize, thus remaining confined to the distal region of
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, Otx2 is
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.
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D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
Therefore, these results indicate that headless phenotype and abnormal organisation of the primitive streak
may be determined very early at the pre-early-streak
stage by an impairment of AVE properties.
Moreover, as revealed by the mesodermal early marker
Brachyury (T), epiblast cells do not migrate posteriorly
at the site of the primitive streak formation but remain
for an abnormally longer period in a ring close to
embryonic-extraembryonic boundary. Only later, and in
a small number of Otx2 −/ − embryos, the presumptive
AVE cells appear anteriorized. However, although abnormal, a primitive streak forms in all the Otx2 − /−
embryos and mesodermal cells appear to be concentrated
at the posterior third of the embryo (Acampora et al.,
1995, 1998b; Ang et al., 1996).
These findings, therefore, favour a relationship between primitive streak formation and anterior location
of the AVE and suggest that Otx2, normally expressed
in both epiblast cells and AVE, might be required in these
two cell types in order to mediate proper positioning of
the AVE and normal formation of the primitive streak.
However, data deduced from the analysis of chimeric
embryos and further mouse models indicate that early
abnormalities in both AVE and primitive streak of
Otx2 −/− embryos should be ascribed to Otx2 requirement in the VE (Acampora et al., 1998b; Rhinn et al.,
1998; see below).
Interestingly, an intriguing feature of Otx2 − /−
embryos is that lack of anterior neuroectoderm is paralleled by failure of epiblast cells to express the lacZ
reporter gene.
Thus, since Otx2 is already transcribed from the
earliest stages (unfertilised egg in Xenopus and at least
morula in mouse), this observation suggests that maintenance of Otx2 transcription in the epiblast cells requires
at least one normal allele expressed in the AVE while
Otx2 transcription in the latter is independent of the
presence of a normal allele (Simeone, 1998; Acampora
and Simeone, 1999).
These findings also support the existence of Otx2-mediated signal(s) emitted from the AVE and directed to the
epiblast cells. Nevertheless, it is still unclear whether the
same signal operates to mediate both specification of
anterior patterning and Otx2 transcription in epiblast
cells or these two events are independently controlled.
3.3. The role of Otx2 in the induction and maintenance
of rostral CNS identities
In order to understand the role of Otx2 in the
specification of the anterior patterning, it is of particular
relevance to define its functional contribution to the
different tissues where it is expressed during gastrulation.
A direct evidence proving a role for Otx2 in the AVE
has been recently provided by generating murine
chimeric embryos and new mouse models (Acampora et
al., 1998b; Rhinn et al., 1998; Acampora et al., unpublished results). Chimeric embryos containing Otx2−/−
epiblast and wild-type VE rescue an early Otx2−/−
neural plate but subsequently fail to develop a brain,
suggesting that Otx2 is firstly required in the AVE for
induction of rostral neural plate and, then, in the
epiblast-derived tissues for specification of forebrain and
midbrain regional identities. Conversely, when chimeric
embryos consist of an Otx2− /− VE and an Otx2+/+
epiblast, none of the phenotypic features of Otx2−/−
embryos are rescued (Rhinn et al., 1998). This latter
result also argues, as previously suggested by Otx2−/−
mice (Acampora et al., 1995), that impaired ame of
Otx2− /− embryos is a consequence of Otx2 requirement at earlier stages in the VE.
Mice replacing Otx2 with Otx1 were originally generated in order to assess whether the two proteins shared
functional equivalence or, alternatively, displayed unique
properties specified by their limited amino acid divergence. Murine OTX 1 and OTX 2 gene products, in fact,
share extensive sequence similarities even though in
OTX1, downstream of the homeodomain, these regions
of homology to OTX2 are separated by stretches of
additional amino acids including repetitions of alanine
and histidine residues (Simeone et al., 1993).
Interestingly, homozygous mutant embryos replacing
Otx2 with the human Otx1 (hOtx1) cDNA (hOtx12/
hOtx12) recover anterior neural plate induction and
normal gastrulation but show a headless phenotype from
9 d.p.c. onwards (Fig. 1F –H). A combined analysis of
both hOtx1 RNA and protein distribution during early
gastrulation has revealed that while the hOtx1 mRNA
is detected in the VE and epiblast, the hOTX1 protein
is revealed only in VE (Fig. 1F). Nevertheless, this
VE-restricted translation of the hOtx1 mRNA is sufficient to recover early anterior neural plate but fails to
maintain fore-midbrain identities (Fig. 1G), and, consequently, hOtx12/hOtx12 embryos display a headless
phenotype with a normal body plan (Fig. 1H) (Acampora et al., 1998b).
In fact, at early-streak stage, hOtx12/hOtx12 embryos
recover anteriorization of the AVE, normal primitive
streak and proper expression of Lim1, cer-l, Hesx1, gsc
and T. At late gastrula stage, the anterior patterning of
the rostral neural plate is correctly defined by the
expression of fore-, mid- and hindbrain markers such as
Six3, Pax2, Gbx2 and Hoxb1 and the ame properly
expresses Lim1, Noggin, cer-l and Hesx1. These findings
support the idea, as indicated by chimeric studies (Rhinn
et al., 1998) that Otx2 is required in the AVE for
initiating the specification of the anterior patterning and
demarcation of forebrain and midbrain territories
(Acampora et al., 1998b).
Further analysis of hOtx12/hOtx12 embryos reveals
that at the early somite stage the forebrain markers
BF1 and the hOtx12 mRNA are not detected in the
rostral neuroectoderm where mid-hindbrain markers
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
such as Wnt1, En1, Fgf8, Gbx2 and Pax2 appear to be
co-expressed.
These observations lead to argue that the OTX2 gene
product is required from the headfold stage onwards to
maintain the anterior patterning previously established
by the AVE. In this respect, the phenotype observed at
the early somite stage appears to be the consequence of
an A/P repatterning process involving the entire anterior neural plate (fore-midbrain) which, in the absence
of any OTX gene product, adopts a more posterior fate
(hindbrain) (Acampora et al., 1998b; Acampora and
Simeone, 1999). Therefore, the hOtx12/hOtx12 mouse
model allows to uncouple two distinct phases both
requiring Otx2 early induction of anterior neural patterning that is under the control of AVE and its subsequent maintenance that is likely mediated by
epiblast-derived cells (the ame and neuroectoderm).
However, since Otx2 is normally transcribed and
translated in both ame and rostral neuroectoderm,
while the hOtx12 mRNA is transcribed but not translated either in the ame or in the rostral neuroectoderm,
it cannot be deduced from this model whether Otx2 is
required in one or both of these tissues to mediate
maintenance of the anterior identity and whether hOtx1
is functionally equivalent to Otx2 also in these tissues.
A cell-autonomous role of Otx2 in the neuroectoderm
has emerged from the analysis of chimeras with a
moderate contribution of Otx2 −/ − cells (Rhinn et
al., 1999). It has been shown that genes such as Wnt1,
Hesx1 and R-cadherin are selectively, not expressed in
Otx2 − /− cells whereas other genes such as En2 and
Six3 are uniformly expressed in neuroectodermal cells
of both genotypes.
How Otx2 is integrated in the network of other
genetic functions which have been shown to be fundamental for anterior neural patterning is still an open
question. The overall phenotype of Lim1 −/ − and
hOtx12/hOtx12 embryos, for example, show impressive
phenotypic similarities. Recently, the stage and the cell
type in which Lim1 is required for head specification
have been approached by chimera and tissue recombination experiments (Shawlot et al., 1999). These experiments have revealed that Lim1 is required in the AVE
for inducing anterior neural patterning and, subsequently, in the ame to refine and maintain the anterior
identity. Therefore, since maintenance of anterior pattern requires Otx2 in the ame or in the anterior neuroectoderm or in both tissues, it can be speculated that
Lim1 might contribute to mediating the release of ame
signal(s) instructing maintenance of anterior character
and Otx2 might confer to neuroectoderm the competence to respond to this signal(s) emitted from the
mesendoderm. This possibility is also compatible with a
cell-autonomous role within the neuroectoderm and
does not exclude an additional role also in the ame.
79
A recent in vitro study supports the possibility of a
direct protein –protein interaction between OTX 2 Cterminus and Lim1 homeodomain (Nakano et al.,
2000).
3.4. AVE-restricted OTX2 function is necessary for
proper expression of Lim1 and bone morphogenetic
protein (BMP) antagonists in the ame
In hOtx12/hOtx12 (Acampora et al., 1998b) and
chimera experiments (Rhinn et al., 1998), a tight correlation exists between correct specification of anterior
patterning and the recovery of normal gastrulation.
Moreover, in Otx2− /− embryos failure of anterior
specification always coincides with a distal location of
the AVE and abnormal gastrulation. In particular, in
these embryos ame is no longer identifiable, and expression of Nog, Chd and Lim1 is strongly abnormal or
absent (Acampora et al., 1998b; Rhinn et al., 1998). In
hOtx12/hOtx12, general morphology as well as expression of these genes in the node and ame appears to be
normal, and additionally, cer-l expression in the definitive endoderm emerging from the node also appears to
be unaffected.
As previously mentioned, recent studies have shown
that (i) anterior midline tissue including ame and ventral neuroectoderm is required for maintenance and
regionalization of forebrain (Camus et al., 2000); (ii)
BMP antagonists such as Nog and Chd are required in
a temporally coherent manner in the node and ame for
forebrain development (Bachiller et al., 2000); and (iii)
Lim1 is required in the ame for stabilising anterior
patterning (Shawlot et al., 1999). On this basis, it can
be hypothesised that anterior specification by AVE-restricted OTX function might act through ame-restricted
non-cell autonomous properties of Chd, Nog and Lim1.
This hypothesis does not exclude the existence of a
direct inducing signal for anterior patterning emanating
from the AVE. Rather it indicates that the VE is also
important for normal development of the primitive
streak (Beddington and Robertson, 1999). In this respect, it is noteworthy that chimera experiments performed to assess the role of HNF3b have indicated that
this gene is necessary in the VE to promote proper
primitive streak elongation (Dufort et al., 1998).
Since, contrary to OTX2, hOTX1 protein is not
detected in the ame, it cannot be excluded a priori that
OTX2 also plays a role in these cells. Noteworthy is
also the fact that in the absence of any OTX protein,
hOtx12/hOtx12 embryos exhibited anterior patterning
at least until 7.75 –8 d.p.c., while they subsequently fail
in proper positioning of the IsO at the MHB. Chd −/
− ; Nog − /− embryos lacked anterior brain structures
and retained a relatively normal midbrain (Bachiller et
al., 2000). Together, these findings suggest that while
early forebrain maintenance and/or induction is under
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D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
the control of Chd/Nog signalling in the ame, OTX 2 is
required for late maintenance by specifying midbrain
territory and allowing the correct positioning of the
IsO.
3.5. Specific and interchangeable roles between Otx1
and Otx2
Mutant phenotypes, expression patterns (see above)
and amino acid sequence comparison support two possibilities, (i) the functional properties of Otx1 and Otx2
largely overlap and differences in their temporal and
spatial transcriptional control are responsible for the
highly divergent phenotypes of Otx1 −/ − and
Otx2− /− mice; or (ii) OTX1 and OTX2 gene products display unique functional properties specified by
their limited amino acid divergence. In order to distinguish between these two possibilities, mice models that
replace Otx1 with the human Otx2 (hOtx2) cDNA and
vice versa were generated.
Homozygous mice in which Otx1 is replaced with the
human Otx2 cDNA (hOtx21/hOtx21), despite a reduced expression of the transgenic alleles, no longer
show epilepsy and corticogenic abnormalities caused by
the absence of Otx1. This rescue is due to a restored
normal cell proliferation activity in the dorsal neuroepithelium, as revealed by BrdU labelling experiments at
9.5 d.p.c. (Acampora et al., 1999a). This is particularly
relevant when considering the different expression patterns of Otx1 and Otx2 genes in the dorsal telencephalon from the 9.5 d.p.c. stage onwards. Indeed, at
this stage, while Otx1 is expressed throughout the
entire dorsal telencephalon, Otx2 is expressed only in
the mediodorsal area and basal neuroepithelium,
whereas it disappears completely from the mediodorsal
area by 11 d.p.c. Therefore, the rescue observed in
hOtx21/hOtx21 mice suggests that Otx1 and Otx2 have
interchangeable roles in the cortex. To complement the
information derived by the loss-of-function and gene
replacement experiments, it would be particularly interesting to see also the effects of an OTX over-dosage on
the brain and, specifically, on cortical development.
hOtx21/hOtx21 mice also show a significant improvement of mesencephalon, eye and lachrymal gland defects. Some of the Otx1 −/ − inner ear abnormalities
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,
homozygous mutant mice in which Otx2 has been
replaced with the human Otx1 (hOtx1) cDNA (hOtx12/
hOtx12) recover the anterior neural plate and proper
gastrulation but fail to maintain fore-midbrain identities, displaying a headless phenotype from 9 d.p.c.
onwards (Acampora et al., 1998b). A deeper analysis
has revealed that despite RNA distribution in both the
VE and epiblast, the hOTX1 protein was synthesised
only in the VE, where it was sufficient to rescue VE-restricted Otx2 functions.
In sum, these mouse models support an extended
functional equivalence between OTX1 and OTX2
proteins and provide evidence that Otx1−/−;
Otx2− /− contrasting phenotypes stem from differences in expression patterns rather than in amino acid
sequences.
The clearest exception to the overall Otx functional
equivalence is provided by the lateral semicircular canal
of the inner ear, that in hOtx21/hOtx21 mice is never
restored (Morsli et al., 1999) as well as in mice in which
Otx1 is replaced with the Drosophila otd gene (Acampora et al., 1998a). These findings, discussed below in
evolutionary terms, suggest that the ability to specify
the lateral semicircular canal of the inner ear may be,
indeed, an Otx1-specific property (Acampora and
Simeone, 1999).
However, it is noteworthy that these studies do not
address the question of whether hOTX1 might be functionally equivalent to OTX2 in the embryonic neuroectoderm and ame, due to the lack of hOTX1 protein
in these tissues. Genetic replacement studies carried out
by Suda et al. (1999) provide some evidence that OTX1
might be able to partially rescue the headless phenotype
that was observed in hOtx12/hOtx12 mutant animals.
However, it is unclear whether the amount of OTX1
was similar to that of OTX2 in these experiments (Suda
et al., 1999). This is an important aspect since a reduction of OTX gene products below a critical threshold is
always reflected in a mis-specification of fore- and
midbrain regions (Acampora et al., 1997, 1998b; Suda
et al., 1997; Broccoli et al., 1999; Millet et al., 1999;
Simeone, 2000). In this context, it has been recently
discovered that different roles attributed to two Hox
genes belonging to the same paralogous group are the
result of quantitative modulations in gene expression
rather than of qualitative properties depending on
protein sequence (Duboule, 2000; Greer et al., 2000).
Thus, even if OTX1 and OTX2 do share a certain
degree of functional equivalence in the neuroectoderm,
the limited partial recovery of the phenotype might be
ascribed either to a reduced level of OTX1 protein or to
an OTX2-specific function. Further investigation is
needed to address this important evolutionary aspect.
4. Brain patterning depends on a critical Otx gene
dosage
4.1. Morphogenetic origin of the IsO
The initial patterning of the primitive neuroepithelium is established during early gastrulation, when signals originating from both the underlying mesoderm
and the VE or present within the ectoderm itself,
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
roughly divide the future CNS into few main subdivisions (Rubenstein and Shimamura, 1997; Beddington
and Robertson, 1998). At the end of this process,
different regions have acquired their own identity expressing a unique set of transcription factors. Later on,
potent signalling centres are generated at the boundary
between adjacent territories, which express diffusible
molecules that refine and polarise the neighbouring
neural tissue (Meinhardt, 1983; Rubenstein et al.,
1998). The best studied centre is located at the MHB in
the isthmic region. Over the last years, elegant tissue
transplantation experiments in chick have addressed its
role in neural patterning (Martinez et al., 1991, 1995;
Marin and Puelles, 1994). In fact, when isthmic tissue is
grafted to ectopic sites in the brain, as the caudal
prosomeres or some rhombomeres, it can not only
maintain its own identity, but can also induce the
surrounding tissue to change its programmed fate giving rise to midbrain and cerebellar structures. Due to
this potent non cell-autonomous function, the MHB
signalling centre has been re-named IsO. However,
within the forebrain, the territory rostral to the ZLI,
the boundary between prosomeres 2 and 3, is not
competent to respond to midbrain inducing signals
released by the IsO (Martinez et al., 1991). In the last
years, different proteins specifically expressed in the
isthmic region and required for its development have
been characterised. They include the transcription factors EN1, EN2, PAX2, PAX5 and two secreted
molecules, WNT1 and FGF8 (Rowitch and McMahon,
1995; Joyner, 1996). Although some of these factors
like EN2 and PAX5 appear to be general markers of
the region, the dynamic expression profiles of the others
(EN1, PAX2, WNT1 and FGF8) parallel the progressive refinement of specification taking place within the
midbrain-hindbrain domain. In fact, if their expression
domains are initially broad encompassing the presumptive midbrain and rostral hindbrain (in 1 – 5 somite old
embryo), later on they become progressively restricted
to narrow transverse rings that encircle the neural tube
in the isthmic area (Bally-Cuif and Wassef, 1995). In
particular, Wnt1 ring of expression is almost completely overlapped and localised at the caudal end of
the prospective midbrain rostral-adjacent to the Fgf8
positive ring that lies on the isthmic-cerebellum anlage
(Hidalgo-Sanchez et al., 1999b, Fig. 2). The functional
role of all these genes (En, Pax, Fgf8 and Wnt1) has
been tested in great detail in different animal models,
clearly showing their requirement for the normal development of this region. In fact, mice homozygous for a
Wnt1 null allele completely lack all the midbrain and
isthmic derivatives (McMahon and Bradley, 1990;
Thomas and Capecchi, 1990). Mutations in En1 cause a
similar but milder phenotype (Wurst et al., 1994),
whereas, mutations in En2 only lead to subtle defects in
cerebellum cyto-organisation (Joyner et al., 1991).
81
However, En compound mutant mice lack the entire
region resembling the Wnt1 mutant phenotype (W.
Wurst and A.L. Joyner, unpublished results) and En2
can rescue the En1 mutant brain defects when it is
expressed in place of En1, revealing overlapping functions between the two genes (Hanks et al., 1995). A
similar brain deletion has been found also in mice
carrying both Pax2 and Pax5 null alleles (Schwarz et
al., 1997; Urbanek et al., 1997) and in the zebrafish noi
mutants that harbour an induced mutation in the Pax2
orthologue (Brand et al., 1996; Lun and Brand, 1998).
Likewise, Fgf8 mutant mice and zebrafish display an
early brain deletion due to degeneration of the midbrain and isthmic tissue (Meyers et al., 1998; Reifers et
al., 1998). In each mutant outlined so far, it has been
reported that the expression of the other genes described is correctly initiated but no longer maintained.
This is probably the cause that leads to the described
loss of the brain structures (McMahon et al., 1992;
Reifers et al., 1998; V. Blanquet and W. Wurst, unpublished results). These important findings strongly suggest a close functional connection among these genes,
where the inactivation of a gene is able to feedback
negatively on the transcription of all the other genes,
leading to the degeneration of brain structures. The
genetic maintenance loop among these genes is the
molecular origin of the IsO and subsequent midbrain –
hindbrain specification and morphological shaping
(Fig. 3). This hypothesis is also supported by the striking phenotypes obtained in gain of function experiments. When, in fact, acrylic beads soaked with
recombinant FGF8b protein are implanted in different
areas of the caudal forebrain, the nearby regions are
induced to change their fate into midbrain, cerebellar
and isthmic structures (Crossley et al., 1996; Irving and
Mason, 1999; Martinez et al., 1999). These results are
mediated by the de-novo induction of Wnt1, Pax2,
En2, En1 in the site of bead implantation. The same
phenotype is obtained by inducing En gene expression
ectopically in the caudal prosomeres either in zebrafish
(Ristoratore et al., 1999) or chick (Shamim et al., 1999).
Thus, single genes can trigger the activation of the
entire genetic loop formed by Wnt, Fgf, Pax and En
genes to initiate the neural fate re-specification.
4.2. Otx genes are required for correct positioning of
the isthmus
An important question to be answered is how all
these genes are induced in this particular region of the
developing neural tissue. Why not more anteriorly in
the forebrain or more caudally in the spinal cord?
Grafting or tissue ablation experiments in chick have
shown that Fgf8 expressing tissue with properties of the
IsO is generated when midbrain and rhombomere 1
tissue are juxtaposed but not when midbrain contacts
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D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
Fig. 2. A schematic gene expression network in mice with different Otx2, Gbx2 or Fgf8 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 Fgf8 and
Gbx2 activity in the same region, that positions the MHB more caudally (En1Otx2/ + ). In Gbx2 mutant embryos (Gbx2 −/ −), Otx2 and Wnt1
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
Fig. 3. Genetic hierarchy determining MHB formation. In the induction phase (7.5– 8.5 d.p.c.), Fgf8 expression is induced where the
Otx2 and Gbx2 expression domains face each other. Recently, several
reports have been shown that Fgf8 is activated when mesencephalic
and metencephalic fated tissues are placed in contact. This secreted
molecule is able to activate several transciption factors like PAX2,
PAX5, EN1, EN2 and others that are necessary for the future
specification and patterning of the region. All these molecules seem to
maintain each other into an auto-regulatory loop. Moreover, FGF8
can re-induce Gbx2 expression, and inhibit Otx2 activity.
any other rh or neural tissue (Hidalgo-Sanchez et al.,
1999a; Irving and Mason, 1999). The genetic identity of
the tissue rostral and adjacent to the Fgf8 expression
domain is depending on Otx1 and Otx2 transcription
factors (Simeone et al., 1993; Simeone, 1998). As previously mentioned, Otx2 −/ − embryos fail to specify
forebrain, midbrain, and anterior hindbrain. The last
region is specified by the IsO and never expresses Otx2
(Acampora et al. 1995; Matsuo et al., 1995; Ang et al.,
1996). This finding argues in favour of an involvement
of the Otx genes in the early IsO generation. Moreover,
a more direct involvement of the Otx function in the
anterior neuroepithelium specification is provided by
the phenotype analysis of Otx compound mutants carrying only one Otx2 functional allele. In fact, while
Otx1 −/− ; Otx2+/ − embryos do not display
mesendoderm and gastrulation defects, they show
molecular and morphological transformation of the
caudal prosomeres 1 – 2 and mesencephalon into a strikingly enlarged metencephalon (Acampora et al., 1997).
This brain re-patterning is the primary consequence of
an abnormal enlargement of the Fgf8 expression do-
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.,
1999) that describes a strong repression of Otx2 expression in the chick mesencephalon around the place
where a FGF8 secreting source is implanted. Moreover,
the ectopic expression of the Fgf8b isoform under the
control of the Wnt1 regulatory regions, inhibits the
endogenous Otx2 expression in the mesencephalic anlage and tranforms this area in hindbrain-fated tissue
(Liu et al., 1999; Fig. 2). An even stronger re-location
of the IsO in the neuroepithelium is present in the
homozygous hOtx12/hOtx12 embryos described previously (Acampora et al., 1998b). In this mouse model,
where no OTX protein is present in epiblast derivatives,
embryos normally undergo specification of the anterior
neural plate which, however, fails to maintain its identity and acquires a more posterior fate, yielding a
headless phenotype at late embryogenesis as assessed by
the rostral mislocalisation at the rostral tip of the
neuroectoderm of all the genes normally present in the
IsO and rostral hindbrain, like Wnt1, Fgf8, Pax2, En1
and Gbx2, (Acampora et al., 1998b; Fig. 2). This
finding provides a strong evidence for an Otx gene
dosage requirement in anterior neural fate determination and following maintenance throughout a continued repression on the posteriorizing genetic
determinants (Simeone, 1998). Is the action played by
Otx genes only necessary or even sufficient to induce
anterior fate in the neural plate? Gain of function
experiments have been carried out, in the last years, to
answer this question. As already mentioned, over-expression of Otx2 RNA in Xenopus results in an abnormal expansion of the head coupled with a strong
reduction of the tail and trunk and with ectopic forma-
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D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
tion of the most anterior structure of the Xenopus
head, that is, the cement gland (Blitz and Cho, 1995;
Pannese et al., 1995).
To get a more specific and controlled Otx2 ectopic
expression inside the neuroepithelium a knock-in approach has been devised for targeting the Otx2 coding region into the En1 genomic region (Broccoli et
al., 1999). Therefore, the exogenous Otx2 allele becomes expressed ectopically in the rostral metencephalon where En1 expression domain is normally
found, thus encompassing the isthmic region (Wassef
and Joyner, 1997). The mice carrying this newly-engineered Otx2 allele (En1Otx2/ + ) display a striking alteration in the ratio between mesencephalic and
cerebellar tissue. In fact, while the rectum is enlarged
up to one-third, affecting in particular the inferior
colliculi, the cerebellum loses almost completely its
medial structure, called the vermis. Because of this
brain rearrangement, the mice develop a strong ataxia
during the early postnatal life, due to the compromised cerebellar functions (Broccoli et al., 1999). This
malformation, already detectable in 10.5 d.p.c. embryos, is the final morphological outcome of a backward expansion of mesencephalic tissue at the
expenses of the cerebellum. This repatterning process
is driven by the knocked-in Otx2 allele that activates
anteriorly expressed genes like En1, Wnt1 and
EphrinA5 in its ectopic expression domain, whereas
hindbrain molecular determinants such as Fgf8 and
Gbx2 result repressed parallely. As a consequence, the
IsO is shifted slightly caudally (Broccoli et al., 1999;
Fig. 2). How far from its normal position can the IsO
be shifted and up to what level of the A/P axis is
neural tissue competent to respond to an Otx2 ectopic expression are the questions to be answered in
the near future. While Otx2 seems to play a key role
in determining the anterior fate of the neural plate,
another homeobox gene, named Gbx2, has been individuated as the major molecular determinant in conferring the metencephalic identity (Bouillet et al. 1995;
Chapman and Rathjen, 1995; von Bubnoff et al. 1995;
Wassarmann et al., 1997). Gbx2 is expressed at very
early stages of neuraxis development starting at 7.5
d.p.c., where it is expressed in all three germ layers of
the gastrulating embryo in a region that extends rostrally from the posterior end of the embryo into the
prospective hindbrain. Interestingly, its anterior expression boundary moves forward as the Otx2 expression domain retracts progressively into the perspective
fore-midbrain territories (Conlon, 1995). This mutually excluding expression of these genes suggests a
repression mechanism, either direct or indirect, between OTX 2 and GBX2 gene products. By 9.5 d.p.c.,
Gbx2 RNA is confined to a sharp transverse ring that
is caudal immediately to the midbrain, and co-localises with Fgf8 and Pax2 expression domains. At
this stage, Gbx2 expression is also detected in two
longitudinal columns in the hindbrain and spinal cord
(Bulfone et al., 1993; Wassarmann et al., 1997;
Shamim and Mason, 1998; Hidalgo-Sanchez et al.,
1999b). Its pivotal role in determining the rostral
hindbrain neural identity has been assessed by gene
targeting inactivation experiments (Wassarmann et al.,
1997). In fact, Gbx2 null mutants die perinatally, lack
the cerebellum, the locus coeruleus and the IV and V
motor nuclei. These defects can be traced back to an
early stage of neuroectoderm development (at least 8
d.p.c.) and are likely due to a failed specification of
rhombomeres 1–3 and isthmic area that are replaced
by a small undetermined region (Wassarmann et al.,
1997; Fig. 2). Moreover, the midbrain is enlarged,
extending caudally over its normal limit. This event is
preceded by an abnormal caudal extension of the
Otx2 expression domain, indicating Gbx2 as one of
the factors that confines the Otx2 acitivity in the
anterior neural plate. However, other unidentified factor(s) prevent Otx2 expression from expanding further
caudally. To test the hypothesis that Gbx2 is involved
in inhibiting Otx2 expression, Gbx2 was expressed in
the midbrain at early somite stages under the control
of the Wnt1 enhancer (Millet et al., 1999). Consistent
with the initial assumption, the Otx2 caudal limit was
shifted rostrally between the diencephalon and the
mesencephalon, leading to an enlargement of the presumptive anterior hindbrain, and a rostral repositioning of the gene expression network that generates the
IsO (Fgf8, Wnt1, En1 and Pax2; Fig. 2). Unfortunately, due to the Gbx2 transient ectopic expression,
the transgenic embryos recover a normal proportion
between the mesencephalon and the metencephalon by
12.5 d.p.c. (Millet et al., 1999). In conclusion, this
large body of results strongly suggests that the reciprocal repression between Otx2 and Gbx2 is the first
molecular event leading to the inductive process which
will specify the midbrain and hindbrain (Fig. 3). In
particular, this mutual repression is instrumental to
three subsequent events occurring between 7.5 and 8.5
d.p.c. embryos, (i) a sharpening and positioning of the
Otx2 and Gbx2 expression borders; (ii) the establishment of the mesencephalic and metencephalic identity,
and (iii) the induction of the molecular cascade underlying the IsO genesis and its maintenance.
Finally, the generation of conditional mouse mutants for Otx2 and Gbx2 as well as the isolation of
their regulatory regions will shed more light in the
near future on the mechanisms by which reasonably
few transcription factors talk to each others to lay
down the determination program leading to the final
complex pattern of the CNS.
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
5. otd/Otx conserved functions throughout evolution
5.1. Otx genes in the animal kingdom
Genes related to Drosophila otd and vertebrate Otx
have been isolated from a wide range of organisms
(Fig. 4). Most of them, up to protochordates have only
one member, with few exceptions where duplication
seems to have occurred in independent lineages (Li et
al., 1996; Umesono et al., 1999).
An Otx related gene is present already in Cnidarians,
primitive metazoans with a defined body plan and
radial symmetry. In these organisms, the Otx function
is associated to cell movements involved in the formation of new axes rather than in the formation of the
head (Smith et al., 1999). Rising in the evolutionary
scale, Otx has been found in animals with primitive
85
bilateral symmetry such as planarians (Stornaiuolo et
al., 1998; Umesono et al., 1999). In the planarian
Dugesia tigrina, Otx expression has been found in regeneration blastemas after transverse sectioning, with
an asymmetric pattern of transcripts more abundant in
head regenerating tissues (Stornaiuolo et al., 1998). The
expression of the Otx genes in these early metazoans
reveals some features in common with chordate Otx
genes. Although not directly correlated with a defined
anterior structure, the ancient function seems to deal
with patterning body axis and making tissues competent to respond to anteriorizing signals (Smith et al.,
1999), at least in budding and regeneration processes
which involve cell movements. Two Otx-related genes
have been identified only on the basis of homeodomain
sequence homology in the nematode C. elegans (Galliot
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.
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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, relegation 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
(synapomorphy) because of their embryonic and larval
bilateral symmetry (Lowe and Wray, 1997). Thus, the
highly divergent and non-head specific Otx expression
in sea urchin can be justified as a consequence of the
highly modified body plan of this phylum.
Comparative studies have demonstrated the existence
of Otx-related genes in all chordates (Simeone et al.,
1992, 1993; Frantz et al., 1994; Li et al., 1994; Mori et
al., 1994; Bally-Cuif et al., 1995; Blitz and Cho, 1995;
Mercier et al., 1995; Pannese et al., 1995; Kablar et al.,
1996) including urochordates (Wada et al., 1996),
cephalochordates (Williams and Holland, 1996), and
agnathan vertebrates (Ueki et al., 1998), where they are
expressed in the rostralmost CNS, independently of the
complexity acquired by this area during evolution. The
critical evolutionary position of lampreys will be discussed below, mainly with regard to Otx gene
duplication.
In urochordates and cephalochordates, only one Otx
gene has been identified so far that may be related to
Otx2 (Wada et al., 1996; Williams and Holland, 1998).
Indeed, in addition to similarities in amino acid sequence and expression, they are both expressed during
gastrulation in endoderm cells which suggests that their
oldest and primary role might be to mediate signals
required to specify anterior neuroectoderm. Restriction
of an early widespread expression of Otx2-like genes to
the anterior CNS is a remarkable feature of all vertebrates, which can be already observed in Ascidians and
Amphioxus. This led to hypothesise the homology between fore-midbrain territory of vertebrates to the less-
Fig. 5. Schematic comparison of expression domains of Otx-related
genes (grey) during embryogenesis in some representative protochordates (Ascidia and Amphioxus) and vertebrates (Lamprey and
mouse). Both lampreys Otx-related genes are shown because of their
highly divergent expression patterns. Abbreviations — D, diencephalon; Ep, epiphysis; Ey, eye; F, forebrain; FE, frontal eye; HB,
hindbrain; IO, infundibular organ; ll, lower lip; M, midbrain; MHB,
mid-hindbrain boundary; NC, nerve cord; Oe, olfactory epithelium;
Os, optic stalk; RV, rhomboencephalic vesicle; SC, spinal cord; SV,
sensory vesicle; T, telencephalon; ul, upper lip; and VG, visceral
ganglion.
evoluted sensory vescicle and cerebral vescicle of
Ascidians and Amphioxus, respectively (Fig. 5)
(Williams and Holland, 1998). Some authors have also
extended this homology further backwards in evolution. As mentioned previously, it has been proposed
that a primitive Otx function could be associated only
with cell movement rather than with anterior patterning
(Smith et al., 1999). Similarly, functional data in frog
(Blitz and Cho, 1995; Pannese et al., 1995) and mouse
mutants (Acampora et al., 1995; Matsuo et al., 1995;
Ang et al., 1996) suggest a possible involvement of
Otx2 in controlling the cell movements occurring during gastrulation. Recently, an early role of the ascidian
Otx2-related gene Hroth has been suggested in the
inhibition of notochord and muscle cell fate or in the
interference with notochord cell movements (Wada and
Saiga, 1999). Conservation of the expression domains
among Otx2-like orthologues from low chordates
(Wada et al., 1996; Williams and Holland, 1996; Ueki
et al., 1998), to vertebrates (Acampora and Simeone,
1999 and references therein) is consistent with the low
rate of divergence of their alignable sequences.
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
The duplication event generating the Otx1 branch
from the ancestor Otx2-like gene in gnathostome vertebrates cannot be dated precisely and will be discussed
below. However, comparative sequence analysis indicates clearly that Otx1-like genes evolve more rapidly,
as also shown by a further duplication event occurred
in both Xenopus (Kablar et al., 1996) and Zebrafish
(Mori et al., 1994) and by a ratio of sequence divergence higher than Otx2-like genes (Williams and Holland, 1998).
These data can be reinforced by the profound
changes in the expression domains of different vertebrate Otx1-like genes (Simeone et al., 1993; Mori et al.,
1994) which underlie rapid evolution of regulatory sequences as well.
5.2. A common genetic program in insect and
6ertebrate head de6elopment
Cloning of several master genes conserved throughout evolution and their comparative expression analysis
has been used largely as a means to identify homologous body regions and molecular mechanisms
among animals of different phyla (Abouheif et al.,
1997).
The most sensational example of such a functional
conservation is represented by the Pax-6/eyeless gene,
which in both Drosophila and vertebrates controls eye
development (Callaerts et al., 1997).
Until recently, it was assumed widely that the insect
and vertebrate CNS had evolved independently
(Garstang, 1928; Lacalli, 1994). This was due to their
position on opposite body sides of the dorso-ventral
(D/V) axis. One theory, the so-called ‘auricularia hypothesis’, postulated the homology between the outer
ectoderm of the insect embryo and the chordate CNS,
based on both anatomical studies made in echinoderms
(auricularia larvae) and urochordates, and on comparative expression of the HOX genes.
Starting from an ancient hypothesis of about two
centuries ago postulated by Geoffroy Saint-Hilaire (De
Robertis and Sasai, 1996), which has been object of
controversial debate (Arendt and Nübler-Jung, 1994;
Lacalli, 1995; Peterson, 1995), an increasing body of
evidence has now accumulated which supports a common evolutionary origin of insect and vertebrate CNS.
The first clues have been provided when it was shown
that the overall arrangement of neuroblasts in three
longitudinal columns on either side of the midline in
both insect and vertebrate CNS (Doe and Goodman,
1985; Chitnis et al., 1995) was paralleled by conserved
topographical expression (although with inverted D/V
polarity) of pairs of homologous genes (NK-2/NK-2.2;
ind/Gsh and Msh/Msx; Arendt and Nübler-Jung, 1996;
D’Alessio and Frasch, 1996; Weiss et al., 1998).
87
Even more convincing was the similar expression of
short gastrulation/chordin and decapentaplegic/Bmp4
pairs of homologous genes, which are antagonistic key
molecules of the D/V patterning (Piccolo et al., 1997) in
both Drosophila and vertebrates. As far as their expression is inverted with respect to the D/V axis in
Drosophila and Xenopus, injection experiments have
shown that these molecules are functionally equivalent
(De Robertis and Sasai, 1996), thus supporting the
existence of a homologous mechanism of D/V patterning in a common ancestor of both Drosophila and
vertebrates and reinforcing the ancient hypothesis of an
inversion of the D/V axis during evolution (De Robertis and Sasai, 1996; Arendt and Nübler-Jung, 1999).
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 positions in evolution which imply intermediate anatomies,
as in hemichordates.
Along the A/P axis, in both Drosophila and vertebrates, the conserved families of HOM/HOX and otd/
Otx genes play a fundamental role in the regional
specification of the neuroectoderm destined to form
nerve cord/posterior brain and anterior brain, respectively. This gross similarity can be refined further when
looking at the precise coincidence of gene-expression
domains with the boundary of metameric units or
neuromeres (Reichert and Simeone, 1999) and, as for
the HOX genes, also according to the phenomenon of
the ‘spatial colinearity’ (Lumsden and Krumlauf, 1996).
Over the last 10 years, functional experiments have
substanciated the equivalence suggested by the insect/
vertebrate comparative expression data. Most of these
studies carried out in Drosophila have shown that mammalian HOX genes could either partially rescue phenotypes due to mutations of their fly orthologues or elicit
responses similar to those elicited by their endogenous
counterparts when transiently overexpressed (Malicki et
al., 1990, 1993; Zhao et al., 1993; Bachiller et al., 1994).
To further sustain the idea that some of the molecular mechanisms underlying brain development were
conserved and comparable, a final, more challenging,
proof remained to be verified. Could the Drosophila otd
gene substitute for Otx genes in the mouse? That is, can
a gene working in a structure as simple as the insect
CNS substitute for homologous genetic functions in an
enormously more complex organ such as the brain of
mammals?
The embryonic Drosophila brain is composed of two
supraesophageal ganglia, each subdivided into three
neuromeres. The anterior ganglion is subdivided into
protocerebral, deutocerebral, and tritocerebral neuromeres. The gap gene otd is expressed mainly in the
anteriormost (protocerebral) neuromere, which is
deleted almost entirely in otd null embryos (Finkelstein
and Perrimon, 1990; Cohen and Jürgens, 1991; Hirth et
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al., 1995; Younossi-Hartenstein et al., 1997). This phenotype is due to the failure of expression of the otd
downstream gene lethal of scute, one of the proneural
genes regulating the three-column arrangement of neuroblasts mentioned above. As a consequence, in the
absence of otd, proneural genes are not activated and a
gap-like deletion of the rostral brain neuromere (protocerebrum) results in late mutant embryos (Hirth et al.,
1995). Other defects are also observed in the ventral
nerve cord and in non-neural structures. Flies that are
homozygotes for ocelliless (oc), a different otd allele, are
viable and lack the ocelli (light sensing organs) and
associated sensory bristles of the vertex (Finkelstein et
al., 1990). Moreover, in cephalic development, different
levels of OTD protein are required for the formation of
specific subdomains of the adult head (Royet and
Finkelstein, 1995). Despite the overall morphological
differences, however, expression pattern and mutant
phenotypes of Drosophila otd and mouse Otx genes can
be paralleled (see previous sections) easily. Nevertheless, OTD and OTX proteins are highly conserved only
in the homeodomain, which represents roughly onetenth of the whole OTD protein and about one-sixth
and one-fifth of OTX 1 and OTX 2 proteins, respectively (Simeone et al., 1993).
Outside the homeodomain, homology is restricted to
a few very short sequences. Drosophila OTD protein
also lacks the so-called ‘‘OTX tail’’ (Freud et al., 1997),
a conserved motif of about 20 amino acids which is
present in single copy in echinoderm, Ascidian and
Amphioxus Otx-related genes, whilst is tandemly duplicated in all vertebrate OTX proteins at the COOH
terminus (Williams and Holland, 1998).
The limited sequence homology shared by OTD and
OTX proteins underlines the importance that the homeodomain might have in triggering the CNS developmental programmes in both insects and vertebrates.
Outside the homeodomain, the proteins could either
have diverged, to allow mammals to acquire new and
specific functions or might be more homologous in
terms of tertiary structure than what the primary sequence alignment indicate. To gain insight into the
possibility that otd and Otx genes might share conserved genetic functions during CNS development, we
undertook experiments in which a Drosophila otd
cDNA replaces both mouse Otx1 and Otx2 genes.
When a full-coding Drosophila otd cDNA is introduced into a disrupted Otx1 locus by homologous
recombination many abnormalities of the Otx1 − /−
mice are rescued, regardless of a lower level of OTD
(about 30%) as compared with the endogenous OTX 1
level (Acampora et al., 1998a). Homozygous knock-in
otd mice (otd 1/otd 1) show no significant perinatal death
(with respect to a 30% death of Otx1 −/ − newborns)
and, most importantly, they have neither the abnormal
behaviour nor EEG characteristics observed in the
Otx1 null mutants.
In otd 1/otd 1 adult mice, brain size as well as the
thickness and cell number of the temporal and perirhinal cortices, both reduced in Otx1− /− mice, are very
similar to wild-type. The rescue is likely to be ascribed
at least in part, to a restored normal proliferating
activity of the dorsal telencephalic neuroepithelium at
9.75 d.p.c. (Acampora et al., 1998a). As described in a
previous section, Otx1 and Otx2 co-operate in brain
morphogenesis and a minimal threshold (corresponding
to either two copies of Otx2 or to one copy of Otx1
and one copy of Otx2) of their gene products is required for correct brain regionalization (Acampora et
al., 1997). When otd is expressed in place of both copies
of Otx1 and in the presence of one functional copy of
Otx2 (otd 1/− ; Otx2+ /− or otd 1/otd 1; Otx2+/−),
brain patterning abnormalities of the Otx1−/−;
Otx2+ /− mutants are rescued partially in a otd dosedependent manner, both at morphological and molecular level (Acampora et al., 1998a). The extent of this
rescue decreases progressively along the A/P axis, being
the posterior mesencephalon still severely affected with
respect to a normal telencephalon and to ameliorated
diencephalic and anterior mesencephalic structures. All
of these data indicate strongly that regionalization of
the brain requires levels of OTX proteins that increase
along the A/P axis and are particularly critical at the
MHB (Acampora et al., 1999b).
A partial rescue is also observed in some sensory and
sensory-associated structures such as iris, ciliary process
and Harderian glands.
On the contrary, the lateral semicircular canal of the
inner ear (last to be established in evolution) (Fritzsch
et al., 1986; Torres and Giraldez, 1998) is never restored in otd 1/otd 1 mice, suggesting that, either it requires higher levels of protein or that specification of
this structure is dependent upon an Otx1-newly established function. This seems indeed to be the case since,
as previously mentioned, in hOtx21/hOtx21 mice, Otx2
is able to rescue some ear defects in the regions where
both Otx genes are transcribed but not in the lateral
semicircular canal, where only Otx1 is normally expressed (Acampora et al., 1999a; Morsli et al., 1999).
The absence of the lateral canal in the inner ear of
Otx1 − /− mice might represent a back-evolutionary
mutation that can indicate the presumptive date of
duplication of the Otx genes. This event should have
occurred after that the lineage leading to cephalochordates had split from that leading to vertebrates
(Williams and Holland, 1998) and probably after the
latest passage leading from Agnatha to Gnathostomes.
In fact, in lampreys, extant agnathan vertebrates, only
two semicircular ducts (anterior and posterior) are
present in the inner ear, and despite two Otx-like genes
have been identified, none of them is clearly related to
Otx1. However, it cannot be excluded that the Otx
genes evolved by duplication in a common ancestor of
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
lampreys and gnathostomes (Fig. 4) and that the unclassified lamprey Otx represents a still evolutionary
unstable version of an Otx1-like gene (Ueki et al.,
1998).
More recently, we have generated a mouse model in
which it is the Otx2 gene to be substituted by the
Drosophila otd cDNA (otd2), using the same targeting
strategy as in the replacement with lacZ (Acampora et
al., 1995) and with hOtx1 cDNA (Acampora et al.,
1998b). Also in this case, the otd mRNA is detected in
both AVE and epiblast, whereas the protein only in the
VE. The OTD protein, exactly as the hOTX1 protein
does, is able to take over all of the Otx2 functions in
the AVE, thus recovering both gastrulation defects and
absence of an early anterior neural plate due to the lack
of Otx2. Later on, as in the hOtx12/hOtx12 mutants,
however, otd 2/otd 2 embryos fail to maintain the anteriormost identities of the brain and become headless.
These results, as far as limited to the AVE, provide a
further proof of functional equivalence, shared by otd/
Otx genes, via the activation of the same basic genetic
pathway(s).
Similar conclusions have been drawn by complementary experiments in Drosophila (Leuzinger et al., 1998;
Nagao et al., 1998). Heat-shock induced expression of
hOTX1 and hOTX2 proteins in the fly is able to rescue
the CNS defects of otd null mutant embryos (Leuzinger
et al., 1998) as well as cephalic defects of the ocelliless
mutations, both at the morphological and molecular
level (Nagao et al., 1998), whereas in a wild-type background, OTX overexpression leads to induction of ectopic neural structures (Leuzinger et al., 1998).
As previously mentioned, even though these crossphylum rescues highlight the fundamental role of the
homeodomain because it is the only recognisable region
conserved between OTD and OTX proteins, they do
not exclude that functional equivalence may be extended to the overall structure of OTD/OTX proteins.
The fact that OTD and OTX proteins are able to
drive cephalic development through the activation of
genetic pathways conserved between the two taxa, reinforces the idea that insect and chordate CNS are indeed
homologous structures originated from a common ancestor and controlled by a common basic genetic program (Sharman and Brand, 1998; Acampora and
Simeone, 1999; Reichert and Simeone, 1999).
Interestingly, substitution of a different key gene
involved in brain patterning processes with its
Drosophila orthologue has led to very similar conclusions (Hanks et al., 1998). In fact, Drosophila engrailed
was able to substitute for mouse Engrailed 1 (En1)
functions in mid- and hind-brain regions but not in
limb development. Therefore, despite a higher degree of
homology between EN proteins with respect to OTD/
OTX proteins, this rescue reinforces the idea that some
of the biochemical properties of highly conserved regu-
89
latory genes may be conserved across the two phyla and
indicates that new functions have been acquired by the
vertebrate genes during evolution.
5.3. Otx genes in brain e6olution
One of the most interesting observations raised by
our mouse models is the existence of a differential
post-transcriptional control of the hOtx1/otd and Otx2
mRNAs between VE and epiblast cells.
In heterozygous hOtx12/Otx 2 and otd 2/Otx 2 embryos, Otx2 mRNA and protein always colocalize,
while the hOtx1 and otd mRNAs are translated only in
the VE, thus suggesting that the knocked-in mRNAs
detected in epiblast cells are post-transcriptionally regulated by an Otx2-independent cis-acting control
(Acampora etal., 1998b).
In Otx2+ /− embryos, the same Otx2 region that is
replaced by the hOtx1 and otd cDNA is substituted
with the lacZ gene (Acampora et al., 1995). In these
embryos the lacZ mRNA is correctly detected in both
AVE and epiblast while the b-gal staining is, again,
heavily reduced in the epiblast at early-mid streak stage
(Acampora et al., 1995).
Therefore, three different mouse models replacing the
same Otx2 genomic region with three different genes
(lacZ, hOtx1 and otd) are suggestive of a different
post-transcriptional control between AVE and epiblast
cells. One or more Otx2 regions deleted in these models
(introns, untranslated or coding sequences) should be
responsible for this differential control.
Loss of mRNA translation in the epiblast might be
also influenced by abnormal molecular event(s) affecting RNA stability, processing and transport of the
chimeric knocked-in transcripts. Whatever the impairments are, since in the AVE the mRNA is correctly
translated, the absence of protein should be considered
a peculiar event occurring in epiblast cells and their
derivatives.
The finding that Otx2 escapes this post-transcriptional control suggests that this is necessary for the
maintenance of fore-midbrain territory specified by the
AVE (Acampora et al., 1998b) and might have evolutionary implications. Indeed, the architectural components of the vertebrate brain (telencephalon,
diencephalon and mesencephalon) are less clear in protochordates and a vertebrate-type brain, which is composed of a midbrain and six prosomers, firstly appears
in Petromyzontoidea. It may be hypothesized that the
specification of this prosomeric brain might have required the presence of a sufficient level of OTX2
protein in the early neuroectoderm cells and this event
might have been acquired by modifying post-transcriptional control of Otx2 transcripts rather than its coding
sequence which would have retained common functional 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 divergence among the species fits with this hypothesis.
Therefore, although this conservation in expression
pattern and coding sequence might favour a remarkable
role and functional equivalence of otd/Otx genes, it is
unclear why the brain vescicles of protochordates have
been so deeply and suddenly modified in a prosomeric
territory that has been maintained in its basic topography until mammals (Rubenstein et al., 1998).
A rather obvious answer to this question is that new
genetic functions have been gained and recruited in
developmental pathways. Indeed, it might be that conserved genes such as the Otx acquired different roles
even while retaining an evolutionary functional equivalence. Based on this hypothesis, it is expected that
drastic evolutionary events should act on the regulatory
control (transcription and translation) of Otx-related
genes rather than on their coding sequences. Interestingly, the findings that, (i) the Drosophila otd rescues in
mouse most of the Otx1 −/ − impairments (Acampora
et al., 1998a); (ii) the human Otx1 and Otx2 rescue
most of the otd defects in flies (Leuzinger et al., 1998;
Nagao et al., 1998; Sharman and Brand, 1998); (iii)
Otx1 rescues Otx2 requirements in VE (Acampora et
al., 1998b); (iv) Otx2 rescues most of the Otx1 − /−
defects (Acampora et al., 1999a); (v) the Drosophila otd,
similarly to Otx1, rescues Otx2 requirements in the VE
(Acampora et al., unpublished results), indicate that
Otx1, Otx2 and otd genes show a high degree of
functional equivalence in the tissues and body regions
where they are properly expressed. Therefore, these
data support the notion that otd/Otx functions have
been established in a common ancestor of fly and
mouse and retained throughout evolution, while copy
number and regulatory control of their expression have
been modified and re-adapted by evolutionary events
that have led to the specification of the increasingly
complex vertebrate brain (Sharman and Brand, 1998;
Simeone, 1998; Acampora and Simeone, 1999; Hirth
and Reichert, 1999; Reichert and Simeone, 1999). Gene
duplication may allow the duplicated gene to acquire
new specific function(s) either retaining or losing ancestral properties, and similarly, modification of the regulatory control of gene expression may establish new
expression patterns which might alter pre-existing cellfates by generating new specialised cellular functions.
A likely consequence of both increased genomic complexity and modification of regulatory control of gene
expression may result in an greater number of molecular interactions. This may contribute to modifying relevant morphogenetic processes that, in turn, can confer
a change in shape and size of the body plan as well as
in the generation of cell-types with new developmental
potentials. On this basis, Otx gene duplication and
subsequent or contemporary modification of regulatory
control might have contributed to the evolution of the
mammalian brain, for example by increasing the extent
of neuroectodermal territory recruited to form the
brain. This event might involve an improvement of
proliferative activity of early neuronal progenitors
(Acampora et al., 1998a, 1999a) and/or the positioning
of the MHB (Acampora et al., 1997, 1998b; Suda et al.,
1997). Additional property(ies) may be also conferred
to the duplicated gene by altering its coding sequence.
Thus, the limited amino acidic divergence between
OTX1 and OTX2 might underlie modifications of their
functional properties, as shown by the non-rescued
absence of the lateral semicircular canal of the inner ear
in mice replacing Otx1 with Otx2 (Acampora et al.,
1996, 1999a; Morsli et al., 1999).
6. Conclusions
In the last decade an impressive amount of knowledge has contributed to shed some light on the complexity of molecules and mechanisms involved in the
development of the vertebrate brain. In this context
otd/Otx genes play a cardinal role. Previous mouse
models have indicated that Otx1 and Otx2 are required
for normal brain and sense organ development and,
together with the Drosophila orthodenticle (otd) gene
they share a high degree of recriprocal functional equivalence. As far as the Otx genes are concerned, their
interchangeability indicates that the differential transcriptional control between Otx1 and Otx2 is the major
molecular reason responsible for generating Otx1 −/−
and Otx2 − /− divergent phenotypes. Nevertheless,
one of the most interesting observations raised by
mouse models replacing Otx2 with hOtx1 or otd is the
existence of a differential post-transcriptional and/or
translational control between the VE and epiblast. Indeed, hOtx1 or otd mRNA were detected in VE,
epiblast and its derivatives while hOTX1 or OTD
proteins in the VE only. This event results in the failure
of maintenance of fore-midbrain identities in the mutant embryos.
In our opinion one of the most interesting perspectives will be the understanding of the molecular mechanism(s) underlying this phenomenon, and its potential
relevance in the morphogenetic changes leading to the
evolution of the mammalian brain.
Acknowledgements
We thank A. Secondulfo for manuscript preparation.
This work was supported by the the EC BIOTECH
programme, the MRC Grant programme (c
G9900955), the Italian Association for Cancer Research
(A.I.R.C.) and the ‘MURST-CNR Biotechnology Programme Legge 95/95’.
D. Acampora et al. / Progress in Neurobiology 64 (2001) 69–95
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