BACKGROUND
the lgi <dig> gene  <cit>  predisposes to the development of a rare form of partial epilepsy with autosomal dominant inheritance and high penetrance  <cit> , referred to both as autosomal dominant partial epilepsy with auditory features   <cit>  and autosomal dominant temporal lobe epilepsy   <cit> .
it has also been shown recently that lgi <dig> is the auto antigen responsible for limbic encephalitis, previously thought to be caused by autoantibodies against voltage gated potassium channels  <cit> .
these patients are part of the group of autoimmune synaptic encephalopathies and exhibit seizures and neuropsychiatric disorders.
lgi <dig> is a secreted protein  <cit>  and has also been implicated in cell movement and invasion in glioma cells  <cit>  through suppression of the erk signaling pathway that results in down regulation of matrix metalloproteinases  <cit> .
recent studies have shown that lgi <dig> interacts with a number of different proteins that are related to synaptic function  <cit> , notably members of the adam family of proteins , which represent some of the lgi <dig> receptors  <cit> .
most recently  <cit> , lgi <dig> was also shown to interact with the nogo receptor  <dig>  and enhances neuronal growth on myelin based inhibitory substrates and antagonizes myelin-induced growth cone collapse.
mutant null mice for lgi <dig> show early onset seizures  <cit> , as do mice null for adam <dig> and adam <dig>  <cit> .
thus, lgi <dig> represents the only hereditary epilepsy predisposition gene which is not a channelopathy and offers the opportunity to understand different mechanisms behind seizures in these individuals.
analysis of mutant null mice has demonstrated that lgi <dig> is clearly involved in synapse transmission  <cit> , although, whether it also plays a role in normal brain development is not clear.
although abnormal structures within the brains of adlte patients have been reported by several groups  <cit> , the level of resolution afforded using current imagining technologies precludes detailed evaluations of these potential abnormalities.
clearly, a better understanding of the cell lineages which express lgi <dig> during mammalian brain development will be important in defining its overall function.
extensive analysis of gene expression in the brain of adult mice demonstrated that the hippocampus and cortex were the predominant regions expressing lgi <dig>  <cit> , even though the resolution of the cell lineages involved was relatively low using this approach.
immunohistochemical studies in humans have confirmed these broad observations, although many of the antibodies used in these studies have either been shown to cross react with other members of the lgi <dig> family of proteins  <cit>  or have not been tested for cross reactivity.
to overcome this potential limitation, we developed a bac transgenic mouse in which the gfp reporter gene was placed under the control of lgi <dig> cis-regulatory elements  <cit> .
in these bac transgenics the gfp gene is expressed in cells which have the transcriptional capability of expressing the endogenous lgi <dig> gene, and these cells can be identified using conventional immunohistochemistry.
this system has been extensively used as part of the gensat program to define expression patterns of genes expressed in the central nervous system of adult mice  <cit> .
to date, however, there have been no reported studies of lgi <dig> expression patterns at the cellular level during embryonic development.
in this report, we describe an immunohistochemical study of the developing brain and associated structures of the nervous system from embryonic days e <dig> -e <dig>  and postnatal days p1-p <dig>  these studies show that lgi <dig> expression is widespread in the developing brain.
more interestingly, co-staining studies shown that lgi <dig> is expressed in migratory neuroblast cell lineages.
RESULTS
the lgi <dig> bac transgenic system
bac transgenic technology is now well established  <cit>  and has been used extensively to define gene expression patterns through the insertion of a gfp reporter into the target gene in the context of its endogenous cis-regulatory elements  <cit> .
thus, with the caveat that regulatory elements that lie some distance away from the promoter may not be present on the bac, any cell capable of activating the endogenous promoter of a given gene will also activate the reporter gene carried on the transgenic bac clone.
this system has been developed to overcome many shortcomings of antibodies, whose limitations may include, low specificity for the target protein, cross reactivity with homologous proteins, or poor recognition of the endogenous protein in its native conformation.
it should be noted, however, that gfp is only a reporter for endogenous gene expression patterns and does not define the intracellular localization of the protein it reports.
as such, the intracellular fluorescence signal reflects the ubiquitous distribution of gfp protein.
typically, multiple tandem bac integrations occur during construction of the transgenic animals which fortuitously provides a strong gfp signal.
in our case, > <dig> copies of the bac are present in mice used in this study  <cit> .
this reporter system, therefore, has the added advantage of allowing the visualization of expression patterns for genes which normally show low levels of expression or which, like lgi <dig>  <cit> , are normally rapidly secreted and so may be more difficult to visualize using traditional antibodies.
importantly, since the bac shows multiple tandem integration events  <cit> , the intensity of the gfp fluorescence does not reflect the intensity of the endogenous gene expression, although relative expression levels between different cells in the same field can be used as a means of determining comparative lgi <dig> expression levels.
lgi <dig> expression during embryonic development
to investigate the distribution of lgi <dig> expression in the mouse brain during development, we collected different embryonic/postnatal stage brains from the egfp-reporter bac transgenic mice, which we previously showed can be used to define lgi <dig> expression patterns at the cellular level  <cit> .
in this analysis we focused on the brain regions that have been implicated in epilepsy, but did not restrict the analysis to the development of specific systems.
if lgi <dig> influences normal development, then establishing its overall expression pattern during brain development would be important.
most of the sections used in this study were mid-coronal and mid-sagittal, since these planes allow a symmetrical view of the tissue that permits a comprehensive visualization of areas of interest, such as neocortex, hippocampus, nerve ganglia, and sensory organs.
using this strategy, we characterized lgi <dig> expression patterns during early development .
a summary of temporal expression patterns in major regions of the brain is given in figure  <dig> and specific details at each developmental stage are described below.
e <dig>  represents the earliest embryonic stage in our analysis, partly because of the technical difficulties associated with processing earlier embryos, and partly because prior to this stage, brain development is still rudimentary.
at e <dig> , the neural tube is still developing and the structures that will eventually give rise to the brain are largely absent.
at this embryonic stage, lgi <dig> was prominently expressed in the primitive eye .
lgi <dig> was also detected in the skin ectoderm , the mesenchymal connective tissue, the optic vesicle, and the neuroepithelium lining the neural tube .
following neural tube closure at embryonic stage e <dig> , lgi <dig> expression was found in sensory and secretory organs, such as olfactory placode  and the peripheral margin of rathke's pouch , which gives rise to the anterior pituitary.
additionally, strong expression could be seen along the roof of the hindbrain  and midbrain mesenchymal connective tissue .
other notable areas of lgi <dig> expression at e <dig>  included ganglionic tissues, such as facio-acoustic ganglion  and dorsal ganglia .
in the forebrain, lgi <dig> showed strong expression in the skin ectoderm, lamina terminalis, and the mandibular component of the first branchial arch , which gives rises the glands of the anterior two thirds of the tongue and ossicles of the middle ear.
lgi <dig> expression became notably more extensive throughout the developing brain with increased expression in the forebrain, primarily along the wall of the telencephalic vesicle, although weak expression of lgi <dig> was seen in the primitive neocortex.
at embryonic stage e <dig> , several cranial nerve ganglia showed prominent expression, particularly in the relatively large trigeminal ganglion .
in addition, lgi <dig> expression was strong in the optic stalk and optic cup .
as the brain developed, lgi <dig> expression at e <dig>  was observed in the roof of the neopallial cortex , choroid plexus, and areas of the midbrain, such as the pretectal area, the anterior ventral tegmentum, tectum, and superior colliculus .
more caudal structures, such as the inferior colliculus and areas of the hindbrain  showed moderate levels of lgi <dig> expression.
by embryonic stage e <dig> , lgi <dig> expression was observed in the roof of the neopallial cortex, areas of the diencephalon and cranial nerve ganglia, such as trigeminal, facio-acoustic, and vagus.
strong expression was found in the periventricular zone, ganglionic eminence , and medulla oblongata .
expression in the ganglionic eminence was now detected for the first time.
the ganglionic eminence contains progenitor cells that are destined to become part of the olfactory cortex as well as the striatum  <cit> .
lgi <dig> was also found to be expressed in several non neural tissues, such as the tongue  and lower molar region  which represents the first signs of ossification.
lgi <dig> was now strongly expressed in the choroid plexus .
e <dig>  lgi <dig> expression began to subside in the olfactory lobes by this embryonic stage, as soon as the bulbs became apparent.
lgi <dig> was also detected in cells of the emerging hippocampus, which appeared as a thin line of cells  surrounding the cortical neuroepithelium.
within the telencephalon, high levels of expression were found in the basal telencephalic plate, specifically in the posterior region .
cells from this area give rise to the corpus striatum, which becomes part of the basal ganglia.
moderate lgi <dig> expression was seen in the cortical neuroepithelium and periventricular zone .
the superior sagittal sinus mesenchymal connective tissue and skin ectoderm, as well as the thalamus, was generally devoid of lgi <dig> expression at this stage, although several structures immediately superior to the thalamus, including the pretectum, showed moderate levels of lgi <dig> expression.
expression in the developing cerebellum at this stage of development was weak.
in contrast, strong levels of lgi <dig> expression were seen in septal neuroepithelium, cranial nerve ganglia of the vestibulocochlear and vagus, vomeronasal, olfactory and maxillary nerves as well as the choroid plexus.
at this point of the mouse development, isolated patches of expression were also seen in non-neuronal organs, such as the cartilage primordium of the vertebra, the tongue and molar tooth, demonstrating that lgi <dig> expression is not restricted to neuronal tissues as suggested previously in adult animals  <cit> .
by e <dig>  , lgi <dig> expression patterns largely reflected those observed at e <dig>  and e <dig> , although as embryonic growth continued, the regions showing lgi <dig> expression became more extensive with the increasing size of the brain.
deep within the telencephalon, the ganglionic eminence  continued to show lgi <dig> expression.
strong expression was also seen in the ventricular zone .
thalamic cells appear not to express lgi <dig>  although some areas showed relatively weak expression.
robust lgi <dig> expression was found in the lower tegmental neuroepithelium, mammalary body, and posterior hypothalamus neuroepithelium .
high levels of lgi <dig> expression were now found within the subpial and parenchyma layers of the medulla oblongata .
expression in the eye , tongue  , merckel's cartilage, jaw, and ganglia, as well as in the sphenoid palatine ganglion , remained strong at this stage.
by embryonic day  <dig>  , lgi <dig> expression patterns became more defined in specific regions, such as the subventricular zone, hippocampus, choroid plexus, sensory organs, and nerve ganglia.
strong expression was seen in the hypothalamus regions, including the anterior hypothalamic area, paraventricular hypothalamic nucleus, and zona incerta .
the cerebral cortex showed low levels of expression, although a thin layer of cells expressing lgi <dig> could be seen above the genu of the corpus callosum, which marked the formation of the subplate.
figure 7a.
<dig> shows expression of lgi <dig> in the cortical plate, intermediate cortical zone, and subventricular zone.
by this time, the hippocampus had also undergone further differentiation, but retains lgi <dig> expression, albeit at lower levels, remaining highly expressed in the dentate gyrus .
lgi <dig> had been weakly expressed in thalamus in the earlier embryonic stages and still appeared weak in the later embryonic stages.
strong expression continues to be seen in the choroid plexus.
lgi <dig> expression in the early postnatal stages of the murine brain
at postnatal day one , moderate to low expression of lgi <dig> was present in several areas described above .
the lowest levels of lgi <dig> expression were seen along the neocortex, and were almost undetectable in the frontal and cingulated cortices.
expression levels were weak in the basal ganglia, although the stria terminalis showed higher levels of lgi <dig> expression .
thalamic and hypothalamic areas also showed very weak to absent expression, even though low-level expression was detected around the third ventricle, which represents the medial habenular nuclear.
the hippocampus now showed weak expression in ca <dig> and ca <dig> areas, although moderate expression was retained in the dentate gyrus as well as in the inferior tectal neuroepithelium and superior cerebellar peduncle .
within the cerebral cortex at stage p <dig>  moderate expression levels were seen in layers ii/iii and v of the neocortex.
expression in the hippocampus was maintained unchanged at this stage, with moderate levels seen in the dentate gyrus.
we also observed lgi <dig> expressing cells in the striatum , as shown in figures 8b.
<dig> and 8b.
<dig>  within the thalamus, lgi <dig> protein levels were still weak, although the paraventricular thalamic region showed strong expression at this stage .
in the midbrain, expression was seen in the pretectal and tectal regions, tegmentum, and both superior and inferior colliculi, which became more diffuse at this stage.
within the pons, cerebellum and medulla oblongata the expression was now either weak or absent.
the choroid plexus, however, still shows high levels of lgi <dig> expression .
lgi <dig> expression in the later postnatal stages of the murine brain
in later postnatal stages , lgi <dig> expression levels were essentially the same , with strong expression in the choroid plexus, but in regions such as the neocortex, hypothalamus, midbrain, hippocampus, and cerebellum, expression was greatly reduced compared with embryonic stages described above .
expression in the medulla oblongata was absent compared to earlier embryonic stages where its expression was strong.
the cerebral cortex now generally showed weak lgi <dig> expression, although lgi <dig> expressing cells were seen in areas of the frontal cortex  and cingulum .
the globus pallidus showed little or no expression, while the striatum  showed relatively low levels of lgi <dig> expression.
within the hippocampus, an area typically showing high levels of lgi <dig> in the dentate gyrus, lower expression levels were observed throughout the region at these stages.
both the hypothalamus and thalamus showed little or no lgi <dig> expression.
sagittal sections through the brain at p <dig> and p <dig> demonstrated that, although the thalamus expressed lgi <dig> weakly overall, the paraventricular thalamic region appeared to have stronger lgi <dig> expression .
in the dorsal midbrain, the pretectal nuclei showed weak expression as did the periaqueductal gray, an area surrounding the cerebral aqueduct, which showed moderate expression.
more posteriorly, juxtaposed to the unstained superior colliculi, the inferior colliculi showed moderate levels of lgi <dig> expression.
in these stages, the pons and medulla oblongata did not express lgi <dig>  the cerebellum, although nearly completely devoid of lgi <dig> expression, showed scattered staining in the purkinje cells as we described previously  <cit> .
figure 9c.
<dig> shows lgi <dig> expression in the granular cell layer of the cerebellar cortex in p <dig> brain.
weak or no lgi <dig> signal was seen in the molecular layer of the cerebellar cortex.
the choroid plexus continued to show high expression levels at these stages.
co-expression of lgi <dig> with nestin, doublecortin, and beta-iii tubulin
in the developing brain at embryonic stages e <dig>  and e <dig> , co-expression of lgi <dig>  was seen with nestin , dcx , and beta-iii tubulin  in neural structures such as the optic vesicle epithelium , cephalic mesenchyme , and pretectal neuroepithelium .
as the brain developed , co-expression of lgi <dig> and nestin/dcx was observed in the ganglionic eminence , basal telencephalic plate , and medulla oblongata .
at embryonic stage e <dig> , co-expression of lgi <dig>  dcx and nestin was seen within the subpial and parenchyma layers of the medulla oblongata .
this nestin/dcx/lgi <dig> co-expression is typical of migrating neuroblasts.
beta-iii tubulin expression was found for the first time in the cortical plate at embryonic stage e <dig>   and overlapped with lgi1-expressing cells.
beta-iii tubulin has been suggested to be one of the earliest markers to signal neuronal commitment in primitive neuroepithelium.
co-expression of slug , rc <dig> , ng <dig> , and map <dig>  was not found in lgi1-expressing cells at the time points used in this study.
discussion
the underlying mechanism behind the genetic predisposition to epilepsy conferred by mutations in the lgi <dig> gene is still poorly understood.
although evidence derived from focused electrophysiological studies implicate abnormalities in synaptic transmission leading to hyperexcitability  <cit> , there have also been suggestions that lgi <dig> may also have a fundamental role in brain development  <cit> , which may contribute to the seizure phenotype.
in a bac transgenic model expressing a truncated lgi <dig> protein, for example, mutant lgi <dig> led to neuronal restructuring involving inhibition of dendritic pruning and an increase in spine density  <cit> .
gross anatomical analysis of the brain in lgi <dig> mutant null mice  <cit>  however, did not reveal any major structural defects, although subtle changes could not be ruled out at this level of analysis.
in humans, although imaging studies have been inconsistent  <cit> , there are suggestions of abnormalities and reduced focal brain mass in adtle patients.
also, in our zebrafish studies, knockdown of lgi <dig> in embryos resulted in increased apoptosis in subregions of the brain, leading to an overall reduction in brain size  <cit> .
with the possibility that lgi <dig> may play a role in overall brain development, and neuronal cell positioning in particular, it became important to define the structures in the developing brain that express lgi <dig>  since these are the cell lineages that are potentially most affected by loss of function.
in this study we have broadly defined the temporal and spatial expression pattern in the developing brain and have demonstrated that, unlike the more restricted pattern seen in the early postnatal and adult mouse brain, expression of lgi <dig> is widespread and highly orchestrated in a variety of cell types and is not restricted to differentiated neurons.
evidence from our previous in vitro gene expression studies suggested that lgi <dig> may have a role in axon guidance and cell migration  <cit> .
consistent with this suggestion, high levels of lgi <dig> expression were found in regions of the developing brain such as the ganglionic eminence , medulla oblongata , ventricular zone  and telencephalon where mitotic neuronal precursors are located, and from which they migrate to populate the cortex and other areas.
lgi <dig> expressing cells in the pretectal area, ge, mo and telencephalon showed coexpression of lgi <dig> with dcx  and nestin .
nestin is expressed transiently during development and does not persist through adulthood, except in neuroprogenitor cells of the subventricular zone  <cit>  which still showed lgi <dig> expression in e <dig>  embryos.
interestingly, the presence of cells co-expressing all three genes in this area suggests that these cells are migrating immature neurons.
also, the presence of oriented gfp-positive fibers in the subpial and parenchyma layers of the medulla oblongata suggest that lgi <dig> may be involved in the relocation of immature neurons in the mouse subpial medullary region and may play a role in the migration of medullary precerebellar neurons in the caudal medulla  <cit> .
evidence from studies in adult brain  <cit> , suggest that expression of lgi <dig> is largely restricted to the hippocampus and cortical neurons.
however, in our study we have shown that lgi <dig> expression can be detected in the cephalic neuroepithelium at e <dig> , which coincides with the first signs of neurogenesis in the mouse  <cit> .
the appearance of lgi <dig> expression in the cerebral cortex begins at ~e <dig> , and peaks at e <dig> .
as development progresses, expression levels in the cortex progressively diminish.
on the other hand, lgi <dig> appears not to be involved in early differentiation and maturation of glial lineage since lgi1-expressing cells do not coexpress rc <dig>  or ng <dig> .
even thought differentiated neurons expressed lgi <dig>  many of these cells do not express map <dig> at the observed stages.
we speculate that once these cells have migrated to their final destinations, lgi <dig> expression levels decay.
lgi <dig> was also found to be strongly expressed in the basal telencephalic plates that will develop into the basal ganglia associated with motor functions.
dcx-expressing cells in the ventral portion of the basal telencephalic plates also express lgi <dig>  suggesting that these dcx-lgi1-expressing cells migrate and will populate cortical and subcortical regions.
in addition, marín et al  <cit>  argue that patterning of the basal telencepahlon is crucial for the growth of cortical axons as well as for guiding cortical projections involved in sensory-motor information.
the presence of lgi <dig> in migrating neuroblasts is consistent with a function in cortical and medullar axon guidance, as suggested by our gene expression studies in model cell systems  <cit> .
beta-iii tubulin is suggested to be one of the earliest markers to signal neuronal commitment in primitive neuroepithelium, and it is accepted as a neuron-specific protein marker, which is highly expressed in cortex at birth and then its expression levels decrease with increasing postnatal development  <cit> .
during brain development, the maturation of neuroblasts into neurons is accompanied by up-regulation of beta-iii tubulin.
coexpression of lgi <dig> and beta-iii tubulin was detected in the ventricular and subventricular zones, as well as the cortical plate, suggesting a conserved neurogenic program in these cells from embryonic stages to adult life, which further implies that lgi <dig> may participate in normal neuronal development.
we also observed lgi <dig> expression in many areas of the developing sensory system, for example, the tongue , eye, and olfactory bulb.
the olfactory system is anatomically connected to the amygdala, lateral septum, hypothalamus and the hippocampus, which are part of the limbic system  <cit>  and which also express lgi <dig>  this correlation is consistent with a role for lgi <dig> in the development of limbic encephalitis  <cit> .
within the hippocampus, expression levels are found primarily in the dentate gyrus and scarcely within the ca1-ca <dig> region of the pyramidal cell layer.
there is evidence that lgi <dig> also regulates intra-hippocampal circuit formation and the dentate gyrus plays a critical role in seizures, since it appears to have low epileptogenic thresholds  <cit> .
although thalamic protein levels are weak overall, the paraventricular thalamic area expresses lgi <dig>  according to huang et al  <cit> , the thalamic paraventricular nucleus receives the highest innervation from hypothalamic hypocretin/orexin neurons, generating substantial excitation of the medial prefrontal cortex.
major inputs into the anterior thalamic group are from the hypothalamus, the hippocampus, and the tectum while output from this region is mainly to the cingulate gyrus.
in the developing mouse eye, lgi <dig> expression was seen in the optic vesicle, optic stalk and optic cup, as well as the neural retina and the retinal pigment epithelium.
lgi <dig> expression is seen in the retinal ganglion layer, and dominant-negative mutations in transgenic mice lead to abnormal postnatal pruning of retinal axons in the visual relay thalamus  <cit> .
in zebrafish studies, lgi <dig> expression was seen in the epithelium surrounding the lumen of the optic vesicle  <cit>  and interestingly, in early zebrafish embryos, loss of lgi <dig> results in abnormal eye development  <cit> , supporting its critical role in development of this organ.
CONCLUSIONS
several general patterns of lgi <dig> distribution were apparent during embryonic brain development.
lgi <dig> expression appears to spread throughout the dorsal-most brain structures with only weak levels in the ventral regions in early embryo stages within the medulla oblongata showing the strongest expression.
this dorsoventral differentiation also appears in the hippocampus, where the dentate gyrus showed more robust expression.
lgi <dig> expression also appears to follow a superficial to deep, as well as a caudal to cephalic axis.
at e <dig> , the strongest signal was found in the surface ectoderm, as well as the infundibular recess of the diencephalon and dorsal midbrain in the pretectal neuroepithelium.
as development progresses, lgi <dig> expression moves interiorly into the neocortex and other more caudal structures, expression in the pretectum and diencephalon areas begins to subside, as shown by weak expression in basal ganglia and thalamic nuclei, although expression in the paraventricular thalamic nucleus remains strong at p <dig>  overall, the spatial and temporal expression profile for lgi <dig> during embryogenesis and postnatal stages is consistent with the idea that it plays an important role in normal brain development, especially neuronal migration and synapse formation.

 the lgi gene predisposes to the development of a rare form of partial epilepsy with autosomal dominant inheritance and high penetrance, referred to both as autosomal dominant partial epilepsy with auditory features and autosomal dominant temporal lobe epilepsy. it has also been shown recently that lgi is the auto antigen responsible for limbic encephalitis, previously thought to be caused by autoantibodies against voltage gated potassium channels. these patients are part of the group of autoimmune synaptic encephalopathies and exhibit seizures and neuropsychiatric disorders. lgi is a secreted protein and has also been implicated in cell movement and invasion in glioma cells through suppression of the erk signaling pathway that results in down regulation of matrix metalloproteinases. recent studies have shown that lgi interacts with a number of different proteins that are related to synaptic function, notably members of the adam family of proteins, which represent some of the lgi receptors. most recently, lgi was also shown to interact with the nogo receptor and enhances neuronal growth on myelin based inhibitory substrates and antagonizes myelin-induced growth cone collapse. mutant null mice for lgi show early onset seizures, as do mice null for adam and adam. thus, lgi represents the only hereditary epilepsy predisposition gene which is not a channelopathy and offers the opportunity to understand different mechanisms behind seizures in these individuals. analysis of mutant null mice has demonstrated that lgi is clearly involved in synapse transmission, although, whether it also plays a role in normal brain development is not clear. although abnormal structures within the brains of adlte patients have been reported by several groups, the level of resolution afforded using current imagining technologies precludes detailed evaluations of these potential abnormalities. clearly, a better understanding of the cell lineages which express lgi during mammalian brain development will be important in defining its overall function. extensive analysis of gene expression in the brain of adult mice demonstrated that the hippocampus and cortex were the predominant regions expressing lgi, even though the resolution of the cell lineages involved was relatively low using this approach. immunohistochemical studies in humans have confirmed these broad observations, although many of the antibodies used in these studies have either been shown to cross react with other members of the lgi family of proteins or have not been tested for cross reactivity. to overcome this potential limitation, we developed a bac transgenic mouse in which the gfp reporter gene was placed under the control of lgi cis-regulatory elements. in these bac transgenics the gfp gene is expressed in cells which have the transcriptional capability of expressing the endogenous lgi gene, and these cells can be identified using conventional immunohistochemistry. this system has been extensively used as part of the gensat program to define expression patterns of genes expressed in the central nervous system of adult mice. to date, however, there have been no reported studies of lgi expression patterns at the cellular level during embryonic development. in this report, we describe an immunohistochemical study of the developing brain and associated structures of the nervous system from embryonic days e -e and postnatal days p1-p these studies show that lgi expression is widespread in the developing brain. more interestingly, co-staining studies shown that lgi is expressed in migratory neuroblast cell lineages.  the lgi bac transgenic system bac transgenic technology is now well established and has been used extensively to define gene expression patterns through the insertion of a gfp reporter into the target gene in the context of its endogenous cis-regulatory elements. thus, with the caveat that regulatory elements that lie some distance away from the promoter may not be present on the bac, any cell capable of activating the endogenous promoter of a given gene will also activate the reporter gene carried on the transgenic bac clone. this system has been developed to overcome many shortcomings of antibodies, whose limitations may include, low specificity for the target protein, cross reactivity with homologous proteins, or poor recognition of the endogenous protein in its native conformation. it should be noted, however, that gfp is only a reporter for endogenous gene expression patterns and does not define the intracellular localization of the protein it reports. as such, the intracellular fluorescence signal reflects the ubiquitous distribution of gfp protein. typically, multiple tandem bac integrations occur during construction of the transgenic animals which fortuitously provides a strong gfp signal. in our case, > copies of the bac are present in mice used in this study. this reporter system, therefore, has the added advantage of allowing the visualization of expression patterns for genes which normally show low levels of expression or which, like lgi, are normally rapidly secreted and so may be more difficult to visualize using traditional antibodies. importantly, since the bac shows multiple tandem integration events, the intensity of the gfp fluorescence does not reflect the intensity of the endogenous gene expression, although relative expression levels between different cells in the same field can be used as a means of determining comparative lgi expression levels. lgi expression during embryonic development to investigate the distribution of lgi expression in the mouse brain during development, we collected different embryonic/postnatal stage brains from the egfp-reporter bac transgenic mice, which we previously showed can be used to define lgi expression patterns at the cellular level. in this analysis we focused on the brain regions that have been implicated in epilepsy, but did not restrict the analysis to the development of specific systems. if lgi influences normal development, then establishing its overall expression pattern during brain development would be important. most of the sections used in this study were mid-coronal and mid-sagittal, since these planes allow a symmetrical view of the tissue that permits a comprehensive visualization of areas of interest, such as neocortex, hippocampus, nerve ganglia, and sensory organs. using this strategy, we characterized lgi expression patterns during early development. a summary of temporal expression patterns in major regions of the brain is given in figure and specific details at each developmental stage are described below. e represents the earliest embryonic stage in our analysis, partly because of the technical difficulties associated with processing earlier embryos, and partly because prior to this stage, brain development is still rudimentary. at e, the neural tube is still developing and the structures that will eventually give rise to the brain are largely absent. at this embryonic stage, lgi was prominently expressed in the primitive eye. lgi was also detected in the skin ectoderm, the mesenchymal connective tissue, the optic vesicle, and the neuroepithelium lining the neural tube. following neural tube closure at embryonic stage e, lgi expression was found in sensory and secretory organs, such as olfactory placode and the peripheral margin of rathke's pouch, which gives rise to the anterior pituitary. additionally, strong expression could be seen along the roof of the hindbrain and midbrain mesenchymal connective tissue. other notable areas of lgi expression at e included ganglionic tissues, such as facio-acoustic ganglion and dorsal ganglia. in the forebrain, lgi showed strong expression in the skin ectoderm, lamina terminalis, and the mandibular component of the first branchial arch, which gives rises the glands of the anterior two thirds of the tongue and ossicles of the middle ear. lgi expression became notably more extensive throughout the developing brain with increased expression in the forebrain, primarily along the wall of the telencephalic vesicle, although weak expression of lgi was seen in the primitive neocortex. at embryonic stage e, several cranial nerve ganglia showed prominent expression, particularly in the relatively large trigeminal ganglion. in addition, lgi expression was strong in the optic stalk and optic cup. as the brain developed, lgi expression at e was observed in the roof of the neopallial cortex, choroid plexus, and areas of the midbrain, such as the pretectal area, the anterior ventral tegmentum, tectum, and superior colliculus. more caudal structures, such as the inferior colliculus and areas of the hindbrain showed moderate levels of lgi expression. by embryonic stage e, lgi expression was observed in the roof of the neopallial cortex, areas of the diencephalon and cranial nerve ganglia, such as trigeminal, facio-acoustic, and vagus. strong expression was found in the periventricular zone, ganglionic eminence, and medulla oblongata. expression in the ganglionic eminence was now detected for the first time. the ganglionic eminence contains progenitor cells that are destined to become part of the olfactory cortex as well as the striatum. lgi was also found to be expressed in several non neural tissues, such as the tongue and lower molar region which represents the first signs of ossification. lgi was now strongly expressed in the choroid plexus. e lgi expression began to subside in the olfactory lobes by this embryonic stage, as soon as the bulbs became apparent. lgi was also detected in cells of the emerging hippocampus, which appeared as a thin line of cells surrounding the cortical neuroepithelium. within the telencephalon, high levels of expression were found in the basal telencephalic plate, specifically in the posterior region. cells from this area give rise to the corpus striatum, which becomes part of the basal ganglia. moderate lgi expression was seen in the cortical neuroepithelium and periventricular zone. the superior sagittal sinus mesenchymal connective tissue and skin ectoderm, as well as the thalamus, was generally devoid of lgi expression at this stage, although several structures immediately superior to the thalamus, including the pretectum, showed moderate levels of lgi expression. expression in the developing cerebellum at this stage of development was weak. in contrast, strong levels of lgi expression were seen in septal neuroepithelium, cranial nerve ganglia of the vestibulocochlear and vagus, vomeronasal, olfactory and maxillary nerves as well as the choroid plexus. at this point of the mouse development, isolated patches of expression were also seen in non-neuronal organs, such as the cartilage primordium of the vertebra, the tongue and molar tooth, demonstrating that lgi expression is not restricted to neuronal tissues as suggested previously in adult animals. by e, lgi expression patterns largely reflected those observed at e and e, although as embryonic growth continued, the regions showing lgi expression became more extensive with the increasing size of the brain. deep within the telencephalon, the ganglionic eminence continued to show lgi expression. strong expression was also seen in the ventricular zone. thalamic cells appear not to express lgi although some areas showed relatively weak expression. robust lgi expression was found in the lower tegmental neuroepithelium, mammalary body, and posterior hypothalamus neuroepithelium. high levels of lgi expression were now found within the subpial and parenchyma layers of the medulla oblongata. expression in the eye, tongue, merckel's cartilage, jaw, and ganglia, as well as in the sphenoid palatine ganglion, remained strong at this stage. by embryonic day, lgi expression patterns became more defined in specific regions, such as the subventricular zone, hippocampus, choroid plexus, sensory organs, and nerve ganglia. strong expression was seen in the hypothalamus regions, including the anterior hypothalamic area, paraventricular hypothalamic nucleus, and zona incerta. the cerebral cortex showed low levels of expression, although a thin layer of cells expressing lgi could be seen above the genu of the corpus callosum, which marked the formation of the subplate. figure 7a. shows expression of lgi in the cortical plate, intermediate cortical zone, and subventricular zone. by this time, the hippocampus had also undergone further differentiation, but retains lgi expression, albeit at lower levels, remaining highly expressed in the dentate gyrus. lgi had been weakly expressed in thalamus in the earlier embryonic stages and still appeared weak in the later embryonic stages. strong expression continues to be seen in the choroid plexus. lgi expression in the early postnatal stages of the murine brain at postnatal day one, moderate to low expression of lgi was present in several areas described above. the lowest levels of lgi expression were seen along the neocortex, and were almost undetectable in the frontal and cingulated cortices. expression levels were weak in the basal ganglia, although the stria terminalis showed higher levels of lgi expression. thalamic and hypothalamic areas also showed very weak to absent expression, even though low-level expression was detected around the third ventricle, which represents the medial habenular nuclear. the hippocampus now showed weak expression in ca and ca areas, although moderate expression was retained in the dentate gyrus as well as in the inferior tectal neuroepithelium and superior cerebellar peduncle. within the cerebral cortex at stage p moderate expression levels were seen in layers ii/iii and v of the neocortex. expression in the hippocampus was maintained unchanged at this stage, with moderate levels seen in the dentate gyrus. we also observed lgi expressing cells in the striatum, as shown in figures 8b. and 8b. within the thalamus, lgi protein levels were still weak, although the paraventricular thalamic region showed strong expression at this stage. in the midbrain, expression was seen in the pretectal and tectal regions, tegmentum, and both superior and inferior colliculi, which became more diffuse at this stage. within the pons, cerebellum and medulla oblongata the expression was now either weak or absent. the choroid plexus, however, still shows high levels of lgi expression. lgi expression in the later postnatal stages of the murine brain in later postnatal stages, lgi expression levels were essentially the same, with strong expression in the choroid plexus, but in regions such as the neocortex, hypothalamus, midbrain, hippocampus, and cerebellum, expression was greatly reduced compared with embryonic stages described above. expression in the medulla oblongata was absent compared to earlier embryonic stages where its expression was strong. the cerebral cortex now generally showed weak lgi expression, although lgi expressing cells were seen in areas of the frontal cortex and cingulum. the globus pallidus showed little or no expression, while the striatum showed relatively low levels of lgi expression. within the hippocampus, an area typically showing high levels of lgi in the dentate gyrus, lower expression levels were observed throughout the region at these stages. both the hypothalamus and thalamus showed little or no lgi expression. sagittal sections through the brain at p and p demonstrated that, although the thalamus expressed lgi weakly overall, the paraventricular thalamic region appeared to have stronger lgi expression. in the dorsal midbrain, the pretectal nuclei showed weak expression as did the periaqueductal gray, an area surrounding the cerebral aqueduct, which showed moderate expression. more posteriorly, juxtaposed to the unstained superior colliculi, the inferior colliculi showed moderate levels of lgi expression. in these stages, the pons and medulla oblongata did not express lgi the cerebellum, although nearly completely devoid of lgi expression, showed scattered staining in the purkinje cells as we described previously. figure 9c. shows lgi expression in the granular cell layer of the cerebellar cortex in p brain. weak or no lgi signal was seen in the molecular layer of the cerebellar cortex. the choroid plexus continued to show high expression levels at these stages. co-expression of lgi with nestin, doublecortin, and beta-iii tubulin in the developing brain at embryonic stages e and e, co-expression of lgi was seen with nestin, dcx, and beta-iii tubulin in neural structures such as the optic vesicle epithelium, cephalic mesenchyme, and pretectal neuroepithelium. as the brain developed, co-expression of lgi and nestin/dcx was observed in the ganglionic eminence, basal telencephalic plate, and medulla oblongata. at embryonic stage e, co-expression of lgi dcx and nestin was seen within the subpial and parenchyma layers of the medulla oblongata. this nestin/dcx/lgi co-expression is typical of migrating neuroblasts. beta-iii tubulin expression was found for the first time in the cortical plate at embryonic stage e and overlapped with lgi1-expressing cells. beta-iii tubulin has been suggested to be one of the earliest markers to signal neuronal commitment in primitive neuroepithelium. co-expression of slug, rc, ng, and map was not found in lgi1-expressing cells at the time points used in this study. discussion the underlying mechanism behind the genetic predisposition to epilepsy conferred by mutations in the lgi gene is still poorly understood. although evidence derived from focused electrophysiological studies implicate abnormalities in synaptic transmission leading to hyperexcitability, there have also been suggestions that lgi may also have a fundamental role in brain development, which may contribute to the seizure phenotype. in a bac transgenic model expressing a truncated lgi protein, for example, mutant lgi led to neuronal restructuring involving inhibition of dendritic pruning and an increase in spine density. gross anatomical analysis of the brain in lgi mutant null mice however, did not reveal any major structural defects, although subtle changes could not be ruled out at this level of analysis. in humans, although imaging studies have been inconsistent, there are suggestions of abnormalities and reduced focal brain mass in adtle patients. also, in our zebrafish studies, knockdown of lgi in embryos resulted in increased apoptosis in subregions of the brain, leading to an overall reduction in brain size. with the possibility that lgi may play a role in overall brain development, and neuronal cell positioning in particular, it became important to define the structures in the developing brain that express lgi since these are the cell lineages that are potentially most affected by loss of function. in this study we have broadly defined the temporal and spatial expression pattern in the developing brain and have demonstrated that, unlike the more restricted pattern seen in the early postnatal and adult mouse brain, expression of lgi is widespread and highly orchestrated in a variety of cell types and is not restricted to differentiated neurons. evidence from our previous in vitro gene expression studies suggested that lgi may have a role in axon guidance and cell migration. consistent with this suggestion, high levels of lgi expression were found in regions of the developing brain such as the ganglionic eminence, medulla oblongata, ventricular zone and telencephalon where mitotic neuronal precursors are located, and from which they migrate to populate the cortex and other areas. lgi expressing cells in the pretectal area, ge, mo and telencephalon showed coexpression of lgi with dcx and nestin. nestin is expressed transiently during development and does not persist through adulthood, except in neuroprogenitor cells of the subventricular zone which still showed lgi expression in e embryos. interestingly, the presence of cells co-expressing all three genes in this area suggests that these cells are migrating immature neurons. also, the presence of oriented gfp-positive fibers in the subpial and parenchyma layers of the medulla oblongata suggest that lgi may be involved in the relocation of immature neurons in the mouse subpial medullary region and may play a role in the migration of medullary precerebellar neurons in the caudal medulla. evidence from studies in adult brain, suggest that expression of lgi is largely restricted to the hippocampus and cortical neurons. however, in our study we have shown that lgi expression can be detected in the cephalic neuroepithelium at e, which coincides with the first signs of neurogenesis in the mouse. the appearance of lgi expression in the cerebral cortex begins at ~e, and peaks at e. as development progresses, expression levels in the cortex progressively diminish. on the other hand, lgi appears not to be involved in early differentiation and maturation of glial lineage since lgi1-expressing cells do not coexpress rc or ng. even thought differentiated neurons expressed lgi many of these cells do not express map at the observed stages. we speculate that once these cells have migrated to their final destinations, lgi expression levels decay. lgi was also found to be strongly expressed in the basal telencephalic plates that will develop into the basal ganglia associated with motor functions. dcx-expressing cells in the ventral portion of the basal telencephalic plates also express lgi suggesting that these dcx-lgi1-expressing cells migrate and will populate cortical and subcortical regions. in addition, marín et al argue that patterning of the basal telencepahlon is crucial for the growth of cortical axons as well as for guiding cortical projections involved in sensory-motor information. the presence of lgi in migrating neuroblasts is consistent with a function in cortical and medullar axon guidance, as suggested by our gene expression studies in model cell systems. beta-iii tubulin is suggested to be one of the earliest markers to signal neuronal commitment in primitive neuroepithelium, and it is accepted as a neuron-specific protein marker, which is highly expressed in cortex at birth and then its expression levels decrease with increasing postnatal development. during brain development, the maturation of neuroblasts into neurons is accompanied by up-regulation of beta-iii tubulin. coexpression of lgi and beta-iii tubulin was detected in the ventricular and subventricular zones, as well as the cortical plate, suggesting a conserved neurogenic program in these cells from embryonic stages to adult life, which further implies that lgi may participate in normal neuronal development. we also observed lgi expression in many areas of the developing sensory system, for example, the tongue, eye, and olfactory bulb. the olfactory system is anatomically connected to the amygdala, lateral septum, hypothalamus and the hippocampus, which are part of the limbic system and which also express lgi this correlation is consistent with a role for lgi in the development of limbic encephalitis. within the hippocampus, expression levels are found primarily in the dentate gyrus and scarcely within the ca1-ca region of the pyramidal cell layer. there is evidence that lgi also regulates intra-hippocampal circuit formation and the dentate gyrus plays a critical role in seizures, since it appears to have low epileptogenic thresholds. although thalamic protein levels are weak overall, the paraventricular thalamic area expresses lgi according to huang et al, the thalamic paraventricular nucleus receives the highest innervation from hypothalamic hypocretin/orexin neurons, generating substantial excitation of the medial prefrontal cortex. major inputs into the anterior thalamic group are from the hypothalamus, the hippocampus, and the tectum while output from this region is mainly to the cingulate gyrus. in the developing mouse eye, lgi expression was seen in the optic vesicle, optic stalk and optic cup, as well as the neural retina and the retinal pigment epithelium. lgi expression is seen in the retinal ganglion layer, and dominant-negative mutations in transgenic mice lead to abnormal postnatal pruning of retinal axons in the visual relay thalamus. in zebrafish studies, lgi expression was seen in the epithelium surrounding the lumen of the optic vesicle and interestingly, in early zebrafish embryos, loss of lgi results in abnormal eye development, supporting its critical role in development of this organ.  several general patterns of lgi distribution were apparent during embryonic brain development. lgi expression appears to spread throughout the dorsal-most brain structures with only weak levels in the ventral regions in early embryo stages within the medulla oblongata showing the strongest expression. this dorsoventral differentiation also appears in the hippocampus, where the dentate gyrus showed more robust expression. lgi expression also appears to follow a superficial to deep, as well as a caudal to cephalic axis. at e, the strongest signal was found in the surface ectoderm, as well as the infundibular recess of the diencephalon and dorsal midbrain in the pretectal neuroepithelium. as development progresses, lgi expression moves interiorly into the neocortex and other more caudal structures, expression in the pretectum and diencephalon areas begins to subside, as shown by weak expression in basal ganglia and thalamic nuclei, although expression in the paraventricular thalamic nucleus remains strong at p overall, the spatial and temporal expression profile for lgi during embryogenesis and postnatal stages is consistent with the idea that it plays an important role in normal brain development, especially neuronal migration and synapse formation. 
during embryonic brain growth, high levels of lgi expression were found in the surface ectoderm, the neuroepithelium, mesenchymal connective tissue, hippocampus, and sensory organs, such as eye, tongue, and the olfactory bulb. in late postnatal stages, lgi expression continued to be detected in many areas in the brain including, hippocampus, paraventricular thalamic nuclei, inferior colliculus, and the cerebral aqueduct. we also showed that lgi1-expressing cells co-express nestin, dcx, and beta-iii tubulin suggesting that lgi1-expressing cells are migratory neuroblasts. currently, little is known about the temporal and spatial expression pattern of lgi during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses gfp under the control of lgi cis-regulatory elements. lgi was also found in the cranial nerve nuclei and ganglia, such as vestibular, trigeminal, and dorsal ganglia. expression of lgi followed an orchestrated pattern during mouse development becoming more subdued in areas of the neocortex of the mid- and hind-brain in early postnatal animals, although high expression levels were retained in the choroid plexus and hippocampus. these observations imply that lgi may have a role in establishing normal brain architecture and neuronal functions during brain development suggesting that it may be involved in neurogenesis and neuronal plasticity, which become more specifically defined in the adult animal.
 mutations in the lgi gene predispose to a rare, hereditary form of temporal epilepsy. currently, little is known about the temporal and spatial expression pattern of lgi during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses gfp under the control of lgi cis-regulatory elements.  during embryonic brain growth, high levels of lgi expression were found in the surface ectoderm, the neuroepithelium, mesenchymal connective tissue, hippocampus, and sensory organs, such as eye, tongue, and the olfactory bulb. lgi was also found in the cranial nerve nuclei and ganglia, such as vestibular, trigeminal, and dorsal ganglia. expression of lgi followed an orchestrated pattern during mouse development becoming more subdued in areas of the neocortex of the mid- and hind-brain in early postnatal animals, although high expression levels were retained in the choroid plexus and hippocampus. in late postnatal stages, lgi expression continued to be detected in many areas in the brain including, hippocampus, paraventricular thalamic nuclei, inferior colliculus, and the cerebral aqueduct. we also showed that lgi1-expressing cells co-express nestin, dcx, and beta-iii tubulin suggesting that lgi1-expressing cells are migratory neuroblasts.  these observations imply that lgi may have a role in establishing normal brain architecture and neuronal functions during brain development suggesting that it may be involved in neurogenesis and neuronal plasticity, which become more specifically defined in the adult animal. 