BACKGROUND
it has recently become evident that the majority of the mammalian genome is transcribed in a developmentally regulated manner producing tens of thousands of interleaved short and long ncrnas  <cit> .
long ncrnas, defined as greater than  <dig> nt in length  <cit> , exhibit specific temporal and spatial expression patterns in mouse brain  <cit> , embryonic stem cell differentiation  <cit>  and t-cell differentiation  <cit> .
ncrnas are also implicated in neural developmental events and in the pathogenesis of neuropsychiatric diseases  <cit> .
these long ncrnas are being shown to have a wide range of functions, including roles in the regulation of chromosomal architecture and dynamics, transcription, post-transcriptional processing, rna editing, rna trafficking and organelle biogenesis  <cit> .
one major emergent function of long ncrnas is their ability to modulate the epigenetic status of nearby protein-coding genes by recruiting chromatin activator or repressor complexes  <cit> .
here we present the first systematic examination of long ncrna expression profiles during differentiation of embryonic forebrain-derived neural stem cells .
we focused on a subset of cortical gabaergic neurons  and oligodendrocytes  that are initially specified from sonic hedgehog -responsive, nkx <dig> -expressing bipotent neuronal-ol progenitor  species within the ventromedial forebrain.
we have previously reported that these n/ops undergo long-distance tangential migration to cerebral cortex where they sequentially give rise to gabans and ols  <cit> .
we employed custom designed microarrays to examine expression of noncoding and protein-coding transcripts during these neural developmental transitions, finding dynamic expression of ncrnas associated with key neural development genes.
we also observed differential expression of specific ncrnas after treatment of oligodendrocyte progenitors  with trichostatin a , a histone deacetylase  inhibitor that prevents progression of olps into post-mitotic oligodendrocytes  by suppressing oligodendrocyte-specific genes and inducing epigenetic reprogramming  <cit> .
these developmental changes underscore the importance of epigenetic regulation in the elaboration of neural cell types and imply functional relationships between chromatin remodeling and selective deployment of ncrnas.
RESULTS
we conducted gene expression profiling using a custom-designed microarray platform to examine protein-coding and noncoding transcript expression accompanying neural stem cell-mediated fate restriction , neural lineage specification , neuronal-glial fate switching , and neural lineage maturation  .
in total, we found 31%  of probes targeting protein-coding transcripts exhibited expression above background and that  <dig>  of these were significantly differentially expressed  at one or more developmental stages compared to nscs .
we found that 9%  of probes targeting ncrnas were expressed above background, of which  <dig>  were significantly differentially expressed  at one or more developmental stages compared to the nsc control .
genomic analysis of expressed ncrnas
long ncrnas are often organized as part of complex interleaved transcriptional networks that include protein-coding genes  <cit> .
a number of studies have shown that the association of long noncoding transcription with protein-coding genes has functional relevance, often with the ncrna regulating expression of its protein-coding counterpart via epigenetic modifications and/or transcriptional co-activation/repression  <cit> .
therefore, we identified all ncrnas expressed in our developmental models that are transcribed antisense to, bi-directionally, or from within the introns of overlapping or adjacent protein coding genes .
in addition, we identified pairs of protein-coding rnas and ncrnas whose genomic loci are positionally conserved between mouse and human  because this genomic conservation may also indicate a functional relationship between the two transcripts.
in many cases, the associated protein-coding genes were critical neuro-developmental genes.
as an illustrative example, the downregulation of a ncrna  is associated with the gli <dig> locus in all progeny of n/ops, whereas gli <dig> was upregulated in n/ops but downregulated in all progeny of n/ops.
this relationship may be of significant interest given that gli <dig> has been shown to act as a bifunctional transcriptional switch that can either repress or activate shh signaling  <cit>  to help specify distinct neuronal cell types  <cit> .
many ncrnas contain conserved secondary structures that are crucial to their function  <cit> .
these conformational features may be bound by proteins  <cit> , processed into smaller regulatory ncrnas  <cit>  or even fulfill intrinsic catalytic functions  <cit> .
to provide support for the proposed functionality of the expressed ncrnas, we looked for evidence of rna secondary structures within differentially expressed ncrna transcripts using the folding program rnaz  <cit> .
we found  <dig> expressed ncrnas that contained high confidence predicted conserved secondary structures   such as stem-loops , known to be present in mirna precursors, small nucleolar rnas  and other rnas with previously characterized functions.
we then combined these genomic analyses to identify candidate ncrnas for further functional analysis.
these are discussed in more detail below in the context of neuronal and oligodendroglial lineage commitment, neuronal-glial lineage switching and progressive stages of oligodendrocyte lineage elaboration.
lineage restriction of neural stem cells  into bipotent neuronal/oligodendrocyte progenitors
a subset of nestin-immunoreactive nscs within the ventral forebrain give rise to bipotent n/ops following exposure to a gradient of shh signaling  <cit>  which are characterized by expression of two bhlh transcription factors  and their potential to give rise to gabans and ols but not astrocytes  <cit> .
our microarray analysis revealed that the initial fate restriction of regional forebrain nscs to bipotent n/ops was associated with relatively modest changes in gene expression profiles, possibly reflecting the relative similarities between these developmental cell types.
nonetheless, there were differences in factors that may be important for promoting nsc lineage restriction and abrogating self-renewal and maintenance programs, including  <dig> upregulated and  <dig> downregulated mrnas .
not surprisingly, some genes previously implicated in neurogenesis and gliogenesis exhibited dynamic changes in mrna expression including transcription factors  and cell-surface receptor linked signal transduction pathways .
for example, we found upregulated mrnas included genes such as mash <dig>  an important transcription factor that promotes lineage restriction of nscs into n/ops  <cit> .
we also identified dynamically expressed ncrnas that may fulfill functional roles in nsc fate restriction.
similar to mrnas, relatively few changes in ncrna expression were observed during the transition from nscs to n/ops, with  <dig> ncrnas exhibiting differential expression  .
these included a previously described ncrna, gomafu, shown to be localized to a novel subnuclear domain in a distinct subset of differentiating neurons in the mouse nervous system  <cit> , which was exclusively downregulated in n/ops but upregulated in all subsequent stages of ol lineage specification and maturation .
furthermore, in situ hybridization  in the adult mouse brain showed gomafu expression in specific neuronal populations and nuclear localization within purkinje cells .
our results show that gomafu is not only expressed during neurogenesis but also during ol lineage specification.
a number of ncrnas are significantly upregulated only during lineage restriction of nscs, including the small nucleolar rna  host gene  <dig>  and  <dig> , an expression profile supported by the restricted neuronal expression of snhg <dig> in whole brain ish .
snhg <dig> and snhg <dig> host a group of c/d box snornas that serve as guides for the site-specific 2'-o-methylation of ribosomal rnas.
furthermore, two identified ncrna species, neat <dig>  and neat <dig>  are downregulated in n/ops and broadly upregulated in their neuronal and glial progeny .
neat <dig> and neat <dig> associate with nuclear paraspeckles and adjacent sc <dig> speckles, respectively; domains where transcription and co-transcriptional pre-mrna processing occur  <cit> .
these nuclear domains have been associated with modulation of cell growth and differentiation  <cit> , and neat <dig>  has been recently shown to be induced upon cell differentiation and to be required for the structural integrity of paraspeckles  <cit> .
specification of gabaergic neurons
bipotent n/ops can differentiate into either gabans or olps in response to different neural cytokines  <cit> .
we therefore examined changes in gene expression during the elaboration of gabans to identify mrnas and ncrnas that may be involved in this type of neuronal lineage specification.
we observed upregulation of  <dig> mrnas, which were enriched for genes involved in nervous system development , neuronal function  and neurogenesis .
we also identified  <dig> downregulated mrnas .
interestingly, these downregulated mrnas were enriched for predicted targets of let-7b  and mir- <dig> , possibly being subject to repression by mirnas.
indeed, both let-7b and mir- <dig> have previously been implicated in promoting neurogenesis  <cit> .
in addition, we identified  <dig> ncrnas that are upregulated during gaban neurogenesis, including a number of examples such as gtl <dig>  rian, evf <dig>  and copg2as that were previously shown to exhibit neuron-enriched expression profiles in the adult mouse brain  <cit> .
in contrast, we identified only  <dig> ncrnas that were downregulated during gabaergic neurogenesis .
we distinguished ncrnas that may be involved specifically in gaban lineage commitment by selecting ncrnas upregulated during gaban differentiation but downregulated during ol differentiation .
we identified  <dig> ncrnas, including an intergenic ncrna transcript ak <dig> that overlaps a highly conserved brain-specific mirna, mir-124a.
mir-124a accounts for nearly half of all brain mirnas  <cit>   and helps to regulate neuronal specification and differentiation  <cit> .
one mechanism by which mir- <dig> was shown to promote neuronal differentiation is by repression of ptbp <dig>  a negative regulator of alternative splicing, resulting in activation of neuron-specific alternative splicing events  <cit> .
indeed, we found that ptbp <dig> and ak <dig> exhibited complementary expression profiles.
however, the ak <dig> transcript may itself function intrinsically as a full-length ncrna, rather than simply acting as a precursor.
the ak <dig> transcript is subject to post-transcriptional modifications including splicing and polyadenylation, contains a number of high confidence predicted stem-loop structures , and is highly conserved with homologous transcripts present in humans  and rats .
indeed, the ak <dig> transcript was recently identified in a genome wide screen for functional lncrnas, termed lincrnas, that exhibit a distinct chromatin signature indicative of transcriptional regulation and high levels of expression  <cit> .
furthermore, ish in adult mouse brain revealed that the full-length ak <dig> transcript is expressed in the mature central nervous system , suggesting an involvement in neurogenesis independent of mir-124a.
because a large number of developmental gene-related enhancers have been shown to drive transcription in the developing forebrain during early neurogenesis  <cit> , we examined ncrnas expressed in gabans that are associated with highly conserved enhancers.
we identified  <dig> ncrnas that were associated with highly conserved elements driving transcription in the forebrain .
two of these ncrnas were associated with ultraconserved enhancers at the dlx genomic loci, one of which, evf- <dig>  was previously shown to regulate binding of the dlx <dig> transcription factor to the ultraconserved enhancer to activate the expression of the encompassed dlx <dig> gene during neurogenesis  <cit> .
we also identified a similar ncrna antisense to the dlx <dig> gene.
both dlx <dig> and the antisense ncrna, dlx1as, are upregulated during gaban differentiation but downregulated during ol differentiation .
these observations are consistent with published data showing that dlx <dig> and dlx <dig> promote gaban lineage specification by suppressing the important oligogenic bhlh transcription factor, olig <dig>  <cit>  and are necessary for tangential migration of ventral forebrain-derived progenitor cells  <cit> .
dlx1as is transcribed from an ultraconserved enhancer that drives expression in the midbrain, forebrain and olfactory system during development and in regions associated with neurogenesis in the adult mouse brain  <cit> .
the third ncrna, ak <dig>  which is also transcribed from an ultraconserved enhancer driving expression in the developing forebrain, has not been previously described and is not associated with any annotated developmental genes.
nonetheless, expression of this spliced transcript during gabaergic neurogenesis, coupled with its extremely conserved enhancer, suggests it plays a role in the developing forebrain.
specification of oligodendrocyte progenitors
application of pdgf-aa to n/ops for  <dig> days in vitro was sufficient to promote the elaboration of olps .
we confirmed by immunofluorescence microscopy that n/ops express the corresponding receptor  expression prior to and independent of the application of pdgf-aa .
the developmental transition of n/ops to ols was associated with the upregulation of  <dig> mrnas including a number of ol-selective genes, such as olig <dig> and olig <dig>  and the downregulation of  <dig> mrnas .
we also found that  <dig> ncrnas were downregulated, whereas  <dig> ncrnas were upregulated, six of which exhibited highly correlated expression profiles to olig <dig>  having a distinct peak during olp differentiation  .
to identify ncrnas with specific roles in ol lineage commitment, we selected ncrnas that were upregulated in olps but downregulated in gabans.
this analysis identified only one ncrna, a novel  <dig>  kb transcript  organized bi-directionally to the sox <dig> gene .
sox <dig> is a member of the sry-box transcription factor family, many of which play key roles in ol lineage elaboration, including sox <dig> and sox <dig> for ol specification and sox <dig> and sox <dig> for progressive ol maturation  <cit> .
the ncrna we identified, which we termed sox8ot , exhibited concordant expression  to the sox <dig> mrna.
furthermore, a positionally conserved homologous sox8ot transcript is also present in humans  and rats , suggesting the bi-directional relationship between sox <dig> and sox8ot is relevant.
the bidirectional organization of the sox <dig> gene and the sox8ot ncrna suggests that these transcripts are subject to common modes of regulation, possibly by sharing a promoter sequence.
indeed, recently published genome-wide maps of chromatin modifications  <cit>  indicate a shared domain subject to dynamic modifications during the transition from embryonic stem to neuronal progenitor cells .
together, these data suggest sox8ot functions in ol lineage commitment, possibly by regulating the expression of the associated sox <dig> gene.
as an alternative strategy to identify ncrnas with specific roles in ol lineage commitment, we identified ncrnas that were differentially expressed only in olps, and found that  <dig> were downregulated while  <dig> were upregulated including dleu <dig>  a previously described ncrna overlapping but transcribed in the opposite direction of the trim <dig>  gene whose expression is upregulated during early ol maturation and terminal differentiation  <cit> .
progressive stages of ol lineage maturation including myelination
the withdrawal of ol cytokines  from olp culture conditions results in cell cycle exit , though the actions of environmental factors such as triiodothyronine, cyclin dependent kinase inhibitors , inhibitory hlh transcription factors  and other transcription factors  that participate in the combinatorial molecular programs regulating this process  <cit> .
in pmos, we identified  <dig> upregulated mrnas including genes involved in lipid metabolic processes , such as mbp, a structural component of the myelin sheath  <cit> .
we also observed downregulation of  <dig> mrnas including genes involved in cell division , mitosis , dna replication , and chromosome organization and biogenesis .
similarly, olp cell cycle exit was accompanied by the upregulation of  <dig> ncrnas and the downregulation of  <dig> ncrnas.
many of the ncrnas are associated with genomic loci encompassing protein-coding genes with previously described functions in development and maturation.
for example, we found a highly conserved ncrna, ak <dig>  hosted within the intron of bai <dig>  which was downregulated in pmos.
bai <dig>  which exhibits a similar expression profile, is a developmentally expressed angiostatic gene that is downregulated in the early phases of brain ischemia and in high-grade gliomas  <cit>  and has been associated with mental retardation  <cit>  and schizophrenia  <cit> .
ol terminal differentiation is associated with the outgrowth of cytoplasmic processes and the accelerated production of myelin  <cit> , and this process is regulated by neural cytokines, various classes of transcription factors, cdkis , and epigenetic factors  <cit> .
in myos, we found  <dig> upregulated mrnas that were enriched for genes involved in lipid metabolic processes , biopolymer metabolic processes , and actin cytoskeleton organization , including a dramatic increase in the expression of myelin genes such as plp <dig>  mbp and mal.
in addition, we found  <dig> ncrnas that were upregulated during ol terminal differentiation and myelination, including  <dig> that were broadly upregulated during progressive stages of ol lineage elaboration, exhibiting a highly correlated   expression profile with mog, an important glycoprotein found on the surface of myos  <cit> .
in contrast, we found that  <dig> ncrnas were downregulated, including  <dig> ncrnas that exhibited a highly correlated expression profile   to melk, a factor important for promoting nsc proliferation  <cit>  that was downregulated during ol lineage elaboration.
a variety of ncrnas were broadly upregulated in olps, pmos and myos including ak <dig> and ak <dig>  associated with the slc44a <dig> and rab11b genomic loci, respectively.
in these cases, both the ncrnas and the protein-coding transcripts were strongly upregulated during ol lineage elaboration suggesting that they may function coordinately to promote appropriate ol maturation and expression of myelin proteins.
we similarly identified a ncrna antisense to ecsit, an important node between bmp and toll-like receptor  pathways that integrates morphogenetic signaling with pathogen recognition and innate immunity during development  <cit> .
the ncrna, ak <dig>  is upregulated in gabans, olps and myos, in contrast to ecsit, which is downregulated in these species.
the observed inverse expression pattern between ak <dig> and ecsit is similar to a correlation we recently observed in embryonic stem cell differentiation  <cit>  and suggests coordination between the expression profiles of this pair of transcripts.
epigenetic reprogramming of cell fate with trichostatin a
histone deacetylation has been implicated in modulating ol lineage progression, particularly for developmental species undergoing cell cycle exit and progressive stages of cellular differentiation characterized by the acquisition of a branched morphology and the onset of myelin gene expression  <cit> .
treatment with tsa, an hdac inhibitor, prevents progression of olps into pmos with the resulting preservation of a simplified morphological phenotype that does not display secondary branching characteristic of differentiating pmos .
the inhibition of hdac suppresses ol-specific gene expression and reprograms these cells into a less lineage committed state receptive to neurogenic or gliogenic differentiation signals  <cit> .
given that we observed a number of ncrnas exhibiting specific expression profiles during ol lineage progression, we were interested to examine whether their expression was similarly regulated by histone deacetylation.
changes to ncrna expression in response to hdac inhibition would suggest the incorporation of these ncrnas within a genetic program that contributes to progression of either neuronal or oligodendrocyte differentiation.
we employed qrt-pcr to examine the expression of ncrnas identified within this study as strong candidates for functional roles in ol or neuronal differentiation.
broadly, we found parallel changes in cellular phenotype and alterations in ncrna expression profiles induced by tsa.
in addition to examples described above, we included other ncrnas associated with neural developmental genes that exhibited representative developmental expression profiles.
almost all selected ncrnas  exhibited significant  changes in expression at  <dig> or  <dig> h .
the majority of these changes were sustained or developed during later stages , suggesting the expression of these ncrnas is subject to regulation by mechanisms of histone deacetylation.
while we observed both increased and decreased expression of long ncrnas, the majority was downregulated, often dramatically, supporting the putative role of these ncrnas in contributing to the progressive differentiation of ols .
downregulated ncrnas included those associated with protein-coding genes involved in progressive ol differentiation.
in addition to those previously described including sox8ot, gomafu, ak <dig>  and ak <dig> , we also observed dramatic downregulation of ncrna ak <dig> which is hosted within an intron of lhfpl <dig>  a member of the tetraspanin proteins which promote proliferation, migration and differentiation of ols  <cit> .
recent evidence has shown that histone deacetylation is responsible for restricting neuronal gene expression, with the inhibition of hdacs inducing neuronal differentiation  <cit> .
therefore, it is of interest that a number of candidate ncrnas exhibit an increase in expression in response to hdac inhibition, possibly suggesting a contribution to neuronal rather than ol differentiation.
for example, the previously described ncrna gtl <dig> was upregulated after tsa treatment.
indeed, within his study we observed upregulation of gtl <dig> during gaban differentiation.
gtl <dig> is expressed in restricted domains of the developing forebrain including ventral telencephalon, neocortex, hippocampus and diencephalon, suggesting roles in regional neuronal development and differentiation  <cit> .
finally, we found that neat <dig> transcription exhibited complex transcriptional dynamics after tsa treatment, being strongly upregulated after tsa treatment under stochastic  oligodendroglial specification conditions, whereas it was downregulated after tsa treatment under the influence of instructive  signals for ol lineage elaboration .
discussion
in this study, we utilized a custom-designed microarray platform to probe the transcription of both protein-coding and noncoding transcripts during ventral forebrain-derived nsc lineage restriction, neuronal and oligodendrocyte  lineage specification, neuronal-glial fate transitions, and progressive stages of ol lineage elaboration including myelination.
expression profiles for mrnas in our study were similar to previously reported findings  <cit>  showing modulation of key signaling pathways, transcription factors, and epigenetic regulators that help to define the molecular codes governing neural lineage elaboration.
additionally, we also observed expression of components of the myelin sheath.
although we examined protein-coding genes, we focused primarily on characterizing the expression profiles of long ncrnas, which are being increasingly appreciated in the regulation of developmental gene expression programs  <cit> .
previous studies of neural differentiation have reported expression profiles for only a small number of long ncrnas, such as nkx <dig> as and the imprinted h <dig> ncrna  <cit> .
in contrast, we examined a large spectrum of long ncrnas and found dramatic changes in expression that are unique to specific stages of cellular differentiation.
many of these ncrnas are transcribed from complex genomic loci that encompass protein-coding genes with defined roles in neuronal and glial lineage elaboration, and these pairs of ncrnas and associated protein-coding genes often exhibited coordinated expression during developmental transitions.
therefore, we suggest that these ncrnas may influence the expression of the associated protein-coding genes similar to previously characterized examples such as nkx <dig> as  <cit> , hotair  <cit> , p15as  <cit> , p21as  <cit>  and evf <dig>  <cit> .
for example, many ncrnas were associated with transcription factors that were themselves differentially regulated during ol differentiation  <cit> .
long ncrnas, such as those identified in this study, may therefore contribute to the combinatorial transcriptional codes involved in lineage specification and terminal differentiation including myelination.
the coordinated expression of long ncrnas with critical neural developmental protein coding genes suggests that both are subject to shared regulatory mechanisms and that long ncrnas are integrated into complex environmentally-mediated neural and glial developmental gene expression programs.
we, therefore, considered whether the well-documented ability of epigenetic mechanisms to promote decisive changes in neuronal and glial gene expression programs also includes the capacity to regulate long ncrna expression.
indeed, treatment with the hdac inhibitor, tsa, effectively arrests the developmental progression of olps into pmos  <cit>  and also affects the expression of long ncrnas, suggesting that these are not only regulated by epigenetic modifications but also these transcripts are incorporated into a broader lineage specific developmental gene expression program.
furthermore, hdac inhibition also has differential effects on certain ncrnas in an environmentally responsive manner.
for example, treatment of olps with tsa in the presence  or absence  of cntf results in opposing profiles of neat <dig> expression, implying that long ncrnas might be deployed in a context-specific manner to dynamically modulate seminal fate decisions within the ol lineage.
although the use of hdac inhibitors has been essential for interrogating the epigenetic mechanisms that govern ol lineage maturation, the cellular effects of tsa administration are complex and our understanding of the mechanisms mediating ncrna expression are still evolving.
nonetheless, long ncrnas may represent important targets for regenerative therapies in the cns because they exhibit both exquisite site-selective as well as genome wide effects  <cit>  associated with dynamic ol developmental transition states characterized by considerable epigenetic flux  <cit> .
CONCLUSIONS
this study is the first systematic examination of the expression of long ncrnas in forebrain-mediated nsc maturational processes, specifically ol differentiation and development.
it is also the first demonstration that long ncrnas, many of which are involved in regulating epigenetic processes, are also themselves subject to regulation by epigenetic processes.
it adds to a growing consensus that not only are large portions of the mammalian genome transcribed  <cit>  but also expressed in a regulated manner  <cit> .
the specific expression of ncrnas has been recently observed in other developmental programs and comparisons between these studies reveal both common and unique features  <cit> .
because the long noncoding transcriptome is still relatively poorly annotated and only partially interrogated by this study, it seems likely that many more long ncrnas will be shown to be expressed during development and their importance for neuronal and glial biology will be more fully understood and appreciated.
these studies will have particular relevance in the future as the role of noncoding transcripts in neurological disease states becomes increasingly recognized  <cit> .

 it has recently become evident that the majority of the mammalian genome is transcribed in a developmentally regulated manner producing tens of thousands of interleaved short and long ncrnas. long ncrnas, defined as greater than nt in length, exhibit specific temporal and spatial expression patterns in mouse brain, embryonic stem cell differentiation and t-cell differentiation. ncrnas are also implicated in neural developmental events and in the pathogenesis of neuropsychiatric diseases. these long ncrnas are being shown to have a wide range of functions, including roles in the regulation of chromosomal architecture and dynamics, transcription, post-transcriptional processing, rna editing, rna trafficking and organelle biogenesis. one major emergent function of long ncrnas is their ability to modulate the epigenetic status of nearby protein-coding genes by recruiting chromatin activator or repressor complexes. here we present the first systematic examination of long ncrna expression profiles during differentiation of embryonic forebrain-derived neural stem cells. we focused on a subset of cortical gabaergic neurons and oligodendrocytes that are initially specified from sonic hedgehog -responsive, nkx -expressing bipotent neuronal-ol progenitor species within the ventromedial forebrain. we have previously reported that these n/ops undergo long-distance tangential migration to cerebral cortex where they sequentially give rise to gabans and ols. we employed custom designed microarrays to examine expression of noncoding and protein-coding transcripts during these neural developmental transitions, finding dynamic expression of ncrnas associated with key neural development genes. we also observed differential expression of specific ncrnas after treatment of oligodendrocyte progenitors with trichostatin a, a histone deacetylase inhibitor that prevents progression of olps into post-mitotic oligodendrocytes by suppressing oligodendrocyte-specific genes and inducing epigenetic reprogramming. these developmental changes underscore the importance of epigenetic regulation in the elaboration of neural cell types and imply functional relationships between chromatin remodeling and selective deployment of ncrnas.  we conducted gene expression profiling using a custom-designed microarray platform to examine protein-coding and noncoding transcript expression accompanying neural stem cell-mediated fate restriction, neural lineage specification, neuronal-glial fate switching, and neural lineage maturation. in total, we found 31% of probes targeting protein-coding transcripts exhibited expression above background and that of these were significantly differentially expressed at one or more developmental stages compared to nscs. we found that 9% of probes targeting ncrnas were expressed above background, of which were significantly differentially expressed at one or more developmental stages compared to the nsc control. genomic analysis of expressed ncrnas long ncrnas are often organized as part of complex interleaved transcriptional networks that include protein-coding genes. a number of studies have shown that the association of long noncoding transcription with protein-coding genes has functional relevance, often with the ncrna regulating expression of its protein-coding counterpart via epigenetic modifications and/or transcriptional co-activation/repression. therefore, we identified all ncrnas expressed in our developmental models that are transcribed antisense to, bi-directionally, or from within the introns of overlapping or adjacent protein coding genes. in addition, we identified pairs of protein-coding rnas and ncrnas whose genomic loci are positionally conserved between mouse and human because this genomic conservation may also indicate a functional relationship between the two transcripts. in many cases, the associated protein-coding genes were critical neuro-developmental genes. as an illustrative example, the downregulation of a ncrna is associated with the gli locus in all progeny of n/ops, whereas gli was upregulated in n/ops but downregulated in all progeny of n/ops. this relationship may be of significant interest given that gli has been shown to act as a bifunctional transcriptional switch that can either repress or activate shh signaling to help specify distinct neuronal cell types. many ncrnas contain conserved secondary structures that are crucial to their function. these conformational features may be bound by proteins, processed into smaller regulatory ncrnas or even fulfill intrinsic catalytic functions. to provide support for the proposed functionality of the expressed ncrnas, we looked for evidence of rna secondary structures within differentially expressed ncrna transcripts using the folding program rnaz. we found expressed ncrnas that contained high confidence predicted conserved secondary structures such as stem-loops, known to be present in mirna precursors, small nucleolar rnas and other rnas with previously characterized functions. we then combined these genomic analyses to identify candidate ncrnas for further functional analysis. these are discussed in more detail below in the context of neuronal and oligodendroglial lineage commitment, neuronal-glial lineage switching and progressive stages of oligodendrocyte lineage elaboration. lineage restriction of neural stem cells into bipotent neuronal/oligodendrocyte progenitors a subset of nestin-immunoreactive nscs within the ventral forebrain give rise to bipotent n/ops following exposure to a gradient of shh signaling which are characterized by expression of two bhlh transcription factors and their potential to give rise to gabans and ols but not astrocytes. our microarray analysis revealed that the initial fate restriction of regional forebrain nscs to bipotent n/ops was associated with relatively modest changes in gene expression profiles, possibly reflecting the relative similarities between these developmental cell types. nonetheless, there were differences in factors that may be important for promoting nsc lineage restriction and abrogating self-renewal and maintenance programs, including upregulated and downregulated mrnas. not surprisingly, some genes previously implicated in neurogenesis and gliogenesis exhibited dynamic changes in mrna expression including transcription factors and cell-surface receptor linked signal transduction pathways. for example, we found upregulated mrnas included genes such as mash an important transcription factor that promotes lineage restriction of nscs into n/ops. we also identified dynamically expressed ncrnas that may fulfill functional roles in nsc fate restriction. similar to mrnas, relatively few changes in ncrna expression were observed during the transition from nscs to n/ops, with ncrnas exhibiting differential expression. these included a previously described ncrna, gomafu, shown to be localized to a novel subnuclear domain in a distinct subset of differentiating neurons in the mouse nervous system, which was exclusively downregulated in n/ops but upregulated in all subsequent stages of ol lineage specification and maturation. furthermore, in situ hybridization in the adult mouse brain showed gomafu expression in specific neuronal populations and nuclear localization within purkinje cells. our results show that gomafu is not only expressed during neurogenesis but also during ol lineage specification. a number of ncrnas are significantly upregulated only during lineage restriction of nscs, including the small nucleolar rna host gene and, an expression profile supported by the restricted neuronal expression of snhg in whole brain ish. snhg and snhg host a group of c/d box snornas that serve as guides for the site-specific 2'-o-methylation of ribosomal rnas. furthermore, two identified ncrna species, neat and neat are downregulated in n/ops and broadly upregulated in their neuronal and glial progeny. neat and neat associate with nuclear paraspeckles and adjacent sc speckles, respectively; domains where transcription and co-transcriptional pre-mrna processing occur. these nuclear domains have been associated with modulation of cell growth and differentiation, and neat has been recently shown to be induced upon cell differentiation and to be required for the structural integrity of paraspeckles. specification of gabaergic neurons bipotent n/ops can differentiate into either gabans or olps in response to different neural cytokines. we therefore examined changes in gene expression during the elaboration of gabans to identify mrnas and ncrnas that may be involved in this type of neuronal lineage specification. we observed upregulation of mrnas, which were enriched for genes involved in nervous system development, neuronal function and neurogenesis. we also identified downregulated mrnas. interestingly, these downregulated mrnas were enriched for predicted targets of let-7b and mir-, possibly being subject to repression by mirnas. indeed, both let-7b and mir- have previously been implicated in promoting neurogenesis. in addition, we identified ncrnas that are upregulated during gaban neurogenesis, including a number of examples such as gtl rian, evf and copg2as that were previously shown to exhibit neuron-enriched expression profiles in the adult mouse brain. in contrast, we identified only ncrnas that were downregulated during gabaergic neurogenesis. we distinguished ncrnas that may be involved specifically in gaban lineage commitment by selecting ncrnas upregulated during gaban differentiation but downregulated during ol differentiation. we identified ncrnas, including an intergenic ncrna transcript ak that overlaps a highly conserved brain-specific mirna, mir-124a. mir-124a accounts for nearly half of all brain mirnas and helps to regulate neuronal specification and differentiation. one mechanism by which mir- was shown to promote neuronal differentiation is by repression of ptbp a negative regulator of alternative splicing, resulting in activation of neuron-specific alternative splicing events. indeed, we found that ptbp and ak exhibited complementary expression profiles. however, the ak transcript may itself function intrinsically as a full-length ncrna, rather than simply acting as a precursor. the ak transcript is subject to post-transcriptional modifications including splicing and polyadenylation, contains a number of high confidence predicted stem-loop structures, and is highly conserved with homologous transcripts present in humans and rats. indeed, the ak transcript was recently identified in a genome wide screen for functional lncrnas, termed lincrnas, that exhibit a distinct chromatin signature indicative of transcriptional regulation and high levels of expression. furthermore, ish in adult mouse brain revealed that the full-length ak transcript is expressed in the mature central nervous system, suggesting an involvement in neurogenesis independent of mir-124a. because a large number of developmental gene-related enhancers have been shown to drive transcription in the developing forebrain during early neurogenesis, we examined ncrnas expressed in gabans that are associated with highly conserved enhancers. we identified ncrnas that were associated with highly conserved elements driving transcription in the forebrain. two of these ncrnas were associated with ultraconserved enhancers at the dlx genomic loci, one of which, evf- was previously shown to regulate binding of the dlx transcription factor to the ultraconserved enhancer to activate the expression of the encompassed dlx gene during neurogenesis. we also identified a similar ncrna antisense to the dlx gene. both dlx and the antisense ncrna, dlx1as, are upregulated during gaban differentiation but downregulated during ol differentiation. these observations are consistent with published data showing that dlx and dlx promote gaban lineage specification by suppressing the important oligogenic bhlh transcription factor, olig and are necessary for tangential migration of ventral forebrain-derived progenitor cells. dlx1as is transcribed from an ultraconserved enhancer that drives expression in the midbrain, forebrain and olfactory system during development and in regions associated with neurogenesis in the adult mouse brain. the third ncrna, ak which is also transcribed from an ultraconserved enhancer driving expression in the developing forebrain, has not been previously described and is not associated with any annotated developmental genes. nonetheless, expression of this spliced transcript during gabaergic neurogenesis, coupled with its extremely conserved enhancer, suggests it plays a role in the developing forebrain. specification of oligodendrocyte progenitors application of pdgf-aa to n/ops for days in vitro was sufficient to promote the elaboration of olps. we confirmed by immunofluorescence microscopy that n/ops express the corresponding receptor expression prior to and independent of the application of pdgf-aa. the developmental transition of n/ops to ols was associated with the upregulation of mrnas including a number of ol-selective genes, such as olig and olig and the downregulation of mrnas. we also found that ncrnas were downregulated, whereas ncrnas were upregulated, six of which exhibited highly correlated expression profiles to olig having a distinct peak during olp differentiation. to identify ncrnas with specific roles in ol lineage commitment, we selected ncrnas that were upregulated in olps but downregulated in gabans. this analysis identified only one ncrna, a novel kb transcript organized bi-directionally to the sox gene. sox is a member of the sry-box transcription factor family, many of which play key roles in ol lineage elaboration, including sox and sox for ol specification and sox and sox for progressive ol maturation. the ncrna we identified, which we termed sox8ot, exhibited concordant expression to the sox mrna. furthermore, a positionally conserved homologous sox8ot transcript is also present in humans and rats, suggesting the bi-directional relationship between sox and sox8ot is relevant. the bidirectional organization of the sox gene and the sox8ot ncrna suggests that these transcripts are subject to common modes of regulation, possibly by sharing a promoter sequence. indeed, recently published genome-wide maps of chromatin modifications indicate a shared domain subject to dynamic modifications during the transition from embryonic stem to neuronal progenitor cells. together, these data suggest sox8ot functions in ol lineage commitment, possibly by regulating the expression of the associated sox gene. as an alternative strategy to identify ncrnas with specific roles in ol lineage commitment, we identified ncrnas that were differentially expressed only in olps, and found that were downregulated while were upregulated including dleu a previously described ncrna overlapping but transcribed in the opposite direction of the trim gene whose expression is upregulated during early ol maturation and terminal differentiation. progressive stages of ol lineage maturation including myelination the withdrawal of ol cytokines from olp culture conditions results in cell cycle exit, though the actions of environmental factors such as triiodothyronine, cyclin dependent kinase inhibitors, inhibitory hlh transcription factors and other transcription factors that participate in the combinatorial molecular programs regulating this process. in pmos, we identified upregulated mrnas including genes involved in lipid metabolic processes, such as mbp, a structural component of the myelin sheath. we also observed downregulation of mrnas including genes involved in cell division, mitosis, dna replication, and chromosome organization and biogenesis. similarly, olp cell cycle exit was accompanied by the upregulation of ncrnas and the downregulation of ncrnas. many of the ncrnas are associated with genomic loci encompassing protein-coding genes with previously described functions in development and maturation. for example, we found a highly conserved ncrna, ak hosted within the intron of bai which was downregulated in pmos. bai which exhibits a similar expression profile, is a developmentally expressed angiostatic gene that is downregulated in the early phases of brain ischemia and in high-grade gliomas and has been associated with mental retardation and schizophrenia. ol terminal differentiation is associated with the outgrowth of cytoplasmic processes and the accelerated production of myelin, and this process is regulated by neural cytokines, various classes of transcription factors, cdkis, and epigenetic factors. in myos, we found upregulated mrnas that were enriched for genes involved in lipid metabolic processes, biopolymer metabolic processes, and actin cytoskeleton organization, including a dramatic increase in the expression of myelin genes such as plp mbp and mal. in addition, we found ncrnas that were upregulated during ol terminal differentiation and myelination, including that were broadly upregulated during progressive stages of ol lineage elaboration, exhibiting a highly correlated expression profile with mog, an important glycoprotein found on the surface of myos. in contrast, we found that ncrnas were downregulated, including ncrnas that exhibited a highly correlated expression profile to melk, a factor important for promoting nsc proliferation that was downregulated during ol lineage elaboration. a variety of ncrnas were broadly upregulated in olps, pmos and myos including ak and ak associated with the slc44a and rab11b genomic loci, respectively. in these cases, both the ncrnas and the protein-coding transcripts were strongly upregulated during ol lineage elaboration suggesting that they may function coordinately to promote appropriate ol maturation and expression of myelin proteins. we similarly identified a ncrna antisense to ecsit, an important node between bmp and toll-like receptor pathways that integrates morphogenetic signaling with pathogen recognition and innate immunity during development. the ncrna, ak is upregulated in gabans, olps and myos, in contrast to ecsit, which is downregulated in these species. the observed inverse expression pattern between ak and ecsit is similar to a correlation we recently observed in embryonic stem cell differentiation and suggests coordination between the expression profiles of this pair of transcripts. epigenetic reprogramming of cell fate with trichostatin a histone deacetylation has been implicated in modulating ol lineage progression, particularly for developmental species undergoing cell cycle exit and progressive stages of cellular differentiation characterized by the acquisition of a branched morphology and the onset of myelin gene expression. treatment with tsa, an hdac inhibitor, prevents progression of olps into pmos with the resulting preservation of a simplified morphological phenotype that does not display secondary branching characteristic of differentiating pmos. the inhibition of hdac suppresses ol-specific gene expression and reprograms these cells into a less lineage committed state receptive to neurogenic or gliogenic differentiation signals. given that we observed a number of ncrnas exhibiting specific expression profiles during ol lineage progression, we were interested to examine whether their expression was similarly regulated by histone deacetylation. changes to ncrna expression in response to hdac inhibition would suggest the incorporation of these ncrnas within a genetic program that contributes to progression of either neuronal or oligodendrocyte differentiation. we employed qrt-pcr to examine the expression of ncrnas identified within this study as strong candidates for functional roles in ol or neuronal differentiation. broadly, we found parallel changes in cellular phenotype and alterations in ncrna expression profiles induced by tsa. in addition to examples described above, we included other ncrnas associated with neural developmental genes that exhibited representative developmental expression profiles. almost all selected ncrnas exhibited significant changes in expression at or h. the majority of these changes were sustained or developed during later stages, suggesting the expression of these ncrnas is subject to regulation by mechanisms of histone deacetylation. while we observed both increased and decreased expression of long ncrnas, the majority was downregulated, often dramatically, supporting the putative role of these ncrnas in contributing to the progressive differentiation of ols. downregulated ncrnas included those associated with protein-coding genes involved in progressive ol differentiation. in addition to those previously described including sox8ot, gomafu, ak and ak, we also observed dramatic downregulation of ncrna ak which is hosted within an intron of lhfpl a member of the tetraspanin proteins which promote proliferation, migration and differentiation of ols. recent evidence has shown that histone deacetylation is responsible for restricting neuronal gene expression, with the inhibition of hdacs inducing neuronal differentiation. therefore, it is of interest that a number of candidate ncrnas exhibit an increase in expression in response to hdac inhibition, possibly suggesting a contribution to neuronal rather than ol differentiation. for example, the previously described ncrna gtl was upregulated after tsa treatment. indeed, within his study we observed upregulation of gtl during gaban differentiation. gtl is expressed in restricted domains of the developing forebrain including ventral telencephalon, neocortex, hippocampus and diencephalon, suggesting roles in regional neuronal development and differentiation. finally, we found that neat transcription exhibited complex transcriptional dynamics after tsa treatment, being strongly upregulated after tsa treatment under stochastic oligodendroglial specification conditions, whereas it was downregulated after tsa treatment under the influence of instructive signals for ol lineage elaboration. discussion in this study, we utilized a custom-designed microarray platform to probe the transcription of both protein-coding and noncoding transcripts during ventral forebrain-derived nsc lineage restriction, neuronal and oligodendrocyte lineage specification, neuronal-glial fate transitions, and progressive stages of ol lineage elaboration including myelination. expression profiles for mrnas in our study were similar to previously reported findings showing modulation of key signaling pathways, transcription factors, and epigenetic regulators that help to define the molecular codes governing neural lineage elaboration. additionally, we also observed expression of components of the myelin sheath. although we examined protein-coding genes, we focused primarily on characterizing the expression profiles of long ncrnas, which are being increasingly appreciated in the regulation of developmental gene expression programs. previous studies of neural differentiation have reported expression profiles for only a small number of long ncrnas, such as nkx as and the imprinted h ncrna. in contrast, we examined a large spectrum of long ncrnas and found dramatic changes in expression that are unique to specific stages of cellular differentiation. many of these ncrnas are transcribed from complex genomic loci that encompass protein-coding genes with defined roles in neuronal and glial lineage elaboration, and these pairs of ncrnas and associated protein-coding genes often exhibited coordinated expression during developmental transitions. therefore, we suggest that these ncrnas may influence the expression of the associated protein-coding genes similar to previously characterized examples such as nkx as, hotair, p15as, p21as and evf. for example, many ncrnas were associated with transcription factors that were themselves differentially regulated during ol differentiation. long ncrnas, such as those identified in this study, may therefore contribute to the combinatorial transcriptional codes involved in lineage specification and terminal differentiation including myelination. the coordinated expression of long ncrnas with critical neural developmental protein coding genes suggests that both are subject to shared regulatory mechanisms and that long ncrnas are integrated into complex environmentally-mediated neural and glial developmental gene expression programs. we, therefore, considered whether the well-documented ability of epigenetic mechanisms to promote decisive changes in neuronal and glial gene expression programs also includes the capacity to regulate long ncrna expression. indeed, treatment with the hdac inhibitor, tsa, effectively arrests the developmental progression of olps into pmos and also affects the expression of long ncrnas, suggesting that these are not only regulated by epigenetic modifications but also these transcripts are incorporated into a broader lineage specific developmental gene expression program. furthermore, hdac inhibition also has differential effects on certain ncrnas in an environmentally responsive manner. for example, treatment of olps with tsa in the presence or absence of cntf results in opposing profiles of neat expression, implying that long ncrnas might be deployed in a context-specific manner to dynamically modulate seminal fate decisions within the ol lineage. although the use of hdac inhibitors has been essential for interrogating the epigenetic mechanisms that govern ol lineage maturation, the cellular effects of tsa administration are complex and our understanding of the mechanisms mediating ncrna expression are still evolving. nonetheless, long ncrnas may represent important targets for regenerative therapies in the cns because they exhibit both exquisite site-selective as well as genome wide effects associated with dynamic ol developmental transition states characterized by considerable epigenetic flux.  this study is the first systematic examination of the expression of long ncrnas in forebrain-mediated nsc maturational processes, specifically ol differentiation and development. it is also the first demonstration that long ncrnas, many of which are involved in regulating epigenetic processes, are also themselves subject to regulation by epigenetic processes. it adds to a growing consensus that not only are large portions of the mammalian genome transcribed but also expressed in a regulated manner. the specific expression of ncrnas has been recently observed in other developmental programs and comparisons between these studies reveal both common and unique features. because the long noncoding transcriptome is still relatively poorly annotated and only partially interrogated by this study, it seems likely that many more long ncrnas will be shown to be expressed during development and their importance for neuronal and glial biology will be more fully understood and appreciated. these studies will have particular relevance in the future as the role of noncoding transcripts in neurological disease states becomes increasingly recognized. 
here we show that many long ncrnas exhibit dynamic expression patterns during neuronal and oligodendrocyte lineage specification, neuronal-glial fate transitions, and progressive stages of ol lineage elaboration including myelination. we also observed changes in ncrna expression profiles in response to treatment with trichostatin a, a histone deacetylase inhibitor that prevents the progression of ol progenitors into post-mitotic ols by altering lineage-specific gene expression programs. this is the first report of long ncrna expression in neuronal and glial cell differentiation and of the modulation of ncrna expression by modification of chromatin architecture. long non-protein-coding rnas are emerging as important regulators of cellular differentiation and are widely expressed in the brain.
 long non-protein-coding rnas are emerging as important regulators of cellular differentiation and are widely expressed in the brain.  here we show that many long ncrnas exhibit dynamic expression patterns during neuronal and oligodendrocyte lineage specification, neuronal-glial fate transitions, and progressive stages of ol lineage elaboration including myelination. consideration of the genomic context of these dynamically regulated ncrnas showed they were part of complex transcriptional loci that encompass key neural developmental protein-coding genes, with which they exhibit concordant expression profiles as indicated by both microarray and in situ hybridization analyses. these included ncrnas associated with differentiation-specific nuclear subdomains such as gomafu and neat and ncrnas associated with developmental enhancers and genes encoding important transcription factors and homeotic proteins. we also observed changes in ncrna expression profiles in response to treatment with trichostatin a, a histone deacetylase inhibitor that prevents the progression of ol progenitors into post-mitotic ols by altering lineage-specific gene expression programs.  this is the first report of long ncrna expression in neuronal and glial cell differentiation and of the modulation of ncrna expression by modification of chromatin architecture. these observations explicitly link ncrna dynamics to neural stem cell fate decisions, specification and epigenetic reprogramming and may have important implications for understanding and treating neuropsychiatric diseases. 