Otx Genes and Seizure Susceptibility

Homeobox-containing transcription factors crucially regulate the patterning and regionalization of the developing nervous system, as well as the proliferation, survival and differentiation of distinct neuronal populations. Altered expression of these factors markedly impacts the structure and function of the embryonic and adult central nervous system (CNS). In both humans and mice, mutations of homeobox genes have been associated to severe syndromes characterized by developmental CNS defects, which often lead to the occurrence of seizures in the adult life. Among homeobox genes, Otx genes (Otx1 and Otx2) are crucially involved in brain regionalization. Our previous work showed that mice lacking Otx1 present cerebral cortex defects and develop epilepsy, whereas mice with conditional deletion or overexpression of Otx2 show profound alterations in midbrain-to-forebrain circuits, which are accompanied by an abnormal response to experimentally-induced seizures. In this brief review, we highlight and discuss the major findings suppporting the role of Otx genes in sculpting midbrain and forebrain circuits involved in epileptogenesis.


Introduction
The embryonic development of the vertebrate CNS is a complex process requiring sequential morphogenetic events that specify and pattern the neural tube.This process requires a concerted and sequential series of events that regulate the proliferation, survival and differentiation of distinct neuronal populations.
Homeobox-containing transcription factors crucially control all stages of CNS development [1,2] .Mutations in homeobox genes markedly impact brain development, and may lead to severe postnatal neurological dysfunctions, including epilepsy [3] .It is well established that altered function of genes controlling specification of brain areas, neuronal identity and circuit formation can lead to altered seizure susceptibility and epilepsy.The

EXPERT REVIEW
altered expression and function of genes involved in brain development may lead to altered differentiation of selected neuronal populations and improper shaping of neuronal circuitry, thus resulting in imbalance between excitation and inhibition in the postnatal brain [4] .
Otx genes are the murine homologs of the Drosophila orthodenticle (otd) gene, and code for transcription factors containing a bicoid-like DNA-binding homeodomain [5] .Several lines of evidence show that Otx1 and Otx2 control specification and regionalization of midbrain and forebrain areas [6][7][8][9] .Otx genes are specifically expressed in the developing brain: Otx2 is expressed in all dorsal and most ventral regions of the telencephalon, diencephalon and mesencephalon, whereas the expression domain of Otx1 is similar to that of Otx2, but contained within it [10] .Loss-of-function mutations of Otx1 and Otx2 genes have not been described in human epilepsies, with the exception of a case of syndromic microphthalmia with learning deficits and seizures associated to a mutation in exon 3 of the Otx2 gene resulting in a truncated protein [11] .However, classical and conditional gene targeting studies in mice showed that altered levels of Otx gene expression may alter seizure susceptibility, indicating that these genes play an important role in epileptogenesis in forebrain and midbrain structures.The epileptic phenotypes in Otx mutant mice are described in the following paragraphs and summarized in Table 1.Only the phenotypes relevant to seizure disorders are reported.Abbreviations are as in the text.

Altered seizure susceptibility in Otx mutance mice
Otx1.Loss of Otx1 obtained by homologous recombination results in spontaneous epilepsy in mice [12]   .Otx1 -/-mice show a high-speed turning behaviour accompanied by a severe electroencephalographic (EEG) pattern consisting of focal and generalized seizures.In Otx1 -/-mice, focal seizures are characterized by behavioural automatisms (head bobbing and teeth chattering) and EEG hippocampal spikes, while generalized seizures consist in convulsions and occasional status epilepticus accompanied by synchronized EEG activity in hippocampus and cerebral cortex [7,12] .Otx1 -/-brains show abnormal layering, reduced thickness and cell loss in the temporal and perirhinal cortex, and a shrinkage of the hippocampus [7,12] .In the somatosensory cortex, loss of Otx1 results in selective loss of layer V pyramidal neurons, ectopic pyramidal cells in layers II-III and a "patchy" distribution of parvalbumin (PV)positive GABAergic interneurons in the cerebral cortex [13,14] .As a consequence of this anatomical disorganization of the cerebral cortex, increased NMDA-mediated excitation and reduced GABA-mediated inhibition occurs in the cerebral cortex of Otx1 mutants [15] (Fig. 1 and Table 1).Notably, the introduction of otd or human Otx2 into the Otx1 locus completely rescues the epileptic phenotype of Otx1 -/- mice, thus demonstrating the evolutionary conservation of Otx and otd gene functions [7,16] (Table 1).The crucial role of Otx1 in the specification of cortical connectivity has been also confirmed by studies demonstrating that Otx1 controls the refinement of long-distance subcortical projections.Within cortical layer V, Otx1 is expressed by neurons that project their axons to the midbrain and spinal cord, and Otx1 -/-mutants are defective in the refinement of these subcortical projections [17] .
Otx2.The knockout of Otx2 in mice is lethal, and homozygous mutant embryos are characterized by the complete absence of forebrain and midbrain regions [18] .The early expression and function of Otx2 in developing embryos (at gastrulation, before the onset of neural tube formation) [18] can explain the headless phenotype of Otx2 -/-embryos.This extreme phenotype of Otx2 -/- embryos does not allow a detailed investigation of Otx2 function in the developing and postnatal brain.This has been successfully achieved by selectively modifying the expression of Otx2 in restricted brain regions during development through conditional gene targeting.
Inactivation of Otx2 in the expression domain of Otx1 results in abnormal midbrain development and perinatal death [19] .More relevant to epilepsy, Otx1 Cre/+ ; Otx2 flox/- mice show a glutamate to GABA switch in glutamatergic progenitors of the thalamus [20] .During normal brain development, Otx2 represses the transcription factor Mash1 (mammalian achaete-scute homolog; currently known as ASCL1), consequently preventing the expression of GABAergic genes.Otx2 inactivation in neurons expressing Otx1 results in activation of Mash1dependent differentiation cascades, leading thalamic glutamatergic precursors to express Pax3, Pax7 and Lim1 (three genes normally coexpressed with Mash1) together with GABAergic markers such as glutamic acid decarboxylase (GAD-67 isoform) and GABA itself [20] (Table 1).Thus, Otx2 plays a crucial role in the molecular mechanism regulating the identity and fate of thalamic glutamatergic neurons.In the developing brain, some GABA-positive migrating-like neurons, located in the dorsal thalamus, have been also shown to express Otx2, indicating a possible role of Otx2 in thalamic cell migration [21] .These results are of particular interest considering the crucial role of inhibitory thalamic neurons in the onset of absence epilepsy [22] .In addition, it would be interesting to study whether similar Otx2dependent transcriptional networks regulating the fate of glutamatergic neurons take place also in other brain areas expressing Otx2 that have a prominent role in epileptogenesis, such as the developing cerebral cortex and hippocampus.
Conditional overexpression of Otx2 in En1-positive mesDA precursors has different effects.En1 Cre/+ ; tOtx2 ov/+ mice show an increased number of mesDA neurons during embryonic and postnatal development [26,27,32] .According to the increased number of mesDA neurons, En1 Cre/+ ; tOtx2 ov/+ adult mice present an increased DAergic innervation to the striatum and frontal cortex, as indicated by increased staining for the active, glycosylated form of the dopamine transporter (glyco-Dat) [33] .En1 Cre/+ ; tOtx2 ov/+ mice also show increased density of PV interneurons in deep layers of the frontal cortex, a major target area of mesDA cells (Table 1 and Fig. 1).The observation that these PV cells are targeted by DAergic fibers [33] suggests that DA might be responsible of PV cell proliferation.Indeed, DA is known to accelarate PV interneurons maturation both in vitro [34] and in vivo in anterior cortical areas [35] .Otx2 protein secreted from increased DAergic terminals might also drive PV cell maturation in the frontal cortex of Otx2 overexpressing mice.Indeed, Otx2 secreted from subcortical structures (retina, dorsal lateral geniculate nucleus and choroid plexus) accumulates in visual cortical PV cells, regulating their maturation [36][37][38] .Considering that transynaptic action of homeoboxcontaining proteins has been described also in the DA system [39,40] , it is conceivable that Otx2 secretion from  ventral midbrain might exert similar effects onto PV interneurons in the frontal cortex.
The increased number of PV cells observed in En1 Cre/+ ; tOtx2 ov/+ mice might explain the response of limbic circuits to KA detected in these mutants.As compared to controls, En1 Cre/+ ; tOtx2 ov/+ mice do not show an altered behavioral response to KA, but appear to be more resistant to KA-induced damage [33] (Table 1 and Fig. 1).An icreased inhibitory tone in limbic areas might therefore contribute to mitigate the long-term effects of KA in these mutants.

Concluding remarks
Altered expression and function of homeobox genes during embryonic brain development leads to abnormal specification of brain areas, neuronal identity and circuit formation, ultimately resulting in impaired neurological functions.Studies performed in the mouse suggest that inactivation of the two Otx genes, Otx1 and Otx2, may differentially alter seizure susceptibility.Inactivation of Otx1 results in spontaneous seizures and epilepsy, while altered levels of Otx2 in ventral midbrain lead to seizure resistance and neuroprotection.The altered maturation of cortical PV inhibitory neurons seems to be a common trait observed in Otx mutants, with Otx1 and Otx2 having similar effects: a marked reduction of PV cells is observed in Otx1 -/-mice [14,41] , and an increased number of PV cells is detected in mice overexpressing Otx2.Thus, Otx factors seem to be crucial determinants of PV cells maturation in cortical circuits, with functional consequences on seizure susceptibility.Further studies should be addressed to understand whether these mechanisms take place also in the epileptic human brain.

Figure 1 .
Figure 1.Schematic representation of brain anatomical abnormalities relevant to seizure phenotypes in adult Otx mutant mice.A) Distribution of pyramidal (excitatory) and PV-positive (inhibitory) neurons and major DA/5-HT are illustrated for wild-type (WT) mice.B) Otx1 -/-mice.These mutants present a disorganized cerebral cortex characterized by ectopic pyramidal neurons and patchy distribution of PV interneurons.DA/5-HT pathways are not reported since they have not been studied in these mutants.C) Conditional deletion of Otx2 in mesDA precursors results in 5-HT hyperinnervation and resistance to KA seizures.D) Conditional overexpression of Otx2 in mesDA precursors results in DA hyperinnervation and increased number of PV interneurons.Symbols: DA and 5-HT pathways are indicated in purple and cyan, respectively; orange circles indicate PV interneurons; black triangles indicate pyramidal neurons; dashed lines and Roman numbers indicate cortical layers.Abbreviations: CTX, cerebral cortex; Raphe, raphe nuclei; STR, striatum; VMB, ventral midbrain; other abbreviations are as in the text.See text andTable 1 for details and references.

Table 1
for details and references.