Organization, conservation and expression of the Evx1/Evx1as locus

The Evx1 locus is located 50 kb downstream of the HoxA gene cluster on chromosome 6 (Fig. 1a). Evx1 and Evx1as are developmentally regulated, displaying peak and concordant expression during gastrulation5. They are both also highly expressed in vivo in the pre-somitic mesoderm (PSM)32. Whole mount in situ hybridization (WISH) of E7.5 and E9.5 embryos demonstrates the Evx1 and Evx1as are co-expressed in the primitive streak during gastrulation (Fig. 1c). At E7.5, both Evx1 and Evx1as are expressed at the posterior-proximal side of the embryo, which is the location of the primitive streak. At E9.5, both transcripts localize to the tail bud, which contains the embryological remnants of the primitive streak. Thus, Evx1 and Evx1as are co-expressed during gastrulation.

Like many lncRNA-coding gene pairs30, Evx1 and Evx1as are expressed from opposite DNA strands in a sense-antisense configuration (Fig. 1a). Interestingly, there are two other lncRNAs within the distal end of the HoxA cluster, Hox11as and HOTTIP25, which are expressed in an antisense direction with respect to Hox11 and Hox13 coding genes, respectively (Fig. 1a). Evx1as is expressed as at least two different isoforms according to EST and depostied cDNA data; the second exon overlaps with the start of Evx1 by ~70 bp and the first exon resides in the first intron of Evx1 (Fig. 1a). We confirmed only 1% of the Evx1as transcript contains the first exon in D4 EBs (Supplementary Figure 1A). EVX1 is highly conserved throughout the protein coding sequence, whereas Evx1as displays notable conservation at the promoter (Fig. 1a), like many lncRNAs33. The human EVX1-EVX1AS locus is syntenic with the murine locus. EVX1AS has two conserved promoters (intronic or bidirectional), but is also expressed from one additional promoter, located 3′ of EVX1 (Fig. 1b).

EVX1 regulates anterior-posterior patterning during gastrulation

In order to study the function of EVX1 and Evx1as, we used EB differentiation as a model for early in vivo mouse embryonic development. Day 4 EBs grown in serum or serum free media (SFM) with recombinant BMP4 display a very similar transcriptional program to embryos undergoing gastrulation7. Evx1 and Evx1as were most highly expressed when differentiated under BMP4 and WNT3A conditions (Fig. 1d), which suggests that they might be either downstream targets of the BMP4 and WNT3A signalling pathways or that BMP4 and WNT3A enhance the generation of posterior mesoderm cell types, wherein the Evx1 locus is expressed. Evx1 and Evx1as were expressed at a much lower level in EBs grown in SFM with Activin A, which is an anterior/dorsal mesoderm inducing factor34. Our results are consistent with expression of human EVX1 in these same growth factors10. Evx1 and Evx1as display a similar expression pattern to Mixl1 (Fig. 1d), a related homeodomain transcription factor which is also expressed in nascent posterior mesoderm12. Expression of pan-mesoderm genes such as Brachyury (T) and Sp5 was similar in BMP4, WNT3A and Activin A, while expression of anterior markers Cer1 and Sox17 was significantly higher in WNT3A and Activin A (Fig. 1d).

We generated EVX1 ‘knockout’ cell lines using CRISPR-Cas9 gene micro-editing of the homeodomain coding region in exon 2, thus leaving Evx1as unperturbed (Fig. 3a). Two independent compound heterozygous Evx1 gene edited clones were selected based on our previously described NGS screening strategy35. Both clones have small frameshift mutations in both alleles (Fig. 2a), which are predicted to result in a premature stop codons within 20 bps of the deletion site. Thus, no functional DNA-binding domain of EVX1 could be generated by either of these clones. We refer to these CRISPR-edited lines as ‘EVX1-Δfs’ from here on. EVX1-Δfs lines and W9.5 controls were differentiated in serum to D4 under suspension culture conditions to identify a potential regulatory role for EVX1 during gastrulation. EVX1-Δfs D4 EBs appeared phenotypically normal, and indistinguishable from the wildtype controls (Supplementary Figure 1B). However, qPCR analysis revealed that both EVX1-Δfs clones display differences in expression of key anterior and posterior patterning markers, including Mixl1 and Cer1 (Supplementary Figure 1C). There were no significant differences between the two clones, therefore only one was used for subsequent analyses (Clone 14) (Supplementary Figure 1C).

Figure 2: EVX1 is required to regulate anterior-posterior patterning during gastrulation. (a) Visualization of CRISPR-induced mutations in two independent mESC clones (clone 6 and 14) using Integrated Genome Viewer (IGV). The peptide sequence of EVX1 is shown. Red and blue indicate different read strands. (b) Scatterplot of average counts of DEGs from mRNAseq comparing 3 replicates of WT and EVX1-Δfs Day 4 EBs. Known markers and regulators of tissue specification are shown in red. RPM = Reads per million reads.(c) Heatmap of counts for differentially expressed genes corresponding to particular Gene Ontology (GO) terms in (d). Each category is hierarchically clustered. Each value is normalized to its mean expression across all samples. Red indicates higher than average expression, blue indicates lower than average expression. (d) GO analysis (DAVID) of DEGs shows the biological processes disrupted in the EVX1-Δfs D4 EBs. Red line indicates an adjusted p-value of 0.05. (e,f) Expression profiling of EVX1-Δfs in SFM supplemented with (e) Activin A (10 ng/ul) or (f) WNT3A (20 ng/ul). Expression of each gene in each sample was first normalized to Hprt then normalized to WT expression. 4 biological replicates were performed. *Indicates a p-value < 0.05, **indicates a p-value < 0.01, ***indicates a p-value < 0.001 when compared to WT. All error bars indicate SEM. Full size image

Figure 3: Evx1as does not have a function independent of EVX1. (a) Schematic of the strategy for dissecting the Evx1/Evx1as locus modified from USCS Genome Browser. The three different deletions performed are shown on the diagram. Wiggle tracks of WT, EVX1-Δfs and Del#3 mRNAseq from D4 EBs are shown. Corresponding qPCR primers pairs are shown in the same colour. The location of the Evx1 deletion is indicated by the arrow. Del = deletion, T1 = transcript 1, T2 = transcript 2. (b) Expression profiling of EVX1-Δfs and Del#3. Expression of each gene in each sample was first normalized to Hprt then normalized to WT expression. 4 biological replicates were performed. Error bars show SEM. *Indicates a p-value < 0.05, **indicates a p-value < 0.01, when compared to WT. No significant differences were found when comparing EVX1-Δfs and Del#3. N.D = not detected. <10 = less than 10% of WT. (c) Scatter plot of average mRNAseq counts from three replicates comparing EVX1-Δfs and Del #3 D4 EBs. Evx1 and Evx1as are shown in red. A value of 1 was added to all RPM values to improve visualization. R2 was obtained from Pearson Correlation. RPM = Reads per million reads. Full size image

To determine whether EVX1 has a global role in anterior-posterior patterning, mRNA-seq was performed on three biological replicates of W9.5 and EVX1-Δfs EBs differentiated for 4 days in serum without LIF (see Methods). Using DESeq2, we identified 802 differentially expressed genes (DEGs). In the absence of functional EVX1, 282 genes were upregulated and 520 genes were downregulated (Fig. 2b). The upregulated genes included many which are preferentially expressed in anterior visceral endoderm and/or anterior mesendoderm (e.g. Sox17, Cer1, and Foxa2). Many of the down regulated genes are normally expressed in posterior mesoderm (e.g. Mixl1, Mesp1, Wnt5a and Fgf3) (Fig. 2b). This suggests there is an imbalance in cellular composition of EVX1-Δfs EBs with loss of posterior cell types and relative expansion of anterior cell types (both mesoderm and endoderm).

Using Gene Ontology (GO) we found the most highly significantly enriched cell biological process terms among the DEGs were: blood vessel development (35) and morphogenesis (28), pattern specification (38), regulation of the cell cycle (29), gastrulation (16) or formation of primary germ layer (12), cell differentiation (25), cell motion (37) and WNT signalling (20) (Fig. 2d). We undertook hierarchical clustering of the genes within each of the GO Ontology categories (Fig. 2c). Each category contains genes down-regulated (blue) or up-regulated (red) in EVX1-Δfs cells. Within the blood vessel development, patterning and gastrulation categories, there is down-regulation of many ligands, receptors, signalling molecules and transcription factors which are implicated in posterior patterning of mesoderm. These include Bmp4, Wnt2, Fgf10, Kdr (Flk1), Cxcr4, Cited1, Cited2, Cdx2, Hand1, Mixl1, Hes1, Tbx3, Tbx6, Mesp1, Eomes, Snai1 and others. There is also upregulation of a smaller set of key patterning genes which are normally expressed in the AVE, anterior mesendoderm or epiblast. These include Sox17, Nanog, Atm, Zic3, and Foxa2. We also found many genes in the WNT signalling pathway are downregulated in EVX1-Δfs EBs, including Wnt3, Wnt2, Wnt5a, Wnt5b, Lef1, Pitx2, Dvl2, Lrp5 and others. On the other hand, WNT pathway antagonists such as Cer1, Cfc1/cripto and Sfrp1 are upregulated. Interestingly, Evx1 is downregulated in WNT3A KO embryos36, demonstrating that there is a mutual dependence (direct or indirect) between WNT signalling and EVX1. There were no significant changes in expression of any members of the HoxA cluster under these differentiation conditions. Full lists of differentially expressed genes are provided in Supplementary Table 2.

Previously EVX1 has been shown to directly repress GSC in human ESCs under Activin A growth conditions10. Gsc was not upregulated in either EVX1-Δfs clone by qPCR or RNAseq under serum differentiation conditions (Supplemental Table 2, Supplementary Figure 1C). To ensure that the lack of Gsc upregulation was not due to differences in the differentiation conditions used, we differentiated EVX1-Δfs and W9.5 EBs in SFM with Activin A. We did not find significant upregulation of Gsc (Fig. 2e). Importantly, the A-P gene expression defect in EVX1-Δfs persisted in Activin A. Defective expression of Mixl1 and T remained, and additional significant reduction in expression of the pan mesoderm marker Sp5 was also present in EVX1-Δfs EBs (Fig. 2e). The upregulation of AVE and endoderm markers Cer1 and Sox17 was less dramatic in Activin A, presumably because selective expansion of AVE and anterior tissues dilutes the differences in anterior gene expression between wildtype and EVX1-Δfs EBs (Fig. 2f).

From this RNAseq data, it was unclear whether EVX1 exerts its A-P patterning function by directly regulating BMP4 and/or WNT signalling pathways or whether changes in Bmp4/Wnt expression are an indirect result of changes in cellular composition within the EVX1-Δfs EBs. BMP4 and WNT direct EB differentiation towards posterior cell types37, so the aberrant transcriptome in EVX1-Δfs EBs may be primarily due to downregulation of these factors. To address this possibility, we attempted to rescue EVX1-Δfs EBs by culture in SFM with exogenous recombinant WNT3A (20 ng/ml) or BMP4 (10 ng/ml). Under WNT3A conditions, we observed no rescue of Mixl1 gene expression; i.e. it remained significantly down regulated (Fig. 2f). Likewise there was no reversal of the up-regulation of Cer1, suggesting the A-P patterning defect was not rescued by WNT3A (Fig. 2f). Similar results were found for EBs grown in BMP4 (Supplementary Figure 1D). Thus, downregulation of Bmp4 and Wnt genes (Wnt3, Wnt5a and Wnt5b) is likely to be an indirect result of changes in cellular composition of EVX1-Δfs EBs and not due to direct regulation of these factors by EVX1. This data also shows EVX1 is required for appropriate transcriptional responses and differentiation downstream of BMP4 and WNT signalling pathways. This function appears to be conserved in humans, as β-catenin, the major effector of WNT signalling, has been shown to bind to regulatory regions in the EVX1 promoter in differentiated hESCs11. Together, these results confirm EVX1 plays a key role in A-P patterning of nascent mesoderm and endoderm and is essential for regulating the posteriorizing effects of WNTs and BMP4.

CRISPR/Cas9 mediated removal of the Evx1/Evx1as locus results in an identical D-V pattering defect as EVX1-Δfs

Bidirectional/antisense transcription from highly expressed, developmentally regulated genes is a common phenomenon in the mammalian genome30. However the functional significance and possible mechanisms of action of such antisense lncRNAs remain topics of debate38,39. The Evx1 locus contains a stable, highly expressed antisense lncRNA associated with Evx1, so it is an ideal locus to study. Visual inspection of RNAseq data from D4 EBs suggests that the majority of the Evx1as transcript is expressed as Evx1as T2 (Fig. 3a). We further validated this by qPCR using primers to distinguish between the different isoforms of Evx1as. Only 1% of the total transcript derived from the overlapping intronic promoter (Supplementary Figure 1A).

We initially used RNAi, delivered via shRNA containing lentiviral vectors40 to attempt stable knockdown of Evx1as without disturbing the genomic locus (Supplementary Figure 2A) (see Methods). Such an approach has been successful for some lncRNAs but challenging for others41,42. In some cases RNAi has obtained different and more severe phenotypes than constitutive deletions, which may be due to off target or toxicity effects in some cases or due to compensatory events in others43,44. We generated stable clones targeting Evx1as using three independent shRNAs (Supplementary Figure 2A). Unfortunately, none of the clones from any of the three siRNAs displayed knockdown of Evx1as to <50% of WT levels, so we could not make concrete conclusions about the potential function of Evx1as from these experiments. There were no significant changes in expression of key mesoderm genes such as Brachyury, Mixl1, or Evx1 itself (data not shown).

Genetic deletion or mutation remains the gold standard to demonstrate the requirement of a gene’s function43. We therefore performed 3 different CRISPR-Cas9 mediated manipulations of the Evx1/Evx1as locus (Fig. 3a). The first manipulation ‘Del#1’ removed the entire first intronic promoter of Evx1as, which is specific to isoform Evx1as T1 (1% of total transcript) and conserved (Fig. 1a). Removal of this promoter, which generates a small proportion of Evx1as RNA, did not affect expression of Evx1as or Evx1 (data not shown). ‘Del#2’ removed the shared promoter region between Evx1 and Evx1as. This deletion removed 220 bps centred around the beginning of Evx1 and Evx1as transcription, based on ESTs and RNAseq data. This deletion resulted in a dramatic loss of Evx1 reducing the transcript to ~16% of WT levels (Supplementary Figure 2B). However this deletion only decreased Evx1as to ~46% of WT levels. This result may be explained by a more malleable transcriptional start site and/or weaker constraints on transcriptional regulation at the lncRNA promoter. As expected, ‘Del#2’ displayed reduction of Mixl1 and significant upregulation of Cer1, supporting the results from the EVX1-Δfs RNAseq data (Supplemental Figure 2B).

Since Evx1as was still expressed at 46% of WT in ‘Del#2’, we could not draw any definitive conclusions about its potential function. Therefore, we generated a large ~2.6 kb deletion (‘Del#3’) that removed the promoters from both versions of the transcript (to prevent the possibility of isoform switching between the two alternative promoters), as well as the second/third exon (Fig. 3a). Successful deletion was validated by PCR and later by RNAseq (Fig. 3a, Supplementary Figure 2C). This deletion resulted in almost complete ablation of Evx1 by qRT-PCR (Fig. 3b). Since the Evx1as qPCR primers sit inside the deletion site (Fig. 3a), primers were designed for the last exon of the transcript to detect any residual expression. Approximately 15% of WT levels of expression of the last exon of Evx1as remained in the ‘Del#3’ D4 EBs (Supplementary Figure 2D).

To determine whether Evx1as has any function independent of EVX1, we performed a direct comparison between the transcriptome of EVX1-Δfs and the Evx1as/Evx1 double KO (‘Del#3’). We reasoned that if Evx1as has an independent function from EVX1, then differences should be observed between the two transcriptomes, regardless of whether it functions in transcriptional or post-transcriptional regulation. Transcriptional differences, if present, could be either due to direct transcriptional regulation by Evx1as or an indirect consequence of Evx1as regulating post-transcriptional pathways.

No significant differences were observed by qPCR between the two lines for a number of gastrulation and A-P patterning genes (Fig. 3b). Both the EVX1-Δfs and Evx1/Evx1as Δ2.6 kb (‘Del#3’) EBs displayed significantly different expression of Mixl1, T and Cer1 relative to WT EBs (Fig. 3b). To determine whether there was any trans function for Evx1as RNA independent of the function of EVX1, we searched for differences genome-wide by mRNAseq. Remarkably, differential gene expression analysis identified only 3 DEGs, two of which were Evx1 and Evx1as, themselves. In the Evx1/Evx1as double KO (‘Del#3’), Evx1 and Evx1as are dramatically reduced to 1% and 4% of EVX1-Δfs (in which Evx1 and Evx1as are expressed the same as WT under serum conditions). The other DEG (Grb10) has no reported role during gastrulation and is unlikely to be of any biological significance. The lack of differences is reflected in the very high correlation observed between the two samples (R2 = 0.9925, Pearson) (Fig. 3c). We therefore conclude that Evx1as does not have function independent of EVX1, and therefore is unlikely to function in trans. This does not rule out a potential function for Evx1as in the regulation of Evx1 in cis (see Discussion).