BACKGROUND
recent studies have demonstrated that non-protein coding rnas  comprise much of the mammalian transcriptome  <cit> .
several thousand mammalian ncrnas have been identified that span multiple kilobases in length  <cit> .
some of these ncrnas show extensive conservation at the nucleotide level, despite lacking evolutionarily conserved open reading frames .
many also show characteristics reminiscent of protein-coding mrnas, such as splicing, transcription by rna polymerase ii, polyadenylation, and 5'capping  <cit> .
perhaps the best characterized mrna-like ncrnas are the xist/tsix transcripts, which mediate x-inactivation in placental mammals  <cit> .
other long ncrnas such as air and h <dig> have been implicated in genomic imprinting  <cit> .
in both of these cases, ncrnas act locally, coating the nearby genomic loci and inducing the formation of heterochromatin and repression of gene expression  <cit> .
recent studies have identified other long ncrnas that regulate transcription at loci on different chromosomes  <cit> .
some of these ncrnas may function as rna-based transcriptional coregulators  <cit> .
other vertebrate long ncrnas have been implicated in the regulation of protein translation  <cit>  and signal transduction  <cit>  and they are likely to fulfill diverse cellular functions.
previous work has shown that many long ncrnas are selectively expressed in the developing nervous system  <cit> .
no functional data on their role in neuronal development is available for the vast majority of neuronally-expressed lncrnas, however.
two notable exceptions are evf- <dig>  which regulates dlx2-dependent activation of transcription of dlx <dig> and development of gabaergic neurons  <cit> , and tug <dig>  which regulates retinal rod photoreceptor development and survival  <cit> .
the developing retina, in particular, expresses a diverse assortment of lncrnas.
many of these are transcribed in an a divergent, head-to-head fashion with over one third of retinally-expressed transcription factors  <cit> .
other classes of lncrnas, which are not located near protein-coding genes, are also strongly expressed in the retina.
the most abundant of these, which represents nearly  <dig> % of all polyadenylated rna in the neonatal retina was first described as retinal non-coding rna  <dig> , also known as both gomafu and miat  <cit> .
this  <dig> kb ncrna is expressed widely in the developing nervous system in an uncharacterized subset of neural precursor cells and is targeted to an as yet uncharacterized domain of the nucleus.
in this study, we perform a detailed analysis of the cellular expression pattern and the function of rncr <dig> in retinal differentiation.
in addition, we devised an experimental strategy to develop dominant-negative forms of rncr <dig>
RESULTS
evolutionary conservation and genomic organization of rncr2
to determine the extent to which rncr <dig> is evolutionary conserved and to identify functionally important domains, we examined genomic and cdna sequence data from other vertebrates.
rncr <dig>  which does not lie within  <dig> kb of any known protein coding gene, showed extensive sequence conservation and little diversity of splicing among mammals .
though conventional sequence alignment searches did not identify any nonmammalian orthologues of rncr <dig>  <cit> , a synteny-based search identified putative rncr <dig> orthologues from chick and xenopus tropicalis.
multiple clustered sequence repeats of the sequence acuaacc were found in the largest exon of each orthologue, with anywhere from five to eight such repeats found within each transcript.
a genome-wide search of human and mouse cdna sequences did not identify any other transcripts with more than one evolutionarily conserved acuaacc repeat .
this repeat sequence includes the acuaay consensus recognition site for the rna binding protein quaking, which regulates both rna subcellular localization and stability  <cit> , although the functional significance of this is unclear.
cellular expression pattern of rncr <dig> in developing retina
we next performed fluorescent in situ hybridization  for rncr <dig>  rncr <dig> has a strikingly punctuate expression pattern in the developing retina at e <dig> , with cells that strongly express rncr <dig> intermingled with cells that express it weakly or not at all .
rncr <dig> expression declines after the first few days postnatal, and by p <dig> is confined to a subset of cells in the inner nuclear layer and ganglion cell layer of the peripheral retina .
rncr <dig> rna is exclusively nuclear at this stage and excluded from dapi-stained chromatin , as previously reported for the embryonic retina  <cit> .
as previously reported  <cit> , rncr <dig> is not detectably expressed in adult retina.
to determine which cells express rncr <dig> in the developing retina, we used the results of a previous genome-scale analysis of gene expression in the developing mouse retina  <cit>  to perform two-color dissociated cell fish using multiple probes that recognized retinal cell-specific markers at different developmental stages .
at e <dig> , we find that rncr <dig> is expressed in a large fraction of cells that express the progenitor-specific markers chx <dig> and fgf <dig> .
rncr <dig> is also expressed in many cells expressing the amacrine-specific marker lhx <dig> and isl <dig>  which is expressed in developing amacrine and ganglion cells.
a substantial minority of cells at this stage expressing the photoreceptor-specific marker crx are rncr2-positive .
a much smaller fraction of cells expressing the progenitor-specific markers fgf <dig> and chx <dig> expressed rncr <dig> at p <dig>  than did at e <dig> , while expression in amacrine and photoreceptor precursors remained prominent .
finally, at p <dig>  rncr <dig> expression was restricted to a subset of amacrine cells , in line with the observed expression in the inner nuclear layer  and ganglion cell layer  .
loss of function of rncr <dig> inhibits amacrine cell and müller glia differentiation in postnatal retina
given the prominent and selective expression of rncr <dig> in the developing retina, we next employed in vivo electroporation of p <dig>  mouse retinas to determine whether rncr <dig> regulates retinal cell differentiation.
to visualize electroporated cells, all constructs were coelectroporated with plasmids that express gfp from the same cag promoter.
for rncr <dig>  we assembled a  <dig> kb cdna corresponding to the previously reported full-length mouse sequence  <cit>  .
overexpression of rncr <dig> did not give any detectable phenotype in either section or dissociated cell immunohistochemistry , and no changes in morphology or position of electroporated cells were observed at p <dig> .
for knockdown analysis, we tested shrnas for their ability to reduce expression of endogenous rncr <dig> expression by in vivo electroporation of p <dig>  retina.
reduction in rna expression was determined by fish analysis of p <dig>  dissociated cells in conjunction with anti-gfp immunocytochemistry to identify electroporated cells .
a shrna construct was identified that resulted in a >75% reduction in the number of double-labeled electroporated cells at p <dig>  compared to cells electroporated with control shrna.
the shrna-mediated knockdown of rncr <dig> led to a decrease in the fraction of cells expressing the müller glia marker glutamine synthase  at p <dig> .
two weeks later at p <dig>  however, a significant increase in the fraction of both gs-positive müller glia  and syntaxin-positive amacrine cells  was observed at p <dig> .
this finding was confirmed by analysis of immunostained sections of electroporated retina .
a significant increase in the fraction of electroporated cells in the inner nuclear layer  , together with a corresponding decrease in the fraction of electroporated cells in the photoreceptor-containing outer nuclear layer   was detected following rncr <dig> knockdown, in line with the observed increase in cells expressing amacrine and müller glial markers .
no change in the fraction of electroporated cells expressing the rod bipolar marker pkcα was observed following rncr <dig> knockdown .
surprisingly, however, no statistically significant reduction in the number of cells expressing the rod-specific marker rho4d <dig> was observed .
additionally, no increase in apoptotic cell death, as indicated by tunel staining, was observed following rncr <dig> knockdown at p <dig> .
one possible explanation is that a subset of inner retinal cells may continue to express rhodopsin in addition to syntaxin and/or gs, although we have seen no evidence to support this using section immunohistochemistry .
fusion of rncr <dig> to an ires-gfp sequence results in nuclear export and a dominant-negative phenotype in vivo
throughout this study, we explored additional approaches to perturb the function of rncr <dig> and to investigate the specific function of different domains of this molecule.
one strategy used to generate dominant-negative forms of nuclear proteins has been to construct mutants that are sequestered in the cytoplasm, thus blocking nuclear entry of any cofactors that interact with the protein in question  <cit> .
we used an analogous approach to generate dominant-negative forms of rncr <dig>  we adapted this strategy for analysis of rncr <dig>  we hypothesized that lncrnas such as rncr <dig>  like proteins, must interact with cellular effector molecules to carry out their physiological functions and that mislocalization of long ncrnas might then result in mislocalization of the associated effector molecules.
since long ncrnas are not translated, the fusion of ires-gfp sequences to ncrnas would mislocalize these molecules to cytoplasmic ribosomes, and away from their normal cellular sites of action in the nucleus, titrating down the endogenous biochemical effectors of rncr <dig> action and effectively resulting in a dominant-negative phenotype.
figures 5a and 5b illustrate this concept schematically.
we first tested whether fusion of rncr <dig> to ires-gfp would mislocalize the rna to the cytoplasm.
using fish in conjunction with immunostaining for the ribosomal protein s <dig>  we observed that full-length rncr2-ires-gfp constructs are mislocalized from the nucleus to the cytoplasm when expressed in hela cells .
cells transfected with rncr2-ires-gfp showed gfp fluorescence, indicating that these ires-fusion constructs are indeed targeted to the ribosome and undergo translation .
we next tested whether electroporation of ires-gfp fusion constructs into p <dig>  retina produced phenotypes like those seen following shrna-mediated knockdown of rncr <dig>  expression of rncr2-ires-gfp resulted in a significant increase in the fraction of syntaxin-positive cells at p <dig> and p <dig> , as was observed with shrna-mediated knockdown of rncr <dig> at p <dig> , and a significant increase in the fraction of cells in the inl and decrease in cells in the onl  .
an increase in amacrine cells was confirmed by section immunohistochemistry of electroporated retina .
furthermore, expression of rncr2-ires-gfp results in a substantial increase in the fraction of cells that do not express any of the four markers examined .
section immunohistochemistry indicates a roughly two-fold increase in the fraction of electroporated cells in the inner nuclear layer, comparable to that observed following shrna-mediated knockdown of rncr <dig> , and overexpression of rncr2-ires-gfp further resulted in a nearly two-fold decrease in the fraction of rhodopsin-positive cells at both p <dig> and p <dig> .
since the decrease in the fraction of rhodopsin-expressing cells was substantially greater than the observed increase in the fraction of cells in the inl, this data implies that overexpression of rncr2-ires-gfp induces both a shift in electroporated retinal precursors away from a rod photoreceptor fate and a reduction in rhodopsin expression levels, as implied by the dramatic increase in the fraction cells of electroporated with the rncr2-ires-gfp construct that are not labeled by any of the four markers tested.
no increase in apoptotic cell death, as indicated by tunel staining, was observed following electroporation of rncr2-ires-gfp at p <dig>  nor was any increase in staining for the cone-specific marker gtγ <dig> observed at p <dig>  eliminating the possibility these changes in rhodopsin expression levels are a result of apoptosis or a change in cell fate from rod photoreceptors to cone photoreceptors.
to further explore the phenotypes seen following rncr2-ires-gfp expression, we generated domain-specific ires-gfp fusions corresponding to non-overlapping 2- <dig> kb domains of the 5', middle and 3' regions of the  <dig> kb rncr <dig> rna , and overexpressed these in p <dig>  retina using in vivo electroporation.
section immunohistochemistry revealed that the expression of ires-gfp fusion constructs of the 5' domain of rncr <dig>  led to an increase in the fraction of amacrine cells, as was observed following overexpression of the full-length rncr2-ires-gfp construct .
however, ires-gfp fusion constructs of the middle  <dig>  kb of rncr <dig>  led to an increase in both müller glia  and amacrine cells , while a construct including the 3' region of rncr <dig>  led to an increase in müller glia only .
the increase in müller glia that is seen following overexpression of the middle and 3' ires-gfp fusion domains mirrors that observed following shrna-mediated knockdown of rncr <dig> expression .
finally, all three domain-specific ires fusion constructs resulted in an increase in the total fraction of gfp-positive cells in the inner nuclear layer , reflecting the increase in amacrine cells and müller glia like that observed following shrna-mediated knockdown of rncr <dig>  we interpret these results to show that ires-gfp fusion constructs representing distinct domains of rncr <dig> possess different functions in control of retinal differentiation.
since rncr <dig> shrna results in an increase in the fraction of muller glia but the full-length rncr2-ires-gfp construct does not , these findings suggest that domain-specific ires-gfp fusion constructs of rncr <dig> may be more effective at inhibiting differentiation of certain cell types than the full-length construct.
when taken together, these results closely phenocopy the changes in cell fates observed following shrna-mediated knockdown of this rna.
discussion
rncr <dig> negatively regulates development of multiple retinal cell types
our findings demonstrate that the overexpression and knockdown of rncr <dig> in the developing retina regulates the process of cell fate specification.
in keeping with rncr2's prominent and selective expression in mitotic progenitors and postmitotic retinal precursor cells, we observe that rncr <dig> acts to negatively regulate the differentiation of multiple and partially overlapping cell types .
this is in contrast to the role of tug <dig>  the only long ncrna previously investigated in the context of retinal development  <cit> , which positively regulates photoreceptor development and survival.
it has recently become clear that negative regulatory interactions play a prominent and crucial role in retinal cell fate specification  <cit> .
rncr <dig> represents the first long ncrna reported to perform this function.
these findings demonstrate that rncr <dig> negatively regulates amacrine differentiation, as both rncr <dig> knockdown and rncr2-ires-gfp expression result in an increase in gfp-positive cells with amacrine-like morphology, as well as in syntaxin immunoreactivity.
however, it seems at first glance difficult to reconcile this finding with the fact that rncr <dig> is also selectively expressed in immature amacrine cells.
several examples of a similar correlation of phenotype and gene expression have been previously reported in developing retina.
one example is pax <dig>  which is prominently expressed in both retinal progenitors and developing amacrine cells.
when pax <dig> is selectively inactivated in retinal progenitors, a massive overproduction of gabaergic amacrine cells is seen  <cit> .
it is thought that pax <dig> may act in retinal progenitors to inhibit premature amacrine differentiation, but have different functions in cells already committed to the amacrine lineage.
rncr <dig> may play a similar role in retinal progenitor and precursor cells.
in addition to inhibiting the development of amacrine interneurons, rncr <dig> also inhibits the development of müller glia but does not affect the development of other neuronal subtypes such as bipolar interneurons.
interestingly, however, we observe that shrna-mediated knockdown of rncr <dig> results in a reduction of cells expressing the muller marker gs at p <dig>  in contrast to the significant increase in gs-positive cells that is observed at p <dig>  it is possible that rncr <dig> knockdown induces a delay in the differentiation of muller glia, which initiate expression of gs shortly prior to p <dig>  by p <dig>  however, gliogenesis is complete and all differentiated muller glia should be expressing detectable levels of gs.
thus, we hypothesize that these data may result from rncr <dig> knockdown inducing a delay in muller glia differentiation while nonetheless also inducing a shift in retinal progenitor cells towards a glial fate.
taken together, our findings suggest that the effects of rncr <dig> loss of function on retinal cell differentiation seen in these studies are specific and do not represent a general developmental delay or inhibition of neurogenesis.
although these findings demonstrate the functional importance of rncr <dig> in retinal cell differentiation, its molecular mechanism of action still remains unclear.
unlike other nuclear-retained long ncrnas such as xist and air, it shows no association with chromatin and is tightly associated with nuclear matrix  <cit> .
it shows no obvious structural motifs, but is conserved from amphibians to mammals  and contains multiple putative quaking binding sites.
the biochemical mechanism of action by which rncr <dig> controls retinal cell fate specification awaits further characterization.
mislocalization of nuclear-retained lncrnas to the cytoplasm results in a dominant-negative phenotype in vivo
there have been no general strategies yet proposed for the generation of dominant-negative constructs for long ncrnas.
in this study, we demonstrate that fusion of a nuclear-retained lncrnas to an ires-gfp sequence can mislocalize exogenous ncrnas to the ribosome, and produces phenotypes that largely overlap with those seen with shrna-mediated knockdown.
indeed, the overexpression of ires-gfp fusion constructs encoding different domains of rncr <dig> variously increases the number of amacrine cells, müller glia, or both cell types like what is seen in the rncr <dig> shrna-mediated knockdown, depending on the domain of rncr <dig> that is tested.
certain novel phenotypes are observed following overexpression of rncr2-ires-gfp fusions that are not seen following rncr <dig> knockdown, most notably a substantial reduction in the fraction of rhodopsin-expressing cells.
it is possible that rncr <dig> has a role in regulating expression of rhodopsin, and that this function might be inhibited more effectively by overexpression of the ires-gfp fusion than by the rncr <dig> shrna construct.
however, it is also possible that overexpression of the rncr2-ires-gfp fusion construct may introduce a neomorphic phenotype in developing photoreceptors that is not apparent following overexpression of rncr <dig> alone .
further studies will be required to resolve this question.
many of the thousands of mammalian lncrnas are nuclear-retained  <cit> .
although other nuclear-retained lncrnas in addition to rncr <dig> have been shown to be susceptible to shrna-mediated degradation  <cit> , conventional rna interference approaches are in general far less successful at reducing expression of these molecules than cytoplasmic transcripts  <cit> .
the fact that the ires-gfp sequence mislocalizes untranslated lncrnas from the nucleus to the ribosome may serve as a general tool for functional analysis of these molecules.
CONCLUSIONS
we show that the long noncoding nuclear-localized rna rncr <dig> regulates retinal cell fate specification.
we find that shrna-mediated loss of function of rncr <dig> increases the fraction of amacrine interneurons and müller glia at the expense of photoreceptor cells.
we further show that fusion of rncr <dig> to an ires-gfp sequence results in mislocalization of this nuclear rna to the cytoplasm.
this largely phenocopies the effects of knockdown of rncr <dig>  and we further use this approach to define domains of rncr <dig> that inhibit amacrine and müller glial differentiation, respectively.
this ires-gfp fusion technique provides a rapid, general method for functionally analyzing nuclear-localized noncoding rnas in vivo, and can potentially allow one to readily observe more severe phenotypes than are seen using rna interference methodology.

 recent studies have demonstrated that non-protein coding rnas comprise much of the mammalian transcriptome. several thousand mammalian ncrnas have been identified that span multiple kilobases in length. some of these ncrnas show extensive conservation at the nucleotide level, despite lacking evolutionarily conserved open reading frames. many also show characteristics reminiscent of protein-coding mrnas, such as splicing, transcription by rna polymerase ii, polyadenylation, and 5'capping. perhaps the best characterized mrna-like ncrnas are the xist/tsix transcripts, which mediate x-inactivation in placental mammals. other long ncrnas such as air and h have been implicated in genomic imprinting. in both of these cases, ncrnas act locally, coating the nearby genomic loci and inducing the formation of heterochromatin and repression of gene expression. recent studies have identified other long ncrnas that regulate transcription at loci on different chromosomes. some of these ncrnas may function as rna-based transcriptional coregulators. other vertebrate long ncrnas have been implicated in the regulation of protein translation and signal transduction and they are likely to fulfill diverse cellular functions. previous work has shown that many long ncrnas are selectively expressed in the developing nervous system. no functional data on their role in neuronal development is available for the vast majority of neuronally-expressed lncrnas, however. two notable exceptions are evf- which regulates dlx2-dependent activation of transcription of dlx and development of gabaergic neurons, and tug which regulates retinal rod photoreceptor development and survival. the developing retina, in particular, expresses a diverse assortment of lncrnas. many of these are transcribed in an a divergent, head-to-head fashion with over one third of retinally-expressed transcription factors. other classes of lncrnas, which are not located near protein-coding genes, are also strongly expressed in the retina. the most abundant of these, which represents nearly % of all polyadenylated rna in the neonatal retina was first described as retinal non-coding rna, also known as both gomafu and miat. this kb ncrna is expressed widely in the developing nervous system in an uncharacterized subset of neural precursor cells and is targeted to an as yet uncharacterized domain of the nucleus. in this study, we perform a detailed analysis of the cellular expression pattern and the function of rncr in retinal differentiation. in addition, we devised an experimental strategy to develop dominant-negative forms of rncr  evolutionary conservation and genomic organization of rncr2 to determine the extent to which rncr is evolutionary conserved and to identify functionally important domains, we examined genomic and cdna sequence data from other vertebrates. rncr which does not lie within kb of any known protein coding gene, showed extensive sequence conservation and little diversity of splicing among mammals. though conventional sequence alignment searches did not identify any nonmammalian orthologues of rncr, a synteny-based search identified putative rncr orthologues from chick and xenopus tropicalis. multiple clustered sequence repeats of the sequence acuaacc were found in the largest exon of each orthologue, with anywhere from five to eight such repeats found within each transcript. a genome-wide search of human and mouse cdna sequences did not identify any other transcripts with more than one evolutionarily conserved acuaacc repeat. this repeat sequence includes the acuaay consensus recognition site for the rna binding protein quaking, which regulates both rna subcellular localization and stability, although the functional significance of this is unclear. cellular expression pattern of rncr in developing retina we next performed fluorescent in situ hybridization for rncr rncr has a strikingly punctuate expression pattern in the developing retina at e, with cells that strongly express rncr intermingled with cells that express it weakly or not at all. rncr expression declines after the first few days postnatal, and by p is confined to a subset of cells in the inner nuclear layer and ganglion cell layer of the peripheral retina. rncr rna is exclusively nuclear at this stage and excluded from dapi-stained chromatin, as previously reported for the embryonic retina. as previously reported, rncr is not detectably expressed in adult retina. to determine which cells express rncr in the developing retina, we used the results of a previous genome-scale analysis of gene expression in the developing mouse retina to perform two-color dissociated cell fish using multiple probes that recognized retinal cell-specific markers at different developmental stages. at e, we find that rncr is expressed in a large fraction of cells that express the progenitor-specific markers chx and fgf. rncr is also expressed in many cells expressing the amacrine-specific marker lhx and isl which is expressed in developing amacrine and ganglion cells. a substantial minority of cells at this stage expressing the photoreceptor-specific marker crx are rncr2-positive. a much smaller fraction of cells expressing the progenitor-specific markers fgf and chx expressed rncr at p than did at e, while expression in amacrine and photoreceptor precursors remained prominent. finally, at p rncr expression was restricted to a subset of amacrine cells, in line with the observed expression in the inner nuclear layer and ganglion cell layer. loss of function of rncr inhibits amacrine cell and müller glia differentiation in postnatal retina given the prominent and selective expression of rncr in the developing retina, we next employed in vivo electroporation of p mouse retinas to determine whether rncr regulates retinal cell differentiation. to visualize electroporated cells, all constructs were coelectroporated with plasmids that express gfp from the same cag promoter. for rncr we assembled a kb cdna corresponding to the previously reported full-length mouse sequence. overexpression of rncr did not give any detectable phenotype in either section or dissociated cell immunohistochemistry, and no changes in morphology or position of electroporated cells were observed at p. for knockdown analysis, we tested shrnas for their ability to reduce expression of endogenous rncr expression by in vivo electroporation of p retina. reduction in rna expression was determined by fish analysis of p dissociated cells in conjunction with anti-gfp immunocytochemistry to identify electroporated cells. a shrna construct was identified that resulted in a >75% reduction in the number of double-labeled electroporated cells at p compared to cells electroporated with control shrna. the shrna-mediated knockdown of rncr led to a decrease in the fraction of cells expressing the müller glia marker glutamine synthase at p. two weeks later at p however, a significant increase in the fraction of both gs-positive müller glia and syntaxin-positive amacrine cells was observed at p. this finding was confirmed by analysis of immunostained sections of electroporated retina. a significant increase in the fraction of electroporated cells in the inner nuclear layer, together with a corresponding decrease in the fraction of electroporated cells in the photoreceptor-containing outer nuclear layer was detected following rncr knockdown, in line with the observed increase in cells expressing amacrine and müller glial markers. no change in the fraction of electroporated cells expressing the rod bipolar marker pkcα was observed following rncr knockdown. surprisingly, however, no statistically significant reduction in the number of cells expressing the rod-specific marker rho4d was observed. additionally, no increase in apoptotic cell death, as indicated by tunel staining, was observed following rncr knockdown at p. one possible explanation is that a subset of inner retinal cells may continue to express rhodopsin in addition to syntaxin and/or gs, although we have seen no evidence to support this using section immunohistochemistry. fusion of rncr to an ires-gfp sequence results in nuclear export and a dominant-negative phenotype in vivo throughout this study, we explored additional approaches to perturb the function of rncr and to investigate the specific function of different domains of this molecule. one strategy used to generate dominant-negative forms of nuclear proteins has been to construct mutants that are sequestered in the cytoplasm, thus blocking nuclear entry of any cofactors that interact with the protein in question. we used an analogous approach to generate dominant-negative forms of rncr we adapted this strategy for analysis of rncr we hypothesized that lncrnas such as rncr like proteins, must interact with cellular effector molecules to carry out their physiological functions and that mislocalization of long ncrnas might then result in mislocalization of the associated effector molecules. since long ncrnas are not translated, the fusion of ires-gfp sequences to ncrnas would mislocalize these molecules to cytoplasmic ribosomes, and away from their normal cellular sites of action in the nucleus, titrating down the endogenous biochemical effectors of rncr action and effectively resulting in a dominant-negative phenotype. figures 5a and 5b illustrate this concept schematically. we first tested whether fusion of rncr to ires-gfp would mislocalize the rna to the cytoplasm. using fish in conjunction with immunostaining for the ribosomal protein s we observed that full-length rncr2-ires-gfp constructs are mislocalized from the nucleus to the cytoplasm when expressed in hela cells. cells transfected with rncr2-ires-gfp showed gfp fluorescence, indicating that these ires-fusion constructs are indeed targeted to the ribosome and undergo translation. we next tested whether electroporation of ires-gfp fusion constructs into p retina produced phenotypes like those seen following shrna-mediated knockdown of rncr expression of rncr2-ires-gfp resulted in a significant increase in the fraction of syntaxin-positive cells at p and p, as was observed with shrna-mediated knockdown of rncr at p, and a significant increase in the fraction of cells in the inl and decrease in cells in the onl. an increase in amacrine cells was confirmed by section immunohistochemistry of electroporated retina. furthermore, expression of rncr2-ires-gfp results in a substantial increase in the fraction of cells that do not express any of the four markers examined. section immunohistochemistry indicates a roughly two-fold increase in the fraction of electroporated cells in the inner nuclear layer, comparable to that observed following shrna-mediated knockdown of rncr, and overexpression of rncr2-ires-gfp further resulted in a nearly two-fold decrease in the fraction of rhodopsin-positive cells at both p and p. since the decrease in the fraction of rhodopsin-expressing cells was substantially greater than the observed increase in the fraction of cells in the inl, this data implies that overexpression of rncr2-ires-gfp induces both a shift in electroporated retinal precursors away from a rod photoreceptor fate and a reduction in rhodopsin expression levels, as implied by the dramatic increase in the fraction cells of electroporated with the rncr2-ires-gfp construct that are not labeled by any of the four markers tested. no increase in apoptotic cell death, as indicated by tunel staining, was observed following electroporation of rncr2-ires-gfp at p nor was any increase in staining for the cone-specific marker gtγ observed at p eliminating the possibility these changes in rhodopsin expression levels are a result of apoptosis or a change in cell fate from rod photoreceptors to cone photoreceptors. to further explore the phenotypes seen following rncr2-ires-gfp expression, we generated domain-specific ires-gfp fusions corresponding to non-overlapping 2- kb domains of the 5', middle and 3' regions of the kb rncr rna, and overexpressed these in p retina using in vivo electroporation. section immunohistochemistry revealed that the expression of ires-gfp fusion constructs of the 5' domain of rncr led to an increase in the fraction of amacrine cells, as was observed following overexpression of the full-length rncr2-ires-gfp construct. however, ires-gfp fusion constructs of the middle kb of rncr led to an increase in both müller glia and amacrine cells, while a construct including the 3' region of rncr led to an increase in müller glia only. the increase in müller glia that is seen following overexpression of the middle and 3' ires-gfp fusion domains mirrors that observed following shrna-mediated knockdown of rncr expression. finally, all three domain-specific ires fusion constructs resulted in an increase in the total fraction of gfp-positive cells in the inner nuclear layer, reflecting the increase in amacrine cells and müller glia like that observed following shrna-mediated knockdown of rncr we interpret these results to show that ires-gfp fusion constructs representing distinct domains of rncr possess different functions in control of retinal differentiation. since rncr shrna results in an increase in the fraction of muller glia but the full-length rncr2-ires-gfp construct does not, these findings suggest that domain-specific ires-gfp fusion constructs of rncr may be more effective at inhibiting differentiation of certain cell types than the full-length construct. when taken together, these results closely phenocopy the changes in cell fates observed following shrna-mediated knockdown of this rna. discussion rncr negatively regulates development of multiple retinal cell types our findings demonstrate that the overexpression and knockdown of rncr in the developing retina regulates the process of cell fate specification. in keeping with rncr2's prominent and selective expression in mitotic progenitors and postmitotic retinal precursor cells, we observe that rncr acts to negatively regulate the differentiation of multiple and partially overlapping cell types. this is in contrast to the role of tug the only long ncrna previously investigated in the context of retinal development, which positively regulates photoreceptor development and survival. it has recently become clear that negative regulatory interactions play a prominent and crucial role in retinal cell fate specification. rncr represents the first long ncrna reported to perform this function. these findings demonstrate that rncr negatively regulates amacrine differentiation, as both rncr knockdown and rncr2-ires-gfp expression result in an increase in gfp-positive cells with amacrine-like morphology, as well as in syntaxin immunoreactivity. however, it seems at first glance difficult to reconcile this finding with the fact that rncr is also selectively expressed in immature amacrine cells. several examples of a similar correlation of phenotype and gene expression have been previously reported in developing retina. one example is pax which is prominently expressed in both retinal progenitors and developing amacrine cells. when pax is selectively inactivated in retinal progenitors, a massive overproduction of gabaergic amacrine cells is seen. it is thought that pax may act in retinal progenitors to inhibit premature amacrine differentiation, but have different functions in cells already committed to the amacrine lineage. rncr may play a similar role in retinal progenitor and precursor cells. in addition to inhibiting the development of amacrine interneurons, rncr also inhibits the development of müller glia but does not affect the development of other neuronal subtypes such as bipolar interneurons. interestingly, however, we observe that shrna-mediated knockdown of rncr results in a reduction of cells expressing the muller marker gs at p in contrast to the significant increase in gs-positive cells that is observed at p it is possible that rncr knockdown induces a delay in the differentiation of muller glia, which initiate expression of gs shortly prior to p by p however, gliogenesis is complete and all differentiated muller glia should be expressing detectable levels of gs. thus, we hypothesize that these data may result from rncr knockdown inducing a delay in muller glia differentiation while nonetheless also inducing a shift in retinal progenitor cells towards a glial fate. taken together, our findings suggest that the effects of rncr loss of function on retinal cell differentiation seen in these studies are specific and do not represent a general developmental delay or inhibition of neurogenesis. although these findings demonstrate the functional importance of rncr in retinal cell differentiation, its molecular mechanism of action still remains unclear. unlike other nuclear-retained long ncrnas such as xist and air, it shows no association with chromatin and is tightly associated with nuclear matrix. it shows no obvious structural motifs, but is conserved from amphibians to mammals and contains multiple putative quaking binding sites. the biochemical mechanism of action by which rncr controls retinal cell fate specification awaits further characterization. mislocalization of nuclear-retained lncrnas to the cytoplasm results in a dominant-negative phenotype in vivo there have been no general strategies yet proposed for the generation of dominant-negative constructs for long ncrnas. in this study, we demonstrate that fusion of a nuclear-retained lncrnas to an ires-gfp sequence can mislocalize exogenous ncrnas to the ribosome, and produces phenotypes that largely overlap with those seen with shrna-mediated knockdown. indeed, the overexpression of ires-gfp fusion constructs encoding different domains of rncr variously increases the number of amacrine cells, müller glia, or both cell types like what is seen in the rncr shrna-mediated knockdown, depending on the domain of rncr that is tested. certain novel phenotypes are observed following overexpression of rncr2-ires-gfp fusions that are not seen following rncr knockdown, most notably a substantial reduction in the fraction of rhodopsin-expressing cells. it is possible that rncr has a role in regulating expression of rhodopsin, and that this function might be inhibited more effectively by overexpression of the ires-gfp fusion than by the rncr shrna construct. however, it is also possible that overexpression of the rncr2-ires-gfp fusion construct may introduce a neomorphic phenotype in developing photoreceptors that is not apparent following overexpression of rncr alone. further studies will be required to resolve this question. many of the thousands of mammalian lncrnas are nuclear-retained. although other nuclear-retained lncrnas in addition to rncr have been shown to be susceptible to shrna-mediated degradation, conventional rna interference approaches are in general far less successful at reducing expression of these molecules than cytoplasmic transcripts. the fact that the ires-gfp sequence mislocalizes untranslated lncrnas from the nucleus to the ribosome may serve as a general tool for functional analysis of these molecules.  we show that the long noncoding nuclear-localized rna rncr regulates retinal cell fate specification. we find that shrna-mediated loss of function of rncr increases the fraction of amacrine interneurons and müller glia at the expense of photoreceptor cells. we further show that fusion of rncr to an ires-gfp sequence results in mislocalization of this nuclear rna to the cytoplasm. this largely phenocopies the effects of knockdown of rncr and we further use this approach to define domains of rncr that inhibit amacrine and müller glial differentiation, respectively. this ires-gfp fusion technique provides a rapid, general method for functionally analyzing nuclear-localized noncoding rnas in vivo, and can potentially allow one to readily observe more severe phenotypes than are seen using rna interference methodology. 
finally, we use the ires-gfp fusion approach to identify specific domains of rncr that are required for repressing both amacrine and müller glial differentiation. overexpression of rncr2-ires-gfp phenocopies the effects of shrna-mediated knockdown of rncr implying that forced mislocalization of rncr induces a dominant-negative phenotype. we further report that rncr rna, which is normally nuclear-retained, can be exported from the nucleus when fused to an ires-gfp sequence. shrna-mediated knockdown of rncr results in an increase of both amacrine cells and müller glia, indicating a role for this lncrna in regulating retinal cell fate specification. furthermore, we present a novel approach for generating dominant-negative constructs of lncrnas, which may be generally useful in the functional analysis of this class of molecules. we find that the rncr is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. recent work has identified that many long mrna-like noncoding rnas are expressed in the developing nervous system.
 recent work has identified that many long mrna-like noncoding rnas are expressed in the developing nervous system. despite their abundance, the function of these ncrnas has remained largely unexplored. we have investigated the highly abundant lncrna rncr in regulation of mouse retinal cell differentiation.  we find that the rncr is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. shrna-mediated knockdown of rncr results in an increase of both amacrine cells and müller glia, indicating a role for this lncrna in regulating retinal cell fate specification. we further report that rncr rna, which is normally nuclear-retained, can be exported from the nucleus when fused to an ires-gfp sequence. overexpression of rncr2-ires-gfp phenocopies the effects of shrna-mediated knockdown of rncr implying that forced mislocalization of rncr induces a dominant-negative phenotype. finally, we use the ires-gfp fusion approach to identify specific domains of rncr that are required for repressing both amacrine and müller glial differentiation.  these data demonstrate that the lncrna rncr plays a critical role in regulating mammalian retinal cell fate specification. furthermore, we present a novel approach for generating dominant-negative constructs of lncrnas, which may be generally useful in the functional analysis of this class of molecules. 