International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples and , 1 Department of Neurological Sciences, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy
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Abstract |
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Introduction |
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Neocortical areas, unlike other neural structures such as the hindbrain, the diencephalon or the basal telencephalon (Puelles and Rubenstein, 1993; Rubenstein et al., 1994
; Lumsden and Krumlauf, 1996
), do not appear to be determined as compartments defined by anatomical and/or molecular boundaries but rather show a broad developmental potential. Experiments have shown that cell mixing between different cortical regions can occur (O'Rourke et al., 1992
) and that fetal neocortical neurons transplanted ectopically can generate axonal connections appropriate for their new position (Schlaggar and O'Leary, 1991
). Nevertheless, at least some features in the cortex are specified early in development (Cohen-Tannoudji et al., 1994
; Bulfone et al., 1995
). Epigenetic interactions may act to distinguish further the identity of the cortical areas.
Recently, a number of regulatory genes have been identified that may be involved both in the earliest phases of brain development and, later, in corticogenesis.
Many of these genes have been extensively studied, including members of the families Emx (Simeone et al., 1992b; Yoshida et al., 1997
), Otx (Simeone, 1998
), Pax (Stoykova and Gruss, 1994
), Dlx (Bulfone et al., 1993
) and POU (He et al., 1989
) and the genes Id-2 (Neuman et al., 1993
), BF-1 (Xuan et al., 1995
), Gli-3 (Franz, 1994
) and T-Brain-1 (Bulfone et al., 1995
).
The analysis of the expression patterns of these genes made it possible to subdivide the embryonic forebrain into molecularly distinct domains (prosomeres) (Puelles and Rubenstein, 1993) and in some of them, to identify layer-specific expression within the cortex (Frantz et al., 1994a
, 1994b
; Bulfone et al., 1995
).
Like many of the cited genes, the Otx genes (Otx1 and Otx2) represent the vertebrate homologues of a Drosophila gene, namely orthodenticle (Finkelstein et al., 1990), whose absence in the fly causes the loss of anterior head structures (Hirth et al., 1995
).
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Materials and Methods |
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The targeting vectors of the Otx1 and Otx2 knock-out mice have been obtained by cloning two genomic regions of these genes (arms of the homologous recombination) and replacing their first and second exons with the E. coli lacZ gene (Acampora et al., 1995, 1996
). The targeting vectors of the knock-in mice (otd1/otd1, hOtx21/hOtx21 and hOtx12/ hOtx12) were obtained by cloning the same arms of the knock-out molecules and replacing the first two exons of Otx1, with the Drosophila otd (Acampora et al., 1998a
) or the human Otx2 cDNAs (D. Acampora et al.,unpublished data), and replacing the same regions of Otx2 with the human Otx1 cDNA (Acampora et al., 1998b
). Linearized targeting plasmids were electroporated into HM-1 embryonic stem (ES) cells and homologous recombinant clones were identified by PCR reactions and then confirmed by Southern blot. Positive clones were microinjected into c57BL/6 blastocystes. The chimaeric males obtained were crossed to B6D2F1 females and the progeny screened for germ line transmission of the mutations by the presence of the agouti coat colour.
Electroencephalographic Recordings
The electroencephalographic recordings of Otx1/, otd 1/otd1 and hOtx21/hOtx21 mice were obtained by inserting, under Avertin anaesthesia (0.4 mg/g body wt i.p.), a deep electrode (tip diameter 100 µm) in the dorsal hippocampus, a screw electrode over the occipital cortex and a reference electrode in the frontal sinus. Muscular activity was recorded by an electrode inserted in the dorsal muscle. All electrodes were connected to a multiplin socket and secured to the skull by acrylic dental cement. After surgery, animals were allowed a 57 day recovery period. Amplification and recording of the electrical activity were obtained through an ATE apparatus (time constant 0.3 s, high cut-off filter 35 Hz, paper speed 30 mm/s). Subsequently, electrode position was confirmed by anatomical analysis.
Histological Analysis of Brains and Sense Organs
Histological sections (10 µm) were stained with cresyl violet. For cortical fine histology, comparable groups of sections centred on the presubicular area were selected from wild-type, Otx1/, otd1/otd1 and hOtx21/ hOtx21 brains (12 months old). Cell number was determined by counting cell bodies along a cortical area defined by the thickness of the cortex and by a unit length of 200 µm on the ventricular side.
BrdU Labelling and Detection of Apoptotic Cells
Pregnant mice at 9.5 d.p.c. were injected intraperitoneally with BrdU solution (50 mg/kg body weight) and killed after 1 h. After embryo genotyping, BrdU detection was performed (Xuan et al., 1995). The fraction of BrdU-positive cells was determined by dividing the number of BrdU-positive nuclei by the total number of nuclei identified in units of neuroepithelium 100 µm in length (Xuan et al., 1995
). The proportion of BrdU-positive cells in wild type embryos was considered 100%.
To detect apoptotic cells, the sections were processed following the TUNEL method (Gavrieli et al., 1992).
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Results and Discussion |
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Otx1 and Otx2 are expressed at early stages in the developing mouse embryo (Simeone et al., 1993). Otx2 is already transcribed before the onset of gastrulation in the epiblast and visceral endoderm (VE). As gastrulation proceeds, its expression is progressively restricted to the anterior third of the embryo in cells belonging to all germ layers. Later, at 9.5 days post coitum (d.p.c.), Otx2 expression demarcates almost the entire anterior brain, with a very sharp posterior border corresponding to the mesencephalic-metencephalic (mes-met) boundary (Simeone et al., 1992a
; Millet et al., 1996
). From these stages onwards, Otx2 expression disappears from the dorsal telencephalon, while still being expressed in restricted regions of the adult brain, with the exception of the cortex (Simeone et al., 1993
; Frantz et al., 1994b
). Otx1 starts to be expressed at the oneto three-somite stage (8 d.p.c.) throughout the forebrain and midbrain neuroepithelium (Simeone et al., 1992a
). From these stages onwards, the expression of Otx1 largely overlaps with that of Otx2, even though the dorsal telencephalon maintains Otx1 transcripts distributed uniformly across the ventricular zone of the cortex from the onset of corticogenesis up to midto late gestation stages (Simeone et al., 1993
; Frantz et al., 1994b
). Both genes are also expressed in developing olfactory, acoustic and ocular sense organs (Simeone et al., 1993
).
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 organization of the adult cortex. The layers are generated in an inside-out pattern, in which cells of the deepest layers appear first in the ventricular zone (VZ), and those of the upper layers 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., 1994b).
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 14 (Frantz et al., 1994b).
Thus, the progressive down-regulation of Otx1 in the ventricular cells suggests that Otx1 may confer deep-layer 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
).
Otx1 might also be involved in the forming of the cortical areas. Indeed, Otx1 expression is heterogeneous across the regions of the adult cortex, with the expression in layer 5 being more prominent in the posterior and lateral cortex but absent in the frontal, insular and orbital cortices, while Otx1 expression in layer 6 is more uniform throughout the neocortex (Frantz et al., 1994b).
To gain insight into the roles of Otx1 and Otx2 in brain and cortex development, mouse mutant models have been generated (Acampora et al., 1995, 1996
, 1997
, 1998a
,b
) (D. Acampora et al., unpublished data).
Otx1 is Required for Correct Brain Development
Heterozygous (Otx1+/) mice were healthy and their intercross generated homozygous mice (Otx1/) in the expected Mendelian ratio. However, 30% of mutants died before the first postnatal month, and appeared smaller in size. Otx1/ mice exhibited both spontaneous high speed turning behaviour and epileptic behaviour (Acampora et al., 1996). The latter consisted of the combination of: (1) focal seizures characterized by automatisms (head bobbing and teeth chattering) and electroencephalographic (EEG) recording of spikes in hippocampus; (2) generalized seizures characterized by convulsions and high voltage synchronized EEG activity in hippocampus and cortex. Occasionally, convulsions were followed by status epilepticus and exitus (Fig. 1e
and Table 1
).
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To assess whether the overall reduction of the Otx1/ brains was due to reduced proliferation or an increase in cell death, or both, we determined the number of apoptotic and proliferating cells within the neuroepithelium of the developing telencephalon. No differences in apoptosis were observed by comparing 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. A defective proliferation of neuronal progenitors at these early stages may contribute to the adult phenotype of the Otx1 mutant mice.
At the prepubescent stage, Otx1/ mice exhibited transient dwarfism and hypogonadism due to low levels of pituitary growth hormone, follicle-stimulating hormone and luteinizing hormone (Acampora et al., 1998c). They also showed sense organ abnormalities, including absence of the ciliary process of the eye, the lachrymal and Harderian glands and the lateral semicircular canal of the inner ear (Acampora et al., 1996
).
Otx2 is Required in Early Gastrulation and Anterior Neural Plate Induction
Data from expression studies and other evidence suggest that Otx2 might be important in early head specification (Ang et al., 1994; Blitz and Cho, 1995
; Pannese et al., 1995
; Simeone et al., 1995
).
Otx2 null embryos die early in embryogenesis, lack the rostral neuroectoderm fated to become the forebrain, midbrain and rostral hindbrain, and show major abnormalities in their body plan (Acampora et al., 1995; Matsuo et al., 1995
; Ang et al., 1996
). Heterozygous Otx2+/ embryos, depending on their genetic background, show head abnormalities that are reminiscent of otocephalic phenotypes (Matsuo et al., 1995
).
The headless phenotype of Otx2/ embryos could be due to abnormalities in tissues with inducing properties, such as the anterior visceral endoderm (AVE) (Thomas and Beddington, 1996; Varlet et al., 1997
; Beddington and Robertson, 1998
) and the prechordal mesendoderm (Lemaire and Kodjabachian, 1996
), or in responding tissues such as the epiblast and anterior neuroectoderm. In homozygous embryos in which Otx2 was replaced by a lacZ reporter gene (Acampora et al., 1995
), however, the first abnormality was detected at the pre-early streak stage. Indeed, at this stage, lacZ transcripts are detected in both the VE and the epiblast of Otx2+/ embryos, but only in the VE of Otx2/ embryos.
The importance of Otx2 in the AVE has also been supported by the analysis of chimaeric embryos containing Otx2/ epiblast cells and wild type VE, or vice versa (Rhinn et al., 1998). Indeed, only chimaeric embryos containing wild type VE were able to rescue an early neural plate.
In summary, these findings indicate that Otx2 represents an important requirement of the AVE for the specification of an early neural plate (see also below). Unfortunately, due to the early death of Otx2/ embryos, the role of Otx2 during subsequent embryonic development is unclear. Further research in which Otx2 1600 is conditionally inactivated could help to answer this question.
Brain Patterning Depends on a Minimal Threshold of OTX Gene Products
Events underlying the anteroposterior (A/P) patterning of the CNS begin to be established during the early gastrulation stage and lead to the generation of distinct transverse domains along the A/P body axis (Tam and Behringer, 1997; Rubenstein et al., 1998
). It has been proposed that the juxtaposition of differently specified territories can generate organizing centres at their points of contact, where cellular interactions result in the production of signalling molecules with inducing properties (Meinhardt, 1983
).
The morphogenetic fate of distinct brain areas is dependent on inductive signals produced by organizing centres and the ability of target tissues to respond to them.
Transplantation experiments have demonstrated organizing properties of the isthmus at the mes-met junction and the existence of a different territorial competence between regions of the brain located rostrally (prosomeres 36) and posteriorly (mesencephalon and prosomeres 1 and 2) to the zona limitans intrathalamica (ZLI) (Martinez et al., 1991; Marin and Puelles, 1994
). Two important signalling molecules, coded by fibroblast growth factor-8 (Fgf-8) (Crossley and Martin, 1995
; Crossley et al., 1996
; Lee et al., 1997
; Meyers et al., 1998
) and Sonic hedgehog (Shh) (Echelard et al., 1993
; Martì et al., 1995
; Roelink et al., 1995
; Chiang et al., 1996
; Ericson et al., 1996
) are transcribed at the isthmic constriction and zona limitans intrathalamica, respectively. Fgf-8 has been shown to mimic the midbrain-inducing properties of the isthmus (Crossley et al., 1996
), while it can only be postulated that Shh has a similar role in either organizing tissue or in conferring different regional competence between territories rostral and caudal to the ZLI, or both (Martinez et al., 1991
; Figdor and Stern, 1993
; Rubenstein et al., 1994
, 1998
; Bally-Cuif and Wassef, 1995
; Crossley et al., 1996
).
It is possible that the mechanisms underlying specification of adjacent territories with a different identity (e.g. mesencephalon and metencephalon), and the correct positioning of an organizer (e.g. isthmic organizer) are dependent on combinatorial patterns of gene expression and on threshold-levels of some critical proteins. Evidence suggests that Otx genes might indeed play an important role in the development of the mes-met region as well as in the establishment of different thresholds of territorial competence along the antero-posterior axis of the brain. This evidence includes expression data (Simeone et al., 1992a, 1993
), Otx2 null mice (Acampora et al., 1995
; Matsuo et al., 1995
; Ang et al., 1996
), transplantation experiments (Millet et al., 1996
), and retinoic acid-induced phenocopies (Simeone et al., 1995
; Avantaggiato et al., 1996
).
To confirm this theory, the level of OTX proteins was modified by altering the Otx gene dosage in vivo (Acampora et al., 1997; Suda et al., 1997
). Only Otx1/; Otx2+/ embryos showed 100% penetrance of gross brain malformations that included a remarkable reduction of the Ammon's horn, as well as a morphological and molecular transformation of the pretectum, dorsal thalamus and mesencephalon into an enlarged metencephalon. Indeed, mesencephalic molecular features within the dorsal telencephalon of Otx1/; Otx2+/ brains was observed, with Wnt-1 expressed along the telencephalic commissural plate, and En-2 along the dorsal telencephalon (Fig. 2
). The rescue of the abnormal phenotype observed in the presence of an additional copy either of Otx2 or Otx1 indicated that Otx 2000
genes might co-operate in brain patterning through a gene dosage requirement. The origin of the repatterning process has been studied by monitoring the early expression pattern of genes involved in the establishment of the mes-met region such as Wnt-1, En-1 and Fgf-8 (Bally-Cuif and Wassef, 1995
; Joyner, 1996
; Rubenstein et al., 1998
). This analysis suggests that the repatterning process was probably triggered by the early mis-expression of Fgf-8 in response to a critically low level of OTX gene products (Acampora et al., 1997
).
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Furthermore, the fact that the dorsal telencephalon in Otx1/; Otx2+/ embryos acquired mesencephalic molecular features correlates with the absence of the zona limitans intrathalamica (as revealed by anatomical inspection and loss of Shh expression), a region that might act as a morphogenetic barrier to the spreading of posteriorizing signals (Crossley et al., 1996; Martinez et al., 1999
). The incomplete transformation of the telencephalon observed in absence of the ZLI could be the consequence of a reduced ability of the telencephalic territory to respond over time to the mesencephalic inductive signals coming from the anteriorized isthmic-like structure.
otd and Otx1 Share a Conserved Genetic Program of Brain Development
A large body of evidence indicates that the regulatory genes controlling brain development are evolutionarily conserved.
Most of these are homeobox-containing genes and among them, the murine Otx and Drosophila otd genes represent a remarkable example of similarity in homeodomain sequence, embryonic expression pattern and mutant phenotype (Cohen and Jürgens, 1991; Holland et al., 1992
; Finkelstein and Boncinelli, 1994
; Acampora et al.,1995, 1996
, 1997
; Hirth et al., 1995
; Matsuo et al., 1995
; Thor, 1995
; Ang et al., 1996
).
In Drosophila, the otd gene is expressed at the anterior pole of the blastoderm embryo and later predominantly in the developing rostralmost brain neuromere (protocerebrum) (Finkelstein and Perrimon, 1990; Cohen and Jürgens, 1991
; Hirth et al., 1995
). In otdmutants, most protocerebral neuroblasts and some deuterocerebral neuroblasts do not form, giving rise to a dramatically reduced brain (Hirth et al., 1995
; Younossi-Hartenstein et al., 1997
). otd mutants also have pattern deletions in cephalic structures. In ocelliless, a viable otd allele, for example, expression in the vertex primordium is abolished and the ocelli (light sensing organs) and associated sensory bristles (Finkelstein et al., 1990
) are lost. Finally, in cephalic development, different levels of OTD protein are required for the formation of specific subdomains of the adult head (Royet and Finkelstein, 1995
).
In mouse, Otx1 null mutants show spontaneous epileptic seizures and multiple abnormalities affecting the telencephalic dorsal cortex, the mesencephalon, the cerebellum and components of the acoustic and visual sense organs (Acampora et al., 1996). Otx2 null mice mutants are early embryonic lethal and lack the rostral neuroectoderm fated to become the forebrain, midbrain and rostral hindbrain (Acampora et al., 1995
; Matsuo et al., 1995
; Ang et al., 1996
). Moreover, Otx genes may co-operate in brain morphogenesis and a threshold level of OTX proteins is required to allow the correct positioning of the isthmic organizer (Acampora et al., 1997
).
In contrast to the extensive similarities in expression and mutant phenotype between the Drosophila otd and the murine Otx genes, that of the OTD and OTX gene products is quite restricted; sequence homology is confined to the homeodomain and a few flanking amino acids (Simeone et al., 1993). Thus, although the ability to recognize the same target sequence might be evolutionarily conserved, murine Otx genes might have also acquired additional functional features, outside the homeodomain, that are different from those encoded by the Drosophila otd gene. This suggests that some conserved features of the invertebrate OTD gene-product might now coexist in Otx genes together with additional new functions required for specific mammalian developmental processes.
To verify this hypothesis, we produced mice replacing Otx1 with a full-coding Drosophila otd cDNA (Acampora et al., 1998a). The heterozygotes (otd1/Otx1) were healthy and fertile and generated homozygous otd1/otd 2600
1 mice at the expected frequency. Regardless of a lower level of OTD protein (about 30% less) as compared to the OTX1 level in wild type animals, homozygous knock-in otd mice did not show significant perinatal death and lacked behavioural and EEG characteristics of seizures recorded in Otx1/ mice. Anatomical analysis showed that the majority of the defects due to the absence of Otx1 were rescued in otd1/otd1 mice, including brain size and cortical organization (Fig. 3C
and Table 1
). The number of cell bodies in all cortical areas of otd1/otd1 brains was comparable to that of wild type and a normal hexalaminar organization was observed (Fig. 3E, F
). BrdU labelling analysis has shown that the ability of Drosophila otd gene to recover the cortical defects of Otx1/ was probably due to the rescue of the reduced proliferation in the dorsal telencephalic neuroepithelium at 9.75 d.p.c.
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In a complementary experiment performed in Drosophila, overexpression of human Otx1 and Otx2 genes rescued the brain and ventral nerve cord phenotypes of otd mutants (Leuzinger et al. , 1998) as well as the cephalic defects of adult flies carrying the ocelliless mutation (Nagao et al., 1998
). Moreover, ubiquitous overexpression of Otx1 and Otx2 genes in a Drosophila wild type background was able to induce ectopic neural structures (Leuzinger et al., 1998
).
These cross-phylum rescues are surprising not only because of the different anatomy and complexity of insect and mammalian brains, but also because of the very limited region of homology shared by the proteins, restricted essentially to the homeodomain. These two observations imply that otd and Otx genes can trigger a basic programme of cephalic development through conserved genetic interactions possibly involving a homeoboxmediated choice of the same target sequence and, probably, the same target genes (Sharman and Brand, 1998). The incomplete rescue mediated by the Drosophila otd gene may reflect both quantitative (higher level of otd expression) and qualitative (Otx-specific) requirements. In particular, failure to recover the lateral semicircular canal of the inner ear in otd1/otd1 mice (Acampora et al., 1998a
; Sharman and Brand, 1998
) suggests an Otx1-specific function acquired during evolution (see also below).
The extended evolutionary conservation between murine Otx1 and Drosophila otd genes supports the hypothesis that genetic functions required in mammalian brain development evolved in a primitive ancestor of flies and mice (Wray et al., 1996).
Specific and Interchangeable Roles between Otx1 and Otx2
Mammalian OTX1 and OTX2 proteins share extensive similarities in their sequences, even though downstream of the OTX1 homeodomain, the regions of homology to OTX2 are separated by stretches of additional amino acids (Simeone et al., 1993). To determine whether Otx1/ and 3000
Otx2/ divergent phenotypes could derive from differences in the temporal expression or biochemical activity of OTX1 and OTX2 proteins, we generated mice in which the Otx1 gene was replaced by a human Otx2 (hOtx2) full-coding cDNA (hOtx21/hOtx21) (D. Acampora et al., unpublished data) or the Otx2 gene was replaced by a human Otx1 (hOtx1) full-coding cDNA (hOtx12/hOtx12) (Acampora et al., 1998b
).
In homozygous mice in which Otx1 was replaced with the human Otx2 cDNA (hOtx21/hOtx21), despite a reduced expression of the transgenic alleles, cerebral cortical development appears to be normal and the mice do not have epilepsy (Fig. 3D, E, F and Table 1
) while, as in otd, a normal cell proliferation activity in the dorsal neuroepithelium was restored, as revealed by BrdU labelling experiments at 9.5 d.p.c. (D. Acampora et al., unpublished data). This is particularly relevant 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 in the basal neuroepithelium and disappears completely from the mediodorsal area at 11 d.p.c. Considering the absence of the OTX1 gene product and a reduced level of the hOTX2 protein in regions that normally would not express Otx2, the rescue observed in hOtx21/hOtx21 mice suggests Otx1 and Otx2 have interchangeable roles in the cortex. It may therefore be the differential transcriptional control of Otx1 and Otx2, rather than their amino acid divergence, which is responsible for the contrasting phenotypes of Otx1/ and Otx2/.
It would thus be interesting to see the effects of an OTX overdosage on the brain and, specifically, on cortical development. hOtx21/hOtx21 mice also showed a significant improvement in mesencephalon, eye and lachrymal gland defects. In contrast, the lateral semicircular canal of the inner ear was never restored. Interestingly, the rescue observed was comparable with that of mice in which Otx1 was replaced with otd (Acampora et al., 1998a), suggesting that the ability to specify the lateral semicircular canal of the inner ear may be an Otx1-specific property (Acampora and Simeone, 1999
).
Homozygous mutant mice replacing Otx2 with the human Otx1 (hOtx1) cDNA (hOtx12/hOtx12) recovered the anterior neural plate induction and a normal gastrulation but showed a headless phenotype from 9 d.p.c. onwards. A combined analysis of both hOtx1 RNA and protein distribution during early gastrulation has revealed that while hOtx1 mRNA was detected in the VE and the epiblast, the hOTX1 protein was revealed only in VE.This VE-restricted hOTX1 protein translation was sufficient to recover gastrulation defects and induction of an early anterior neural plate. From 8.5 d.p.c. onwards, however, hOtx12/hOtx12 embryos failed to maintain fore-midbrain identities, and at the end of gestation, displayed a headless phenotype in which the body plan showed no detectable defects (Acampora et al., 1998b).
These results, besides reinforcing the head-organizing activity of the VE (Beddington and Robertson, 1998), indicate that at least in this tissue, Otx1 and Otx2 are functionally equivalent. Moreover, these findings indicate that Otx2 is necessary in the mesendoderm or the neuroectoderm at the late gastrulation stage, or both, for the maintenance of anterior patterning of the neural plate.
These data support the idea of an extended functional conservation between OTX1 and OTX2, and suggest that Otx1 and Otx2 null mice contrasting phenotypes originate mostly from their divergent patterns of expression.
Specification of Regional Identities in the Brain Requires Increasing Otx Dosage along the Anteroposterior Axis
Genetic analysis of Otx functions has revealed that Otx1 and Otx2 can co-operate in brain morphogenesis and that a minimal threshold of their gene products is required for correct brain regionalization (Acampora et al., 1997).
The finding that otd rescued most of the Otx1 functions, suggests that like Otx1, otd might also be able to co-operate with Otx2 in brain patterning. To verify this hypothesis, we generated mice carrying a single functional copy of Otx2 and one or two copies of otd under 3600
Otx1 control regions (otd1/; Otx2+/ and otd1/otd1; Otx2+/). The Ammon's horn, dorsal thalamus and pretectum that were lost in Otx1/; Otx2+/ embryos were restored, the anterior mesencephalon was improved and the cerebellum was present in its almost normal position. The analysis of these mice during early embryogenesis revealed that the isthmic organizer was shifted slightly forwards and that the anatomical and molecular recoveries were more evident for anterior structures (such as the telencephalon and the diencephalon), where the ectopic expression of En-2 and Wnt-1 genes was gradually reduced as the otd copy number increased (Acampora et al., 1998a). By contrast, the posterior mesencephalon remained severely affected, probably due to the still abnormal distribution of Fgf-8 transcripts. These data indicate that the Drosophila otd gene can rescue the patterning defects of Otx1/; Otx2+/ embryos in a dose-dependent manner. The extent of this rescue is high for the telencephalic region, intermediate for the dorsal thalamus and pretectum and relatively poor for the mesencephalon. Accordingly, a correct specification and regionalization of the brain seems to require levels of OTX proteins that increase along the anteroposterior axis of the brain. Similar results were also obtained in double mutant mice carrying one or two hOtx2 alleles in place of Otx1 (hOtx21/; Otx2+/ and hOtx21/hOtx21; Otx2+/) (D. Acampora et al., unpublished data). Unfortunately, it is still unclear whether the observed rescue is partial because of the reduced level of the OTD and hOTX2 gene products or because of the intrinsic properties of the OTD and hOTX2 proteins.
Conclusions and Perspectives
The role of Otx genes in the morphogenesis of the brain can be considered from two points of view. The first one regards their early function and contributes to a discussion on the evolution of the brain. The second one regards implications for human pathology(ies) related to the adult phenotype of the Otx1/ mice. Expression of Otx genes in vertebrates and Otx-like genes in lower species has always been associated with the most rostral neural structures. During the phylogenetic development of the brain, the architecture of the Otx-expressing regions of the CNS might have been modified on the basis of new genetic instructions. Some of these might include, for example, the duplication of an ancestor Otx gene (Otx1-related genes have been clearly identified only in gnathostomes) or the ability, gained in evolution, of epiblast-like cells to translate Otx2, or the acquirement of a different spatio-temporal transcriptional control of Otx1 and Otx2. The morphogenesis of the gnathostome-type brain may have been caused by a posterior displacement of the mesencephalicmetencephalic boundary as well as differential proliferative properties of the rostral neuroectoderm (forebrain, midbrain) versus the more posterior neuroectoderm (hindbrain and spinal cord). In this context, the anterior shift of the isthmic organizer in Otx1/; Otx2+/ embryos and the reduced proliferative activity of the dorsal telencephalic neuroepithelium observed in Otx1/ mice underline the relevant developmental processes involving the Otx genes during the evolution of the brain.
By contrast, the epileptic and cortical abnormalities observed in Otx1/ mice raise different considerations. Malformations of the human neocortex are commonly associated with developmental delays, mental retardation and epilepsy. Developmental malformations are estimated to be responsible of >50% of intractable seizures in children and ~15% of intractable seizures in adults (Barkovich et al., 1996). Malformations of cortical development comprise a heterogeneous group of disorders recently classified, according to embryological events, in three subgroups: malformations due to (1) abnormal neuronal proliferation, (2) abnormal neuronal migration and (3) abnormal cortical organization.
Although several aetiological factors have been proposed, defective genes are being recognized with increasing frequency as a cause of cortical malformations (Raymond et al., 1995). In the case of tuberous sclerosis, for example, a disorder due to abnormal neuronal proliferation, two genes have been implicated, TSC1 and TSC2, encoding the HAMARTIN and TUBERIN proteins, respectively (Vinters et al., 1998
). Likewise, in X-linked lissencephaly and the double cortex syndrome, two allelic disorders due to abnormal neuronal migration, the mutation of the doublecortin gene seems responsible (Gleeson et al., 1998
). Similarly, both focal pachypolymicrogyria and bilateral periventricular heterotopia, manifestations of two distinct X-linked disorders due to abnormal neuronal migration, are caused by mutations of as yet unidentified genes (Fink et al., 1997
; Yoshimura et al., 1998
). Finally, schizencephaly, a disorder with abnormal cortical organization, has been recently associated with mutations in the EMX2 gene (Brunelli et al., 1996
).
Experimental evidence suggests that mutations in the Otx1 gene might be responsible for malformations of the human neocortex, though no such mutations have been identified so far. Disruption of the Otx1 gene results in defects of cell proliferation and consequent size reduction in specific cortical areas such as temporal and perirhinal cortices. These alterations strongly correlate with the occurrence of spontaneous epileptic activity. Based on these considerations, Otx1/ mice provide the first animal model of abnormal neuronal proliferation leading to cortical malformation with epilepsy.
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Address correspondence to Antonio Simeone, International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples, Italy. Email: simeone{at}iigbna.iigb.na.cnr.it.
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