Hotair Is Dispensible for Mouse Development
[['Hox genes encode transcription factors with crucial roles in the specification of regional identities along the body axes during development .', 'Mutations affecting specific Hox genes typically lead to homeotic transformations , whereby a particular body part is transformed into the identity of another one [1–4] .', 'In mammals , following the two rounds of genome duplication that occurred at the basis of the vertebrate lineage ( see [5] ) , four distinct clusters of Hox genes are found ( HoxA to HoxD ) ( ref . in [6] ) .', 'During development , Hox genes are transcriptionally activated in a precise temporal and spatial sequence , which follows their chromosomal order [7 , 8] .', 'These collinear patterns of transcription are regulated at multiple levels and studies focusing on the HoxA , HoxB and HoxD loci have revealed the importance of intricate combinations of local and long-range cis-regulatory elements .', 'Also , studies using micro-dissected embryonic material have shown that the transcriptional activation of these genes , in different ontogenetic contexts , is accompanied by major changes in both the epigenetic modifications of the surrounding chromatin and its 3D spatial organization [9–11] .', 'Long non-coding RNAs ( lncRNAs ) have been proposed to represent yet another layer of regulatory control at these important developmental loci ( e . g . [12–15] ) .', 'Increasing evidence indeed suggests that lncRNAs can act as regulators of gene expression , for example by interacting with transcription factors and chromatin modifiers to modulate transcription during development [16] .', 'Several lncRNAs associated with the mammalian Hox clusters have been identified , amongst which Hotair ( Hox transcript antisense intergenic RNA ) , a lncRNA transcribed from the intergenic region between Hoxc11 and Hoxc12 within the HoxC cluster and the founding member of this new class of RNAs .', 'Hotair was proposed to help repress some 5’-located ( posterior ) Hoxd genes in trans , through its association with chromatin modification complexes such as PRC2 , LSD1 and CoREST/REST [13 , 17] .', 'Accordingly , Hotair would recruit or enrich this part of the HoxD cluster with Polycomb ( Pc ) complex , thus contributing to its repressed state before transcription starts .', 'This proposal was substantiated by the knockdown of Hotair in human fibroblasts , which led to a decreased binding of Pc repressive complexes in the HoxD cluster and to a concurrent increase in Hoxd genes expression [13] .', 'This important function for a lncRNA in cultured human fibroblasts was however not supported by the analysis of a mouse line carrying a targeted deletion of the entire HoxC cluster [18] , i . e . including the mouse Hotair lncRNA .', 'This deletion showed little effect in vivo , with no alteration of Hoxd genes expression .', 'Also , the presence and enrichment of H3K27me3 repressive chromatin marks at the HoxD locus was not dramatically modified [19] .', 'This lack of effect was tentatively explained by the concomitant in-cis deletion of all Hoxc genes , which may have masked or compensated a potential alteration caused by the absence of Hotair alone [20] .', 'To alleviate this problem , three alleles were recently produced where the Hotair transcript was specifically targeted ( Fig 1 ) .', 'The first allele is a targeted deletion of the two major exons of Hotair .', 'Mice carrying this deletion were reported to display a malformation of the wrist and homeotic transformations of the spine , either from six lumbar vertebrae ( L6 ) to a L5 vertebral formula , or within the post-sacral region [20] .', 'These phenotypes were associated with a de-repression of Hoxd genes and of a set of imprinted genes by modulation of their chromatin state [20] .', 'These effects were scored in the absence of any change in the transcription of the neighboring Hoxc11 and Hoxc12 genes , supporting a function of Hotair in trans [20] .', 'Two additional Hotair deletion mutant alleles combined with LacZ reporter knock-in were recently reported by Lai and colleagues [21] .', 'The first allele deleted nearly the entire Hotair sequence and the second one comprised a smaller deletion starting in the second exon [21] .', 'In both cases , while a subtle alteration of the 4th caudal vertebra was scored , the wrist and the spine appeared normally formed , without any sign of the lumbar homeotic transformation and wrist alterations previously reported for the deletion of both exons [20] .', 'Due to our long-lasting interest in the transcriptional regulation of Hoxd genes during development ( e . g . [22] ) , we addressed these apparently conflicting results by re-assessing the effects of deleting the Hotair lncRNA during early mouse development , using the largest deletion allele previously described [20] .', 'In agreement with earlier and more recent results [19 , 21] , we find that the deletion of Hotair has no substantial effect , neither on wrist morphology , nor on the vertebral formula at the lumbo-sacral level .', 'In addition , transcriptome analyses reveal that the absence of Hotair does not impact upon Hoxd genes expression in trans , in any of the embryonic tissues analyzed .', 'In contrast , we observe subtle yet significant changes in the expression of the neighboring Hoxc11 and Hoxc12 genes in the mutant mice , indicating an in-cis impact of modifying the genomic locus .', 'Taken together , our results strongly suggest that the Hotair lncRNA has little effect–if any- on mouse embryonic development .'], ['We extended the analysis of a mouse strain that includes a deletion of the two major Hotair exons ( Fig 1A ) [20] .', 'Even though we concluded that this mutation is primarily an allele of the HoxC cluster ( see below ) and hence that it should be referred to as HoxCDel ( Hotair ) , we shall refer to it as Del ( Hotair ) throughout this study for sake of simplicity .', 'We first confirmed the expression pattern of Hotair in vivo by whole mount in situ hybridization ( WISH ) using wild type mice of two distinct genetic backgrounds ( CD1 and CBA/C57/B6 ) as well as Del ( Hotair ) -/- mouse embryos at embryonic day 12 . 5 ( E12 . 5 ) ( Fig 1B ) .', 'Staining of Del ( Hotair ) -/- embryos confirmed the specificity of the Hotair probe as no signal was detected in these embryos ( Fig 1B ) .', 'In contrast , wild type embryos of both genetic backgrounds showed the presence of Hotair transcripts in the genital tubercle , the proximal part of the hindlimbs and in the caudal part of the embryo ( Fig 1B ) , confirming previously published data [19] and consistent with the LacZ staining reported for the Hotair knocked-in allele [21] .', 'In both cases , staining was observed just posterior to the lumbar region and was not scored in developing forelimbs [21] .', 'We quantified the expression levels of Hotair with high coverage RNA-sequencing ( RNA-seq ) ( Materials and Methods , S1 Table ) .', 'Based on the spatial expression pattern of Hotair as determined with WISH and on the skeletal phenotypes reported in mice by Li et al . [20] , we micro-dissected both Del ( Hotair ) -/- and wild type E12 . 5 embryos into six distinct parts for comparative RNA-seq analyses ( Fig 1C ) .', 'We thus separately collected the forelimbs ( FL ) , the hindlimbs ( HL ) , the genital tubercle ( GT ) , a piece of trunk corresponding to the lumbo-sacral region ( T1 ) ; a piece of trunk corresponding to the sacro-caudal region ( T2 ) and finally , a piece of trunk corresponding to the developing caudal region ( T3 , Fig 1C ) .', 'As expected from the WISH experiments , Hotair transcripts were scored in the hindlimbs , the genital tubercle and the trunk samples T2 and T3 .', 'The highest steady-state levels of Hotair RNAs were detected in the GT and the T3 embryonic tissues ( Fig 1D , S1 Dataset ) .', 'As a control , Hotair transcripts were not detected in any tissues derived from homozygous Del ( Hotair ) -/- mutant embryos ( Fig 1D ) .', 'To better compare this dataset with published results , we analyzed in parallel the RNA-seq data obtained from primary tail tip fibroblast ( TTF ) , derived from both wild type and Del ( Hotair ) -/- mice [20] .', 'This analysis revealed that the expression level of Hotair in control TTF was very low when compared to its expression levels in the GT or the posterior T3 trunk sample ( Fig 1E , S2 Dataset ) .', 'Hotair was reported to be important for both the proper establishment of the mouse vertebral column and for the formation of the forelimb mesopodial articulation: the wrist [20 , 21] .', 'To confirm this phenotypic effect , we inter-crossed Del ( Hotair ) +/-heterozygous mice and examined the skeletons of F1 animals at postnatal day 22 ( P22 ) .', 'We investigated in particular the three reported sites of observed alterations in mutant Del ( Hotair ) -/- mice [20 , 21] .', 'We first searched for potential differences in vertebral formulae , as it was reported that 58% of Del ( Hotair ) -/- mice had five lumbar vertebrae , while 100% of wild type CBA/C57/BL6 mice had six lumbar vertebrae [20] .', 'All our mutant alleles at Hox loci ( see e . g . [23] ) are systematically backcrossed onto mixed ( B6xCBA ) F1 animals to maintain heterogeneous but similar backgrounds when comparing experimental crosses .', 'After bringing the Hotair mutant mice [20] onto this genetic background for some generations , we found that 80% of Del ( Hotair ) -/- mice displayed five lumbar vertebrae , similar to wild type littermates ( Chi-square test , p-value 0 . 97 , Fig 2A , Table 1 ) .', 'In both wild type and homozygous mutant animals , the L6 formula was sporadically scored , as well as the mixed L6/S1 vertebral type , often observed in our stocks .', 'Despite a limited number of specimens observed , but together with the fact that we were unable to detect specific transcripts in the embryonic trunk at this vertebral level by two independent methods , we conclude that this lncRNA is very unlikely to have a function in the organization of this very flexible morphological boundary ( see discussion ) .', 'We next analyzed the morphology of caudal vertebrae in the post-sacral region .', 'Previous analyses had concluded that mice with Hotair deletions had longer lateral processes on the fourth vertebra when compared to wild type animals , with full penetrance .', 'In our case , we observed that three out of ten Del ( Hotair ) -/- mutant mice had longer processes on the fifth caudal vertebra , compared to wild type ( Fig 2B ) .', 'This may indeed correspond to a very subtle morphological alteration in this region of the caudal spine , although the penetrance of this light phenotype is not 100% .', 'Unlike the lumbo-sacral and wrist alterations [20] , this particular tail vertebral morphology was also scored by Lai and colleagues when analyzing another mutant allele of Hotair [21] .', 'Finally , and even though we were unable to detect any Hotair transcripts in the forelimbs of E12 . 5 mice embryos , unlike for hindlimbs ( Fig 1B ) , we carefully examined both forelimb and hindlimb skeletons of wild type and Del ( Hotair ) -/- mutant mice .', 'We did not detect any alteration in limb morphology ( Fig 2C and Table 1 ) , in particular in the anatomy of the wrist , where malformations due to the loss of Hotair had been previously reported ( Fig 2C ) .', 'The same conclusion was reached concerning the hindlimbs , even though Hotair transcripts could clearly be scored in the proximal part .', 'Altogether , we could not reproduce the reported phenotypic effects of Hotair deletion at two sites , the wrist and the lumbo-sacral region , where we were also unable to detect any Hotair transcripts .', 'Regarding tail vertebrae , a slight effect could indeed be observed , poorly penetrant and likely dependent on the genetic background ( see below ) .', 'To more globally evaluate the effect of Hotair deletion upon developmental gene regulation , we performed a principal component analysis ( PCA ) using the expression levels of all autosomal protein-coding genes detected in our RNA-seq experiments ( Materials and Methods ) .', 'We observed a good separation of the data according to tissue type , although the T1 and T2 samples clustered together ( Fig 3 ) .', 'Principal component 1 ( PC1 ) , which explained 61 . 6% of the total gene expression variance , separated the trunk segments ( T1 , T2 and T3 ) from the other embryonic tissues .', 'Likewise , the differences between GT , HL and FL were resolved along PC2 , which accounted for 12 . 5% of the total variance ( Fig 3 ) .', 'Part of the variance was also explained by the genotypes and we observed that wild type and Del ( Hotair ) -/- samples were separated along PC2 ( Fig 3 ) .', 'Of note , the same separation between wild type and Del ( Hotair ) -/- on PC2 was observed in all tissues , even in T1 and FL where Hotair is not expressed ( Fig 3 ) .', 'In agreement with the results from the clustering between samples , we observed high expression level correlations among biological replicates for all tissues ( S1 Fig ) .', 'Furthermore , gene expression clustering based on pairwise Euclidean distances between samples ( see Materials and Methods ) showed a clear separation between four different groups: the limbs ( FL and HL ) , the GT , the T3 trunk segment and the remaining T1 and T2 trunk segments ( S2 Fig ) .', 'Using this method , we observed a separation between genotypes only when the T3 sample was considered .', 'Altogether , these results point to the reproducibility of replicates and illustrate the good separation between tissues , with the exception of the T1 and T2 trunk samples .', 'The high similarity in global gene expression between T1 and T2 likely reflects the spatial proximity of these two tissues , even though we cannot exclude some variation in the positioning of the precise T1/T2 boundary during dissection , which is a challenging task in such young embryos .', 'Since Hotair was proposed to act as a repressor of gene expression in cultured fibroblasts [13 , 20] , we conducted tissue-specific differential gene expression analyses between wild type and Del ( Hotair ) -/- samples to assess such a potential function under physiological conditions .', 'We only considered as significant an absolute expression fold change greater than 1 . 5 and we set the false discovery rate ( FDR ) threshold at 5% .', 'By using these parameters , we observed between 64 and 588 protein-coding genes differentially expressed in the various tissues analyzed ( S3 and S4 Datasets ) .', 'We first compared all tissues that express Hotair in the wild type condition , i . e . the T2 , T3 , GT and HL samples , reasoning that potential differentially expressed Hotair targets should be identified in these contexts .', 'However , we were not able to identify any common genes with altered expression between wild type and Del ( Hotair ) -/- samples ( S3 Fig ) , suggesting that the Hotair deletion does not affect the same set of genes in all tissues analyzed .', 'We thus divided the differential expression analysis based on the global expression clustering results .', 'First , we compared the trunk samples T1 ( lacking Hotair expression ) , T2 and T3 .', 'We identified 62 down-regulated genes and 13 up-regulated genes between wild type and Del ( Hotair ) -/- samples , which are shared in all trunk sections ( Fig 4A and 4B ) .', 'Of note , we observed a common trend in gene expression differences in all trunk samples , even though only some of them passed the established thresholds ( Fig 4A ) .', 'Gene ontology ( GO ) analysis for either common down-regulated or common up-regulated genes showed no enrichment of functional terms and the majority of differentially expressed genes were down-regulated , which was unexpected given the previously proposed role of Hotair as a repressor [20] ( Fig 4A and 4B ) .', 'GO analysis of down-regulated genes in distinct Del ( Hotair ) -/- trunk tissues revealed significant enrichment ( FDR<10% ) in functional terms related to organ development and multicellular organismal process for most tissues ( S5 Dataset ) .', 'Up-regulated genes in T3 were enriched for functional terms related to metabolic and biosynthetic processes and a weak enrichment for neuron differentiation genes was observed for T2 ( S5 Dataset ) .', 'Differential expression analysis for FL , HL and GT showed no common genes with altered expression ( Fig 4C and 4D ) .', 'GO analyses for differentially expressed genes in these tissues showed enrichment for functional terms related to development ( S5 Dataset ) .', 'We next asked whether Polycomb target genes were preferentially up or down-regulated upon Hotair deletion .', 'We defined putative target genes using H3K27me3 ChIP-seq data from wild type tail tip fibroblasts [20] , selecting genes with a minimum 5-fold enrichment between H3K27me3 ChIP and input DNA in the gene promoter region ( Materials and Methods ) and thus obtained 861 putative target genes ( S6 Dataset ) .', 'We analyzed their pattern of differential expression in the T3 trunk sample , which includes the fetal tail and thus likely has the cell type composition in vivo most related to tail fibroblasts .', 'Out of the 485 putative Pc targets that were expressed in the T3 segment ( RPKM >1 in at least one wild type or Del ( Hotair ) -/- sample ) , 60 genes were significantly differentially expressed ( absolute fold change > 1 . 5 and FDR <10% ) , including 50 down-regulated and 10 up-regulated in the Del ( Hotair ) -/- samples ( S4 Fig ) .', 'This indicates that only 17% of all differentially expressed Pc targets were up-regulated in Del ( Hotair ) -/- samples , which is slightly lower than the proportion of up-regulated genes among non-targets ( 25% up-regulated genes out of 564 differentially expressed non-target genes , Chi-square test p-value 0 . 18 ) .', 'Thus , we could not detect any enrichment for up-regulation of putative Pc target genes when compared to all other expressed protein-coding genes .', 'Therefore , under these physiological conditions , we could not find evidence supporting a role for Hotair in setting up , maintaining or re-enforcing the repression of this set of Polycomb target genes .', 'While physiologically relevant , our analysis is however difficult to directly compare with the situation in tail fibroblasts , as Polycomb occupancy naturally depends on both the tissue-type and the developmental stage .', 'Interestingly , a subset of imprinted genes including H19 and Meg3 was shown to be up-regulated upon deletion of Hotair in TTF [20] .', 'We thus analyzed the expression status of known imprinted genes transcribed ( RPKM>1 ) in at least one sample ( S7 Dataset ) .', 'To ensure maximum sensitivity , we lowered our FDR threshold to 10% while maintaining an absolute expression fold change greater than 1 . 5 .', 'With these parameters , we observed a total of 21 imprinted genes differentially expressed in our samples ( S5 Fig , S7 Dataset ) .', 'We found that 71% of differentially expressed imprinted genes were down-regulated , while only 29% were up-regulated .', 'Notably , H19 and Meg3 were down-regulated in our samples , in contrast to what was observed in TTF .', 'In conclusion , these global transcriptome analyses comparing Hotair deletion mutant and wild type micro-dissected tissues revealed changes in gene expression upon deletion of the Hotair locus .', 'Noteworthy , we observed numerous expression changes not only in tissues that normally express Hotair at detectable levels , i . e . the T2 , T3 , GT and HL samples , but also in tissues like the anterior trunk ( T1 ) or the forelimb ( FL ) , where Hotair lncRNAs were not detected .', 'This suggests that such observed differences in gene expression cannot be explained by a mere direct effect of the Hotair RNA .', 'Potential explanations to these unexpected observations are discussed below .', 'The original observation , which led Hotair to become the paradigm of lncRNAs acting in trans , was its capacity to regulate several genes members of the HoxD cluster by interacting with components of the Polycomb repressive complex 2 ( PRC2 ) [13] .', 'In contrast , no effect was initially reported upon Hoxc genes expression levels [13 , 20] , despite the fact that Hotair is encoded from within the HoxC locus in both humans and mice [13 , 19] .', 'We re-assessed this issue by analyzing the expression of all Hox genes across our various tissue samples ( Fig 5 ) .', 'The global expression patterns of all four Hox clusters expectedly corresponded to previously described expression patterns for such embryonic stage and body levels ( e . g . [24 , 25] ) .', 'In order to detect even subtle effects of Hotair upon Hox gene regulation , we lowered our FDR threshold to 10% for differential expression analyses .', 'Under these conditions , we detected significant down-regulation of some anterior Hoxa genes ( Hoxa3 , Hoxa5 and Hoxa6 ) in the three trunk samples ( Fig 5 and S6 Fig ) .', 'Interestingly , these differences were present in all trunk samples , including in T1 where Hotair is not expressed .', 'We also observed a slight up-regulation of Hoxb9 in HL and GT ( Fig 5 and S6 Fig ) .', 'Notably , in some of the tissues analyzed , we detected significant expression changes for both Hoxc11 and Hoxc12 , i . e . the two genes in the HoxC cluster that flank the Hotair locus ( see below ) .', 'However , in contrast with previous reports using tail fibroblasts [13 , 20] , we did not detect any significant change in the steady-state levels of Hoxd genes RNAs , in any of the analyzed tissues ( Figs 5 and 6A ) .', 'To clarify this contradictory observation , we re-analyzed the previously published RNA-seq data from both wild type and Del ( Hotair ) -/- TTF [20] .', 'By implementing our analytical pipeline , we could not detect any significant difference in expression for any of the Hoxd genes ( S7 Fig , S4 Dataset ) .', 'Noteworthy , the expression levels of posterior Hoxd genes in this TTF dataset are either barely detectable or not detected at all , as for the Hoxd12 gene , for example , suggesting that previous conclusions were raised based on particularly low transcript levels .', 'The deletion of Hotair was claimed to alter both the expression levels and the spatial transcript distribution of the Hoxd10 and Hoxd11 genes in the trunk [20] .', 'We performed whole mount in situ hybridization ( WISH ) on both wild type and Del ( Hotair ) -/- littermates to appreciate potential variations in the expression domains of these two genes .', 'By using our well established protocol , we found that Hoxd10 and Hoxd11 transcripts showed wild type distributions in Del ( Hotair ) -/- mutant specimen ( Fig 6B ) .', 'To more precisely evaluate any potential difference in these expression domains between homozygous mutant and control littermates , we carried out double WISH for the Hox gene of interest in combination with a probe specific for the MyoD gene , which allowed for unambiguous somite visualization [26] .', 'In both wild type and Del ( Hotair ) -/- embryos , Hoxd10 was expressed in the future spine up to the level of somite 26 , whereas Hoxd11 was scored from somite level 29 and caudally , as previously reported [27] .', 'Neither Hoxd10 , nor Hoxd11 showed any detectable increase in the intensity of the signal or in their spatial expression pattern , confirming the RNA-seq results ( Fig 6A and 6B ) .', 'Taken together , these observations suggest that Hotair has no effect on the regulation of Hoxd genes , at least in the developmental context and at the stage where the function of Hox genes is critical for morphological development .', 'Unlike for Hoxd genes , our differential expression analyses between Del ( Hotair ) -/- and wild type samples revealed modest but significant changes for both Hoxc11 and Hoxc12 , the two genes neighboring the Hotair locus and thus flanking the deletion breakpoint ( Fig 5 ) .', 'To further verify this new observation , we carefully analyzed the expression levels of both Hoxc11 and Hoxc12 in all tissue samples from where RNA-seq datasets had been obtained .', 'As expected from their collinear transcription [28] , Hoxc12 transcripts were mainly detected in the most posterior T3 trunk sample .', 'In this sample , a significant reduction in the level of Hoxc12 RNAs was scored in Del ( Hotair ) -/- specimen ( Fig 7A ) .', 'On the other hand , Hoxc11 transcripts were detected in the hindlimbs ( HL ) , the genital tubercle ( GT ) and the T2 and T3 trunk samples .', 'In these tissues , we observed an up-regulation of Hoxc11 RNAs upon deletion of the Hotair locus , which was statistically significant for both HL and T3 ( Fig 7B ) .', 'In addition , a strong positive correlation between the expression levels of Hotair and Hoxc11 was detected in wild type samples ( S8 Fig ) .', 'The correlation was weaker between Hotair and Hoxc12 expression ( S8 Fig ) .', 'We asked whether these changes in expression of Hoxc genes in some Del ( Hotair ) -/- samples were accompanied by alterations in their spatial expression patterns .', 'We analyzed the expression of both Hoxc12 and Hoxc11 by WISH in Del ( Hotair ) -/- E12 . 5 embryos and wild type littermates .', 'We did not observe any change for Hoxc12 expression , neither in the transcript domain , nor in the intensity of RNA signal ( Fig 7C ) .', 'In contrast , Del ( Hotair ) -/- embryos showed a clear rostral expansion of the Hoxc11 transcript domain in the trunk , as well as an increased signal intensity in both the hindlimb buds and the trunk , in agreement with our RNA-seq data ( Fig 7D ) .', 'To understand more precisely the reason why the deletion of the Hotair locus impacted the transcription of the flanking Hoxc genes , we analyzed in details the RNA-seq profiles of the region comprising Hoxc12 , Hotair and Hoxc11 .', 'We first asked if all transcript isoforms derived from the Hotair locus were abrogated in the Del ( Hotair ) -/- allele and observed that the deleted region almost perfectly coincides with the annotated boundaries of the locus in the mouse .', 'However , the inspection of the RNA-seq profiles in tissues that transcribe Hotair RNA revealed the existence of larger transcripts , extending over at least 2 . 4 kb upstream of the annotated promoter ( Fig 8A ) .', 'Although we cannot determine the precise location of Hotair transcription start site ( s ) , the presence of continuous transcription upstream of the annotated gene boundaries indicates that at least one , and probably two of the possible Hotair promoters were not deleted .', 'Indeed in Del ( Hotair ) -/- tissues , we detected transcripts initiating upstream of the annotated Hotair promoter , for instance within the Hoxc11 intron , and spanning over the deleted region ( Fig 8A ) .', 'In contrast to its multiple start site ( s ) , Hotair displayed a very sharp transcription termination site .', 'In wild type tissues , transcription of Hotair terminated at the annotated site , with virtually no RNA-seq reads mapped downstream of this position ( Fig 8A ) .', 'However , in the Del ( Hotair ) -/- samples , we observed transcription downstream of the deleted locus terminating within 100bp of the Hoxc12 termination site ( Fig 8A ) .', 'The presence of this extended transcript , which likely derives from one of the native Hotair promoters ( as predicted with a de novo transcript assembly procedure , S9 Fig ) , likely resulted from the deletion of the wild type Hotair termination signals ( Fig 8A ) .', 'To quantify this gain of transcription , we counted RNA-seq reads mapping on the region between the annotated Hotair termination site and the Hoxc12 termination site , on the Hotair strand .', 'We referred to this transcript , which only appears upon deletion of Hotair , as Ghost of Hotair ( Ghostair ) .', 'We observed significant gains of Ghostair transcription in Del ( Hotair ) -/- samples , in all tissues that expressed Hotair in the wild type condition , i . e . the hindlimb buds , the genital bud and the two trunk samples T2 and T3 ( Fig 8B ) .', 'Subsequent analyses of the RNA-seq profiles revealed an additional un-annotated promoter sequence , yet located on the Hox DNA strand .', 'This promoter lies between Hoxc12 and Hoxc11 and overlaps with a CpG island ( Fig 8A ) .', 'In the wild type situation , it generates a relatively short , poorly abundant and un-spliced transcript , ca 1 . 8kb in size .', 'The estimated termination site for this transcript was found within the region deleted in Del ( Hotair ) , close to the termination of Hotair itself on the other strand .', 'Accordingly , we refer to this short transcript as Anti-Hotair ( AHotair ) .', 'In Del ( Hotair ) -/- samples , this CpG island promoter was still active , giving rise to a much longer AHotair transcript ( Fig 8A , long AHotair or LAHotair ) , consistent with the deletion of its termination site .', 'We did not observe any clear boundaries between this extended transcript and Hoxc11 , suggesting that this AHotair RNA could leak onto the Hoxc11 transcription unit .', 'This was confirmed by a de novo transcript assembly procedure ( see Materials and Methods , S9 Fig ) .', 'To quantify this gain of transcription from the Hox strand , we further defined two transcribed regions; The first one largely corresponded to the short Anti-Hotair transcript detected in wild type samples , starting at the CpG island promoter and ending at the boundary of the deleted region ( Fig 8A ) .', 'The second one , long AHotair , corresponded to the longer transcript observed in Del ( Hotair ) -/- samples , starting at the deleted region boundary and ending at the annotated Hoxc11 transcription start site ( Fig 8A ) .', 'In agreement with our observations based on the RNA-seq profiles , we detected significant increases in expression for both AHotair and LAHotair in Del ( Hotair ) -/- mutant tissues ( Fig 8C and 8D ) .', 'Therefore , the deletion of the two annotated exons of Hotair [20] had a previously ignored important impact in cis by generating two new transcripts , which may potentially interact with the transcription of neighboring Hoxc genes .'], ['In this study , we have re-investigated the phenotypic and molecular effects of deleting the Hotair lncRNA on mouse development in vivo , as reported in [20] .', 'In this previous study , three phenotypic differences between wild type and Del ( Hotair ) -/- mice were reported , namely wrist malformation , a posterior homeotic transformation from lumbar vertebra L6 to sacral vertebra S1 identity and a mild anterior homeotic transformation of the 4th caudal vertebra .', 'We did not detect any wrist malformation , nor did we see any substantial homeotic phenotype in the lumbar region of mutant animals , thus contradicting two of the three reported phenotypic effects of the Hotair deletion .', 'In Mus musculus , the lumbo-sacral transition shows great variability between L5 and L6 depending on the inbred strain considered and the total number of pre-sacral vertebrae .', 'In fact , this number not only varies between inbred strains but also within the same strain and can even be biased by the sex of the animal [29] .', 'Therefore , this region must be considered with great care before concluding on the presence of a homeotic transformation .', 'We note that another study involving two distinct deletion alleles of Hotair–though of smaller extents- also failed to confirm these latter two phenotypic effects [21] .', 'The lack of effect of Hotair deletion upon wrist morphology is consistent with the absence of any detectable Hotair transcripts in mouse embryonic forelimbs ( Fig 1 ) also reported previously [19] and by [21] using a sensitive lacZ reporter transgene system .', 'Regarding the reported L5 to L6 transition , it is noteworthy that in wild type animals , detectable expression of Hotair in the paraxial mesoderm , i . e . in the mesodermal tissue that will generate the vertebrae , barely reaches the level of the lumbo-sacral transition , a transition labeled by its neighboring Hoxc11 gene [30 , 31] .', 'This makes a more anterior ( at the L5 to L6 transition ) Hotair loss-of-function dependent gain of function phenotype due to Hoxd genes difficult to understand .', 'Our analyses did nevertheless reveal a subtle difference between wild type and Del ( Hotair ) -/- mice in the morphology of the post-sacral caudal vertebrae .', 'Although this observation is in agreement with one of the previously described morphological alterations [20 , 21] , we note that the penetrance of the mutant phenotype
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Despite the crucial importance of Hox genes functions during animal development , the mechanisms that control their transcription in time and space are not yet fully understood .', 'In this context , it was proposed that Hotair , a lncRNA transcribed from within the HoxC cluster regulates Hoxd gene expression in trans , through the targeting of Polycomb and consecutive transcriptional repression .', 'This activity was recently supported by the skeletal phenotype of mice lacking Hotair function .', 'However , other loss of function alleles at this locus did not elicit the same effects .', 'Here , we re-analyze the molecular and phenotypic consequences of deleting the Hotair locus in vivo .', 'In contrast with previous findings , we show that deleting Hotair has no detectable effect on Hoxd genes expression in vivo .', 'In addition , we were unable to observe any significant morphological alteration in mice lacking the Hotair transcript .', 'However , we find a subtle impact of deleting the Hotair locus upon the expression of the neighboring Hoxc11 and Hoxc12 genes in cis .', 'Our results do not support any substantial role for Hotair during mammalian development in vivo .', 'Instead , they argue in favor of a DNA-dependent effect of the Hotair deletion upon the transcriptional landscape in cis .']
['During mammalian embryonic development , Hox genes must be tightly regulated .', 'It was proposed earlier that part of this regulation relies upon Hotair , a long non-coding RNA that recruits repressive protein complexes onto the HoxD gene cluster to keep these genes silent before they become activated .', 'A genetic deletion of Hotair in mice induced homeotic transformations , thus supporting this hypothesis .', 'However , other alleles involving this locus gave controversial results and hence we re-assessed the effect of the full deletion of Hotair in vivo .', 'In our genetic background and using our analytical conditions , we could not confirm the reported morphological alterations , nor could we detect any mis-regulation of Hoxd genes in those fetal tissues where Hotair is detected in control animals .', 'However , the genomic deletion induces the mis-regulation in-cis of the neighboring Hoxc11 and Hoxc12 genes , a side-effect which may underlie a weakly penetrant alteration observed in the shape of some tail vertebrae .']
['medicine and health sciences', 'gene regulation', 'rna extraction', 'long non-coding rnas', 'fibroblasts', 'dna transcription', 'developmental biology', 'connective tissue cells', 'embryos', 'extraction techniques', 'research and analysis methods', 'embryology', 'animal cells', 'gene expression', 'connective tissue', 'biological tissue', 'genetic loci', 'biochemistry', 'rna', 'cell biology', 'nucleic acids', 'anatomy', 'genetics', 'biology and life sciences', 'cellular types', 'non-coding rna']