Stella modulates transcriptional and endogenous retrovirus programs during maternal-to-zygotic transition
Maternally inherited factors in the zygote play a critical role during early development ( Ancelin et al . , 2016; Li et al . , 2010; Wasson et al . , 2016 ) . The maternal-to-zygotic transition ( MZT ) marks the time of transfer of developmental control to the embryo following activation of the zygotic genome ( Lee et al . , 2014; Li et al . , 2013 ) . In mice , the major wave of zygotic genome activation ( ZGA ) occurs at the late 2-cell stage ( Golbus et al . , 1973 ) . The earliest zygotically transcribed genes are preferentially enriched in essential processes such as transcription , RNA metabolism and ribosome biogenesis ( Hamatani et al . , 2004; Xue et al . , 2013 ) . Other events that characterise early pre-implantation development include extensive erasure of global DNA methylation and dynamic changes in histone modifications ( Cantone and Fisher , 2013 ) . Previous studies have shown that Stella , encoded by the Dppa3 gene locus , is a maternally inherited factor that is required for normal pre-implantation development ( Bortvin et al . , 2004; Payer et al . , 2003 ) . Stella is a small basic protein with a putative SAP-like domain and splicing-like factor; it also contains nuclear localisation and export signal and has the potential to bind to DNA and RNA in-vitro ( Nakamura et al . , 2007; Payer et al . , 2003 ) ( Figure 1—figure supplement 1A ) . Expression of Stella is high in oocytes , continues through pre-implantation development , and subsequently occurs specifically in primordial germ cells . Stella is also expressed in naïve embryonic stem cells , but is downregulated upon exit from pluripotency ( Hayashi et al . , 2008 ) . Lack of maternal inheritance of Stella results in developmental defects; these manifest from the 2-cell stage onwards , and result in only a small proportion of embryos developing to the blastocyst stage . Notably , a maternally inherited pool of Stella in zygotic Stella knockout embryos , derived from Stella heterozygous females , allows development to progress normally . This indicates Stella has a key role during the earliest developmental events ( Payer et al . , 2003 ) . Stella was suggested to protect maternal pronuclei ( PN ) from TET3 mediated active DNA demethylation ( Nakamura et al . , 2007 , 2012 ) ( Figure 1—figure supplement 1B ) . In the absence of Stella , zygotes display enrichment of 5hmC in both parental PN ( Wossidlo et al . , 2011 ) , and an aberrant accumulation of γH2AX in maternal chromatin ( Nakatani et al . , 2015 ) . In addition , Stella protects DNA methylation levels at selected imprinted genes and transposable elements ( Nakamura et al . , 2007 ) . However , embryos depleted of maternal effect proteins known to regulate imprinted genes only exhibit developmental defects post-implantation ( Denomme and Mann , 2013 ) , implying that the 2-cell phenotype in Stella maternal/zygotic knockout ( Stella M/Z KO ) embryos is not primarily linked with imprint defects . Moreover , TET3 only partially contributes to DNA demethylation and its absence is compatible with embryonic development ( Amouroux et al . , 2016; Peat et al . , 2014; Shen et al . , 2014; Tsukada et al . , 2015 ) . Thus , what impairs pre-implantation embryonic development in the absence of maternal Stella remains unclear . A significant number of transposable elements ( TEs ) are preferentially activated during early development and in a sub-population of mouse embryonic stem cells ( Gifford et al . , 2013; Macfarlan et al . , 2012; Rowe and Trono , 2011 ) . Notably , at the 2-cell ( 2C ) stage in mouse development , there is selective upregulation of endogenous retrovirus ( ERVs ) , which are a subset of TEs characterised by the presence of LTRs that mediate expression and retrotransposition ( Kigami et al . , 2003; Ribet et al . , 2008 ) . Growing evidence suggests that activation of some TEs has important biological functions during early development ( Beraldi et al . , 2006; Kigami et al . , 2003 ) . TEs can regulate tissue-specific gene expression or splicing through their exaptation as gene regulatory elements , and may also play a key role during speciation ( Böhne et al . , 2008; Gifford et al . , 2013; Rebollo et al . , 2012 ) . TEs additionally drive expression of genes directly by acting as alternative promoters that generate chimeric transcripts , which include both TE and protein-coding sequences ( Macfarlan et al . , 2012; Peaston et al . , 2004 ) . Thus the expression of TEs may be functionally important during early embryo development and understanding the regulation of TEs themselves is therefore of great interest . We adopted an unbiased approach to investigate the role of Stella during mouse maternal-to-zygotic transition , using single-cell/embryo RNA-seq analysis of mutant embryos ( Deng et al . , 2014; Xue et al . , 2013; Yan et al . , 2013 ) . We find Stella M/Z KO 2-cell embryos fail to upregulate key zygotic genes involved in regulation of ribosome and RNA processing . Furthermore , the absence of Stella results in widespread misregulation of TEs and of chimeric transcripts that are derived from these TEs . In particular , Stella M/Z KO embryos exhibit a general failure to upregulate MuERV-L transcripts and protein at the 2-cell stage . Our perturbation data is consistent with MuERV-L playing a functionally important role during pre-implantation embryonic development , implying that MuERV-L is amongst the critical factors affected by Stella in early embryos .
We used Stella knockout mice ( Payer et al . , 2003 ) , to collect mutant oocytes and embryos for single-cell/embryo RNA-seq with modifications ( Tang et al . , 2010 ) . Stella knockout ( KO ) oocytes and Stella maternal/zygotic knockout ( Stella M/Z KO , KO ) 1-cell and 2-cell embryos , lacking both maternal and zygotic Stella , were harvested . Strain matched wild type ( WT ) oocytes and embryos served as controls ( Figure 1A; Figure 1—figure supplement 2A ) . Following normalisation , potentially confounding technical factors were controlled for using Removal of Unwanted Variation ( RUV ) ( Risso et al . , 2014 ) ( Figure 1—figure supplement 2B , Figure 1—source data 1 , Supplementary file 1 ) . 10 . 7554/eLife . 22345 . 003Figure 1 . Stella M/Z KO embryos are impaired in maternal-to-zygotic transition . ( A ) A schematic illustration of the single-cell / embryo RNA-seq experimental setup . The total number of oocytes and embryos collected are indicated . The colour scheme represents the transition from maternal ( green ) to zygotic ( red ) transcripts . Maternal Stella is represented in green circle . ( B ) A score plot of the first three principal components for 66 cells using gene counts . The lower panels represent the score plots of the first two principal components using cells belonging to a specific developmental time point . The developmental time points are indicated by colour and the genotypes for Stella are indicated by shape . ( C ) A Venn diagram illustrating the overlap of differentially expressed genes ( DEG ) between WT and KO oocytes , 1-cell and 2-cell embryos ( adjusted p-value<0 . 05 ) ( Figure 1—source data 2 ) . ( D ) The heatmap represents the log2 ratio of the number of upregulated to downregulated genes in KO compared to WT at oocytes , 1-cell and 2-cell stage , belonging to a given cluster of DBTMEE ( Park et al . , 2015 ) . Fisher’s exact test performed and statistically significant p-values are stated . ( E ) A time-series clustering of gene expression dynamics across oocyte to 2-cell embryo ( see Materials and methods ) . ( Left ) Heatmap shows a cluster of WT maternal transcripts ( ED ) , which are differentially expressed in KO samples ( adjusted p-value<0 . 05 , Figure 1—source data 3 ) . ( Right ) Heatmap shows a cluster of WT zygotically activated genes ( ZAG ) ( EU ) , which are differentially expressed in KO samples ( adjusted p-value<0 . 05 , Figure 1—source data 4 ) . Also see Figure 1—figure supplements 1–2 and Figure 1—source data 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 00310 . 7554/eLife . 22345 . 004Figure 1—source data 1 . Gene counts for WT and KO oocyte , 1-cell and 2-cell embryos . Gene counts for 66 samples from single cell / embryo RNA-seq experiments . Removal of Unwanted Variation analysis has been performed to control for potentially confounding technical factors . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 00410 . 7554/eLife . 22345 . 005Figure 1—source data 2 . List of differentially expressed genes between WT and KO samples at oocyte , 1-cell and 2-cell stage , from single cell/embryo RNA-seq analysis . ENSEMBL ID and names of genes identified as significantly differentially expressed with adjusted P-value ( padj ) <0 . 05 . Log2FoldChange=Log2 ( WT/KO ) . Stage indicates the developmental stage that is analysed . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 00510 . 7554/eLife . 22345 . 006Figure 1—source data 3 . List of maternal transcripts , WT class = ED , which are differentially expressed in KO samples . ‘E’ denotes equally expressed , ‘D’ denotes downregulated or ‘U’ denotes upregulated at a later time point . Adjusted p-value ( padj ) shows significant differential expression between WT and KO class . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 00610 . 7554/eLife . 22345 . 007Figure 1—source data 4 . List of zygotically activated genes ( ZAG ) , WT class = EU , which are differentially expressed in KO samples . ‘E’ denotes equally expressed , ‘D’ denotes downregulated or ‘U’ denotes upregulated at a later time point . Adjusted p-value ( padj ) shows significant differential expression between WT and KO class . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 00710 . 7554/eLife . 22345 . 008Figure 1—figure supplement 1 . Stella protein domains . ( A ) A schematic diagram illustrating the putative Stella protein domains: SAP-like domain , splicing-like factor , nuclear export and nuclear localization signals ( Aravind and Koonin , 2000; Payer et al . , 2003 ) . ( B ) A schematic diagram illustrating the N-terminus of Stella ( amino acids 1–75 ) is required for H3K9me2 binding in maternal pronuclei ( PN ) , while the C-terminus of Stella ( amino acids 76–150 ) is required to exclude TET3 from the maternal PN ( Nakamura et al . , 2012 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 00810 . 7554/eLife . 22345 . 009Figure 1—figure supplement 2 . Quality control of single-cell / embryo RNA-seq . ( A ) A scatter plot of the number of reads mapped to exons against the proportion of reads mapped to mitochondrial genes . The dashed red line indicates threshold of a good quality sample if: ( 1 ) cells have a total number of reads mapped to exons greater than 500 , 000; ( 2 ) cells have less than 10% of the proportion of reads mapped to 37 genes on the mitochondria chromosome . All 66 samples are of good quality . ( B ) A score plot of the first two principle components for cells of each developmental stage . RUV = removal of unwanted variation ( see Materials and methods , Figure 1—source data 1 ) . Top panel shows samples clustering pre-RUV , and bottom panel shows samples clustering post-RUV . Each batch represents independent experiments in which samples are collected , and this is indicated by a different colour ( Supplementary file 1 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 009 Principal component ( PC ) analysis revealed that the first PC represents progression from oocyte to the 2-cell stage ( Figure 1B ) . The largest separation is observed between oocyte/1-cell and 2-cell embryos , consistent with the degradation of maternal transcripts and activation of the zygotic genome at MZT . The second and third PCs capture the separation between WT and KO samples , suggesting distinct genome-wide gene expression changes between WT and KO embryos . Furthermore , although KO oocytes and 1-cell embryos exhibit some separation from their WT counterparts , the difference is more clearly pronounced at the 2-cell stage . Notably , Stella M/Z KO 1-cell and 2-cell embryos clustered separately from each other , suggesting that Stella M/Z KO defects at the 2-cell stage are not simply due to delayed developmental progression ( Figure 1B ) . Differential gene expression analysis between WT and KO samples identified 5881 misregulated genes ( adjusted p-value<0 . 05 ) across the developmental stages with 360 genes overlapping across all stages ( Figure 1C , Figure 1—source data 2 ) . Next , we compared the differentially expressed genes ( DEG ) against a public database of early mouse embryonic transcriptomes ( DBTMEE ) ( Park et al . , 2015 ) . This dataset categorises genes by specific expression pattern at any developmental stage . Strikingly , we found significant enrichment of maternal transcripts ( maternal RNA , minor zygotic genome activation and 1-cell transient genes ) and depletion of zygotic transcripts ( major zygotic genome activation , 2-cell transient and mid pre-implantation activation ) in the Stella M/Z KO 2-cell embryos compared to WT ( Figure 1D ) . While Stella KO oocytes are transcriptionally distinct from WT ( Figure 1B ) , Stella KO oocytes do not exhibit overt abnormalities ( Bortvin et al . , 2004; Payer et al . , 2003 ) . Furthermore , the apparent enrichment in maternal transcripts only manifests at the 2-cell stage , suggesting the observed transcriptional differences are not inherited from aberrant transcripts in the Stella KO oocyte but are likely linked to failed downregulation of these maternal transcripts . To independently characterise differences in gene expression dynamics between WT and KO embryos during MZT , genes were clustered based on their pattern of expression in WT oocytes , 1-cell and 2-cell embryos . We identified a cluster of maternal transcripts ( ED ) , defined as highly expressed in WT oocytes and 1-cell and with reduced expression at the 2-cell stage , that are differentially expressed in KO samples ( n = 849 , adjusted p-value<0 . 05 ) ( Figure 1E , Figure 1—source data 3 ) . Of these genes , 37 . 7% did not show the expected reduction in expression in Stella M/Z KO 2-cell embryos . Furthermore , we detected a group of zygotically activated genes ( ZAG ) ( EU ) with low expression in WT oocytes and 1-cell embryos and increased expression in 2-cell embryos , which are differentially expressed in KO samples ( n = 698 , adjusted p-value<0 . 05 ) . Moreover , 39 . 5% of this group of genes exhibited significantly dampened 2-cell activation in the absence of maternal and zygotic Stella ( Figure 1E , Figure 1—source data 4 ) . The combined analysis suggests that Stella M/Z KO embryos exhibit partial impairment in maternal-to-zygotic transition . Gene ontology analysis of genes more highly expressed in KO than WT 2-cell embryos reveals an enrichment of chromatin modifiers , and genes involved in microtubule based processes and response to DNA damage ( Figure 2A , Figure 2—source data 1 ) , consistent with a previous observation of abnormal γH2AX enrichment in maternal PN ( Nakatani et al . , 2015 ) . In addition , downregulated genes in Stella M/Z KO 2-cell embryos are enriched for RNA processing and ribosome biogenesis , with an overall depletion of genes associated with ribosomes in the KEGG pathway and related gene ontology ( Figure 2B , Figure 2—figure supplement 1 ) . The findings from single-cell/embryo RNA-sequencing were validated by independent qRT-PCR analyses . Consistently , genes associated with chromatin modifiers ( Rbbp7 and Kdm1b ) and DNA damage ( Fam175a and Brip1 ) were more highly expressed in KO embryos ( Figure 2C ) , while those associated with ribosome biogenesis ( Nop16 , Emg1 ) , RNA binding ( Larp1b ) and cell cycle regulator ( Cdc25c ) showed lower expression in KO 2-cell embryos ( Figure 2D ) . To explore potential regulatory targets of Stella , a genome-wide co-expression network analysis was performed across the WT dataset . Overall , 910 genes showed correlated expression patterns with Dppa3 ( Pearson correlation >|0 . 9| ) ( Figure 2—source data 2 ) . Of these , 710 genes are positively correlated while 200 genes are negatively correlated with Dppa3 . Genes with a positive expression correlation with Dppa3 are significantly enriched for RNA processing and RNA splicing ( Figure 2E ) , including Snrpd1 and Snrpb2 , both core components of the spliceosome ( Figure 2F ) . 10 . 7554/eLife . 22345 . 010Figure 2 . Stella is associated with the activation of essential zygotic genes . ( A ) Revigo plots ( Supek et al . , 2011 ) of a selection of gene ontology ( GO ) terms enriched in 2-cell DEG ( Figure 2—source data 1 ) . The colour of the circle represents the log10 p-value . Semantic space clusters GO terms of similar functions together . ( B ) A boxplot of normalised counts of genes belong to the Ribosome KEGG pathway between WT and KO across the developmental stages . Two-sided Wilcoxon rank sum test performed between WT and KO and statistically significant p-value is stated . ( C and D ) shows boxplots of single-embryo qRT-PCR validation of RNA-seq in WT ( white ) and KO ( light blue ) 2-cell embryos . All genes are normalised to housekeeping genes , relative to one WT embryo and log2 transformed . ( C ) shows genes significantly upregulated and ( D ) downregulated in KO relative to WT 2-cell embryos ( p<0 . 05 = *; p<0 . 01 = ** , p<0 . 001 = *** and p<0 . 0001 = **** ) . ( E ) A table of the top 10 GO terms enriched in 710 genes whose expression levels are positively correlated with Dppa3 ( Pearson’s correlation coefficient >0 . 9 ) , identified from the genome-wide co-expression network analysis ( Figure 2—source data 2 ) . ( F ) Scatter plots of the expression of Snrpd1 and Snrpb2 against Dppa3 . Gene expression was log2 transformed ( counts per million + 1 ) and Pearson’s correlation analysis was performed . Also see Figure 2—figure supplement 1 and Figure 2—source data 1–2 . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01010 . 7554/eLife . 22345 . 011Figure 2—source data 1 . List of gene ontology terms enriched in differentially expressed genes between WT and KO 2-cell embryos . Gene ontology terms enriched for biological processes were calculated using DAVID software ( version 6 . 7 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01110 . 7554/eLife . 22345 . 012Figure 2—source data 2 . Genome-wide Dppa3 co-expression network analysis . Table shows ensemble ID and official names of genes exhibiting significant expression correlation with Dppa3 ( Pearson Correlation |r| > 0 . 9 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01210 . 7554/eLife . 22345 . 013Figure 2—figure supplement 1 . Ribosome associated genes are depleted in Stella M/Z KO 2-cell embryos . Comparison of normalised counts of genes belonging to the Ribosome associated gene ontology pathway between WT ( white ) and KO ( blue ) across the developmental stages . Two-sided Wilcoxon rank sum test was performed between WT and KO samples and statistically significant p-value stated . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 013 Overall , our analysis suggests that the Stella M/Z KO 2-cell embryos do not effectively transition from maternal to zygotic control , as judged by the aberrant enrichment of maternal transcripts and depletion of zygotic transcripts . Genes regulating essential processes such as RNA processing are depleted in Stella M/Z KO 2-cell embryos and Dppa3 exhibits positive expression correlation with genes enriched in these processes . Notably , most genes however do undergo appropriate MZT changes in mutant embryos , arguing against a general developmental delay . Taken together , this supports a hypothesis whereby Stella plays an important role in upregulating the expression of a subset of genes that are essential during zygotic genome activation . Stella has been shown to affect the DNA methylation of particular TEs in zygotes ( Nakamura et al . , 2007 ) and primordial germ cells ( Nakashima et al . , 2013 ) . Since many TEs are specifically activated during early development , we investigated their expression in WT and KO oocytes and embryos . We remapped the single-cell/embryo RNA sequencing data to obtain read counts for class I TEs ( retrotransposons ) ( Figure 3—source data 1–2 ) . To get an overview of the extent of TE activation , we calculated the fraction of transcripts mapped to TEs as a ratio of total mapped reads . We found a dramatic increase in the ratio of reads mapped to TEs , primarily of the LTR class , at the 2-cell stage compared to oocyte and 1-cell , suggesting widespread TE activation coincident with MZT ( Figure 3A , Figure 3B ) . Further inspection revealed that activation of the LTR class is dominated by upregulation of ERVL and ERVL-MaLR families , while LINE and SINE classes are primarily accounted for by L1 and Alu families , respectively ( Figure 3—figure supplement 1A ) . Moreover , principal component analysis showed developmental progression can be clearly captured by TE expression alone along PC1 ( Figure 3C ) , similar to recent studies identifying stage-specific transcription initiation of ERV expression in early human embryos ( Göke et al . , 2015; Grow et al . , 2015 ) . In addition , WT and KO oocytes and embryos can be partially separated in individual PCAs along each of the developmental stages , signifying differences in expression of TEs between WT and KO samples . 10 . 7554/eLife . 22345 . 014Figure 3 . TEs are mis-expressed in the absence of Stella . ( A ) Top panel shows boxplots of the ratio of reads mapped to TEs to total reads mapped in WT and KO oocytes , 1-cell and 2-cell embryos . The number of reads mapped to TEs are based on uniquely and multi-mapped counts ( Figure 3—source data 2 ) . ( B ) A heatmap of the relative expression of LTR , LINE and SINE in oocyte ( green ) , 1-cell ( yellow ) and 2-cell embryos ( red ) . Blue indicates higher expression and white indicates lower expression . Samples are clustered by row . ( C ) A score plot of the first three principal components for 66 cells using uniquely mapped TE counts ( Figure 3—source data 1 ) . The lower panels represent the score plots of the first two principal components using cells belonging to a specific developmental time point . The developmental time points are indicated by colour and the genotypes of Stella are indicated by shape . ( D ) A bar chart of the odds ratio of TEs up and downregulated in KO 2-cell embryos compared to WT , intersected with ‘maternal TEs’ and ‘zygotic TEs’ . For maternal TEs enriched in TEs upregulated in KO 2-cell , ***p=5 . 35 × 10−6; for zygotic TEs enriched in TEs downregulated in KO 2-cell , ***p=8 . 13 × 10−7 . ( E ) Bar plots showing the relative expression of TE families ( LTR-ERVL , LINE1-L1 and SINE-Alu ) in WT ( white ) and KO ( light blue ) oocytes , 1-cell and 2-cell embryos , data analysed from single-cell / embryo RNA sequencing . Two-sided Wilcoxon rank sum test performed between WT and KO samples and statistically significant p-values stated . ( F ) Top is an illustration of the structure of the full-length MuERV-L element flanked by 5’ and 3’ LTRs . Bottom is boxplots of the relative expression ( counts per million ) of MuERV-L Int and MT2_Mm transcript in WT ( white ) and KO ( light-blue ) oocytes , 1-cell and 2-cell embryos ( p<0 . 05 corresponds to * and p<0 . 01 corresponds to ** ) . ( G ) Immunofluorescence staining against MuERV-L Gag antibody in 2-cell embryos . ( Left ) The top panel shows bright-field , middle panel is MuERV-L Gag ( green ) and bottom panel are merged images of MuERV-L and DAPI , which counterstains DNA . Representative projections of z-stacks are shown for WT and KO embryos . ( Right ) A boxplot of the z-stack quantifications of the relative fluorescence intensity of MuERV-L Gag protein between WT and KO 2-cell embryos . Also see Figure 3—figure supplements 1–3 and Figure 3—source data 1–2 . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01410 . 7554/eLife . 22345 . 015Figure 3—source data 1 . Uniquely mapped transposable element ( TE ) counts . TE counts for 66 samples from single cell / embryo RNA-seq experiments . Uniquely mapped reads only . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01510 . 7554/eLife . 22345 . 016Figure 3—source data 2 . Uniquely and multi-mapped transposable element ( TE ) counts . TE counts for 66 samples from single cell/embryo RNA-seq experiments . Uniquely and multi-mapped reads . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01610 . 7554/eLife . 22345 . 017Figure 3—figure supplement 1 . TEs are mis-expressed in the absence of Stella . ( A ) Heatmaps of the relative expression of individual families of LTR , LINE and SINE elements in oocyte ( green ) , 1-cell ( yellow ) and 2-cell ( red ) embryos . Samples are clustered by row . Blue indicated relatively high expression while white indicates relatively low expression of TEs . ( B ) Bar charts of relative expression of LTR elements , LINE and SINE in WT ( white ) and KO ( light blue ) oocytes , 1-cell and 2-cell embryos . Two-sided Wilcoxon rank sum test performed between WT and KO samples and statistically significant p-values are stated . ( C ) Validation of MuERV-L Gag antibody . ( Left ) IF staining of an ESC reporter line for MuERV-L – 2C::tdTomato ( Macfarlan et al . , 2012 ) , illustrating the MuERV-L Gag ( GFP ) staining overlaps well with tdTomato+ ESC . ( Right ) IF staining showing robust detection of MuERV-L Gag ( GFP ) in 2-cell embryos , while it is virtually undetectable in metaphase II ( MII ) oocytes . DNA is counterstained with DAPI . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01710 . 7554/eLife . 22345 . 018Figure 3—figure supplement 2 . Dppa3 does not affect MuERV-L expression in mESCs . ( A ) Left shows histogram plots of tdTomato expression in 2C::tdTomato mESCs 48 hr and 96 hr after addition of DOX to induce Dppa3 expression . Red represents tdTomato frequency with Dppa3 over-expression ( OE ) , while blue represents empty vector transfection control . The horizontal bars depict the expression threshold set for dtTomato and the proportion of tdTomato+ cells in each condition is stated . Right shows a bar plot of Dppa3 expression 48 hr and 96 hr after addition of DOX . Dppa3 expression is normalized to a housekeeping gene ( GAPDH ) and relative to empty vector sample . ( B ) Left shows histogram plots of tdTomato expression in 2C::tdTomato mESCs 48 hr and 96 hr after siRNA transfection . Red represents tdTomato frequency with Dppa3 siRNA , while blue represents scramble siRNA control . The bar depicts the expression threshold set for dtTomato and the proportion of tdTomato+ cells in each condition is stated . Right shows a bar plot of Dppa3 expression 48 hr and 96 hr after siRNA transfection . Dppa3 expression is normalized to a housekeeping gene ( GAPDH ) and relative to scramble siRNA sample . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 01810 . 7554/eLife . 22345 . 019Figure 3—figure supplement 3 . Quality control of TE reads mapping and normalization . ( A ) A bar plot of the proportion of multi-mapped TE reads ( Figure 3—source data 2 ) which can be unambiguously assigned at the level of class ( repClass ) , family ( repFamily ) and element ( repName ) based on the RepeatMasker annotation provided by UCSC table browser . ( B ) A heatmap of the expression of LTR , LINE and SINE in oocyte ( green ) , 1-cell ( yellow ) and 2-cell ( red ) embryos based on between-sample normalization method ( See Materials and methods ) . Samples are clustered by row . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 019 We identified ‘maternal TEs’ , defined as TEs expressed higher in WT oocytes than 2-cell embryos and ‘zygotic TEs’ , as TEs expressed higher in WT 2-cell embryos than oocytes . To characterise the disparities between TE expression in WT and KO , we compared differentially expressed TEs at the 2-cell stage , with maternal and zygotic TEs ( Figure 3D ) . Strikingly , TEs upregulated in Stella M/Z KO 2-cells are significantly enriched for maternal TEs while TEs downregulated in Stella M/Z KO 2-cells are associated with zygotic TEs . Furthermore , Stella M/Z KO 2-cell embryos display lower activation of LTR , specifically the endogenous retrovirus ( LTR-ERVL ) family , with small relative enrichment for LINE and SINE classes ( Figure 3E , Figure 3—figure supplement 1B ) . A particular element of interest is MuERV-L ( Kigami et al . , 2003; Ribet et al . , 2008 ) , which encodes a canonical retroviral Gag and Pol , flanked by 5’ and 3’ LTR ( also known as MT2_Mm ) . We observed a selective upregulation of MuERV-L-Int and MT2_Mm transcripts at the 2-cell stage , but this was significantly reduced in Stella M/Z KO embryos ( Figure 3F ) . Many MuERV-L elements have an open reading frame and we therefore tested protein levels using a MuERV-L Gag antibody . This antibody was validated by showing staining overlaps well with a MuERV-L reporter ESC line – 2C::tdTomato ( Macfarlan et al . , 2012 ) , and is specific to 2-cell embryos whilst not detected in metaphase II oocytes ( Figure 3—figure supplement 1C ) . Strikingly , immunofluorescence ( IF ) staining showed a significantly lower signal and loss of perinuclear localisation of MuERV-L Gag ( Ribet et al . , 2008 ) in Stella M/Z KO 2-cell embryos compared to WT , implying Stella influences both transcript and protein levels of MuERV-L ( Figure 3G ) . To assess whether Stella directly binds to specific transcripts / TEs we turned to ESC since obtaining sufficient 2-cell embryos for this analysis is intractable . Chromatin immunoprecipitation-seq ( ChIP-seq ) of HA-tagged Stella resulted in minimal peak calls ( n = 56 ) ( Supplementary file 2 ) , implying Stella does not efficiently bind DNA ( including MuERV-L ) in ESC . Consistently , neither overexpression nor knockdown of Dppa3 in mESC had a marked effect on the expression of MuERV-L ( Figure 3—figure supplement 2 ) . These results suggest that Stella-mediated control of MuERV-L is likely specific to the unique chromatin/cellular context of early embryos . Notably Stella has the potential to bind both RNA and DNA and could thus affect TEs through multiple modes . Overall , our data suggests Stella plays a key role in regulating a subset of TEs specifically in 2-cell embryos , which includes promoting strong activation of MuERV-L elements . Next we explored the relationship between TE and protein-coding gene expression in WT and KO samples . First , we considered the intersection between differentially expressed genes and genes that are neighbours of misregulated TEs at the 2-cell stage , and discovered a highly significant overlap of 12% between the two populations ( p=3 . 19 × 10−87 ) ( Figure 4A ) . Notably , a greater number of TEs downregulated in KO 2-cell embryos are located within 10 kb of the transcriptional start site of zygotically activated genes ( ZAG , n = 698 ) than expected by chance ( p<10−4 ) ( Figure 4B ) . Altogether , this suggests perturbations in TE expression may be linked to neighbouring mRNA expression levels in Stella M/Z KO embryos . To determine if this relationship is applicable genome-wide , we performed global expression correlation analysis between a TE and its nearest gene . To eliminate confounding factors , we excluded TEs that overlap directly with an exon . Notably , we detected 387 TEs whose expression levels were positively correlated with their nearest gene ( Spearman’s correlation >0 . 7 ) while 224 TEs were negatively correlated ( Spearman’s correlation <−0 . 7 ) ( Figure 4—figure supplement 1A ) . This relationship was validated using single-embryo qRT-PCR at four genomic loci , which demonstrated a significant correlation between the expression of a TE and its downstream gene ( Figure 4C ) . These loci were selected to represent a range of LTR class elements; RMER19A , ORR1A2-Int and MT2B are further categorised into ERVK , ERVL-MaLR and ERVL families respectively . Interestingly , 2 of the genes , Chka and Zfp54 , have been implicated in regulation of early embryonic development ( Albertsen et al . , 2010; Wu et al . , 2008 ) . Thus , the expression levels of a subset of TEs are highly correlated with the expression of their nearest gene . 10 . 7554/eLife . 22345 . 020Figure 4 . A positive correlation between the expression of a subset of TEs and their nearest gene . ( A ) A Venn diagram illustrating a significant overlap between differentially expressed genes and genes within ±20 kb of differentially expressed TEs in WT v KO 2-cell embryos . ( B ) A histogram of the mean number of depleted TEs in Stella M/Z KO 2-cell embryos within ±10 kb of the TSS of a group of genes . The red line represents zygotically activated genes ( ZAG , n = 698 ) that belong to WT class EU and differentially expressed in KO samples ( adjusted p-value<0 . 05 ) ( Figure 1—source data 4 ) . The grey bars represent each of 10 , 000 random sets of 698 genes . ( C ) Top is schematic illustrations of the relationship between the TE and downstream gene , where grey box represents the TE and its orientation; white box represents the gene and its orientation . Bottom is scatter plots of qRT-PCR expression in 2-cell embryos . WT ( white circle ) n = 19 and KO ( light blue triangle ) n = 17 . Spearman’s correlation analysis was performed and p<0 . 0001 corresponds to **** . ( D ) Characterisation of chimeric transcripts . ( Middle ) A schematic illustration of the alternative splicing event that occurred between the TE and the downstream gene . SD = splice donor site and SA = splice acceptor site . The endogenous starting codon ( ATG ) is indicated . ( Left ) The PCR product of the chimeric transcript where the forward primer originates from the TE and reverse primer originates from the gene . The PCR product is significantly shorter than the annotated distances between them , confirming a splicing event . ( Right ) Sanger sequencing chromatographs from the PCR product for each chimeric transcript , confirming the site of chimeric junction . * Indicates a different nucleotide to annotation . ( E ) Box plots of single-embryo qRT-PCR of the relative expression of chimeric transcripts in WT ( white ) and KO ( light blue ) 2-cell embryos . p<0 . 01 corresponds to ** and p<0 . 001 corresponds to *** . All expression data are normalised to three housekeeping genes and relative to 1 WT embryo . Also see Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 02010 . 7554/eLife . 22345 . 021Figure 4—figure supplement 1 . A subset of TEs is positively correlated with its nearest genes . ( A ) A histogram of Spearman’s correlation of the expression between a gene and its nearest TE within a distance of 1Mbp . The red line indicates correlation >|0 . 7| . 387 TE / gene pair correlation >0 . 7 while 224 TE/ gene pair correlation <−0 . 7 . ( B ) ( Left ) A bar chart of MuERV-L int transcript expression in individual WT ( white ) and KO ( grey ) 2-cell embryos . Red star indicates 3 KO embryos expressing the lowest MuERV-L transcript ( KO2 , 8 , 1 ) while green circle highlights KO6 , the KO embryo which expresses the highest MuERV-L levels . The right panel shows a heatmap of expression of genes identified to make a chimeric junction with MuERV-L ( Macfarlan et al . , 2012 ) . Heatmap is normalised by row and clustered by column . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 021 Whilst association does not equate to causality , there are a number of ways TEs have been implicated in the regulation of gene expression ( Elbarbary et al . , 2016; Thompson et al . , 2016 ) . One possibility is the establishment of alternative splicing events , which create a junction between an expressed TE and a downstream gene exon , thus leading to the formation of a chimeric transcript ( Peaston et al . , 2004 ) . Indeed , MuERV-L has been shown to form such fusion transcripts with 307 genes at the 2-cell stage ( Macfarlan et al . , 2012 ) . Here , we find Stella M/Z KO embryos with the lowest MuERV-L expression also express lower levels of genes that make a chimeric junction with MuERV-L elements ( Figure 4—figure supplement 1B ) . In contrast , the KO embryo with the highest MuERV-L expression expresses higher levels of these genes . Importantly , we next identified and validated a number of chimeric transcripts that are expressed in 2-cell embryos by PCR and sequenced across the chimeric junction ( Figure 4D ) . Single-embryo qRT-PCR shows most of these chimeric transcripts are expressed at lower levels in KO compared to WT 2-cell embryos ( Figure 4E ) . These chimeric transcripts include Snrpc , which encodes for a component of the ribonucleoprotein required for the formation of the spliceosome , and the RNA binding protein , Rmb8a . Our data implies a link between activation of some TEs and the emergent gene expression network , which is perturbed in Stella M/Z KO embryos due to altered TE expression . This is , in part , mediated by TEs directly regulating gene expression through chimeric transcripts . We propose that the dysregulation in TE expression might directly contribute to the abnormal pre-implantation phenotype observed in Stella M/Z KO embryos . We focused on assessing the function of MuERV-L elements , as they represent one of the most highly activated TEs at the 2-cell stage ( Kigami et al . , 2003 ) , as well as being amongst the most significantly downregulated in Stella M/Z KO embryos ( Figure 3F , G ) . We considered that attenuated MuERV-L activation in Stella M/Z KO embryos could in turn disrupt activation of associated 2-cell transcripts driven by their LTRs ( Figure 4—figure supplement 1B ) . Alternatively , MuERV-L mRNA/protein itself may also have a functional role during early embryonic development . We detected 583 copies of the full-length MuERV-L element , containing the complete gag and pol genes , in the genome . This makes it experimentally challenging to completely eliminate MuERV-L activation in early embryos . Nevertheless , we utilised siRNA to knockdown the expression of MuERV-L elements in 1-cell embryos and monitored the effects on embryonic development ( Figure 5A ) . We designed siRNA to target the consensus sequence of MuERV-L from the Dfam database ( Hubley et al . , 2016 ) ( Figure 5B ) . Computational analysis reveals MuERV-L siRNA targets 80 . 5% of full-length MuERV-L elements with perfect match and 99 . 5% with ≤2 bp mismatches , while scramble siRNA has no targets with ≤2 bp mismatches . At 20 μM siRNA , we observed a slight decrease in MuERV-L transcripts ( Figure 5C ) but a significant reduction in MuERV-L Gag protein staining in 2-cell embryos ( Figure 5D ) . These embryos exhibited a mild developmental delay at 4 to 8 cell stage progression on day 2 , with fewer embryos reaching the blastocyst stage on day 4 , and those that did were smaller and with fewer cells ( Figure 5E ) . This suggests that a modest reduction in MuERV-L at the 2-cell stage has a notable effect on pre-implantation development . 10 . 7554/eLife . 22345 . 022Figure 5 . MuERV-L plays a functional role during pre-implantation development . ( A ) A schematic diagram illustrating the experimental setup . ( B ) Left is an illustration of the structure of the MuERV-L element ( GenBank accession number: Y12713 ) , depicting the exact site of siRNA target . Right is a table showing the number of full-length MuERV-L elements targeted by MuERV-L siRNA and the number of off-targets . ( C ) Box plots of single-embryo qRT-PCR of MuERV-L pol transcript and D ) MuERV-L Gag protein ( fluorescence ) intensity in uninjected controls , 20 µM scramble siRNA or 20 µM MuERV-L siRNA injection 2-cell embryos . ( Right ) Representative z-stack projections of immunofluorescence ( IF ) staining against MuERV-L Gag in 2-cell embryos . Two-tail Wilcoxon Rank Sum Test was performed for both ( C ) and ( D ) and p<0 . 01 corresponds to * , not significant ( n . s . ) or are otherwise stated . ( E ) The effect of MuERV-L siRNA on developmental progression of pre-implantation embryos . ( Left ) Bar graphs illustrating the number of embryos observed at different stages on day 2–4 of in-vitro culture . Blue background indicates area of interest . Experiments were repeated twice and the circle dots represent the data points for each replicate . A total of n = 17 , n = 58 and n = 53 embryos were analysed for uninjected , scramble and MuERV-L siRNA injections respectively . One-tailed Student’s T-test performed and there were no statistical difference between uninjected , scramble or MuERV-L siRNA injected groups . ( Middle ) Day five bright field images of late blastocysts injected with scramble or MuERV-L siRNA . ( Right ) A box plot of IF quantification of number of DAPI+ cells in the late blastocyst stage on day five injected with 20 µM of scramble ( white ) or MuERV-L ( grey ) siRNA . ( F and G ) are box plots of single-embryo qRT-PCR of MuERV-L pol transcript and MuERV-L Gag protein ( fluorescence ) intensity in uninjected control , 80 µM scramble siRNA and 80 µM MuERV-L siRNA injected 2-cell embryos . ( Right ) Representative z-stack projections of IF staining against MuERV-L Gag antibody in 2-cell embryos . Two-tail Wilcoxon Rank Sum Test was performed for both ( F ) and ( G ) and p<0 . 01 corresponds to * , not significant ( n . s . ) or are otherwise stated . ( H ) A bar graph plotting the number of embryos observed at each stage in day two in-vitro culture in uninjected embryos , 80 µM scramble ( white ) or MuERV-L ( grey ) siRNA injection . Experiments were repeated twice and the circle dots represent the data points for each replicate . A total of n = 17 , n = 54 and n = 50 embryos were analysed for uninjected , scramble and MuERV-L siRNA respectively . One-tailed Student’s T-test performed . p<0 . 05 corresponds to * and p<0 . 01 corresponds to ** . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 02210 . 7554/eLife . 22345 . 023Figure 5—figure supplement 1 . Chimeric transcript expression in MuERV-L knockdown embryos . A scatter dot plot of qRT-PCR analysis of chimeric transcripts in uninjected ( circle , n = 4 ) , 80 µM scramble siRNA ( square , n = 12 ) and 80 µM MuERV-L siRNA ( triangle , n = 8 ) treated 2-cell embryos . The median and interquartile range is indicated . All transcripts are normalised to housekeeping genes , relative to one uninjected embryo . Two-sided Wilcoxon rank sum test was performed and statistically significant results are stated . DOI: http://dx . doi . org/10 . 7554/eLife . 22345 . 023 There is nonetheless variability in MuERV-L expression at the 2-cell stage , as shown in other studies ( Ancelin et al . , 2016 ) , and we therefore speculated that a higher concentration of siRNA might increase the repression efficiency of MuERV-L transcripts . Indeed , 80 μM MuERV-L siRNA was more effective ( Figure 5F and G ) , and in turn resulted in >90% embryos arrested at the 2-cell stage ( Figure 5H ) , albeit some indirect effects at higher concentrations of siRNA may contribute to the embryonic arrest . Notably , MuERV-L knockdown resulted in repression of some chimeric transcripts previously characterised in 2-cell embryos ( Figure 4D , Figure 5—figure supplement 1 ) . These data collectively suggest MuERV-L may have a functional role in embryonic development .
The maternal-to-zygotic transition is a complex process that entails extensive global transcriptional and epigenetic changes . Maternally inherited factors such as Stella play a role in this transition in mouse oocytes but the difficulties of obtaining sufficient materials has hindered investigations on how they contribute to MZT . Here , we use single-cell/embryo approaches and find that deletion of maternal and zygotic Stella affects MZT . Furthermore , we observed a failure to activate endogenous retrovirus ( LTR-ERVL ) , and specifically MuERV-L elements . In parallel there is an accumulation of maternal transcripts and a failure to upregulate many zygotic transcripts . These events appear to be partially coupled , with altered LTR promoter activity leading to changes in gene expression , for example through the modulation of chimeric transcripts . In addition , we reveal the biological relevance of TE expression as knockdown of MuERV-L at the 2-cell stage hinders developmental progression , which likely contributes to the phenotype observed following loss of Stella . Maternal mRNA degradation and zygotic genome activation are critical aspects during MZT ( Li et al . , 2013 ) . Maternal mRNA clearance plays both a permissive and instructive role ( Tadros and Lipshitz , 2009 ) , while ZGA also promotes the final degradation of maternal transcripts . Stella has the potential to function as an RNA binding protein , which might facilitate the degradation of maternal transcripts ( Nakamura et al . , 2007; Payer et al . , 2003 ) . In addition , Stella KO oocytes exhibit transcriptional differences compared to WT oocytes . This is consistent with a previous study showing Stella KO oocytes are defective in transcriptional repression ( Liu et al . , 2012 ) , which may be needed for dosage regulation of critical maternally inherited transcripts . Stella has also however been demonstrated to be required beyond oogenesis ( Nakamura et al . , 2007 ) . Maternally inherited factors likely influence the timing and specificity of gene expression during ZGA , which is essentially determined by interplay between the transcriptional machinery and histone modifications that influences chromatin accessibility ( Lee et al . , 2014 ) . The failure of zygotic transcript activation may be linked to the failure of zygotic TE activation . We detected extensive changes in global TE expression during MZT . This is accompanied by strong upregulation of the LTR-ERVL family in 2-cell embryos , which is consistent with a recent study showing that the chromatin surrounding ERVL is in a highly accessible state at the 2-cell stage ( Wu et al . , 2016 ) . LTR-ERVL upregulation is significantly reduced in the absence of maternal Stella , suggesting that Stella may facilitate the activation of many ERVs . The expression changes are validated at the protein level for a specific LTR-ERVL element , MuERV-L . Importantly , the decrease in MuERV-L is unlikely to be a secondary effect of 2-cell embryonic arrest , as maternal Kdm1a ( LSD1 ) mutant embryos , which result in pan 2-cell arrest maintain normal MuERV-L transcript expression ( Ancelin et al . , 2016 ) . Notably , whilst many epigenetic mechanisms have been identified for the suppression of ERVs ( Gifford et al . , 2013 ) , we have identified a factor associated with the activation of a subset of ERVs during mammalian MZT . The question remains , how does Stella regulate TE expression ? In primordial germ cells , Stella directly binds to LINE1 and IAP elements to facilitate DNA demethylation of these targets ( Nakashima et al . , 2013 ) . Whilst experimentally challenging , it would be pertinent to determine potential DNA binding targets of Stella in 2-cell embryos . At the same time , function of Stella at the RNA level cannot be excluded , or it could act by altering the epigenetic status of cells at this time . For example , the H3K9 methyltransferase Setdb1 is aberrantly enriched in Stella M/Z KO 2-cell embryos , and this has been shown to suppress ERVs and chimeric transcripts expression ( Eymery et al . , 2016; Karimi et al . , 2011; Kim et al . , 2016 ) . Since a human orthologue of Dppa3 is expressed in ESCs , germ cells and pre-implantation embryos ( Bowles et al . , 2003; Deng et al . , 2014; Payer et al . , 2003 ) , it would be of interest to know whether DPPA3 plays a similar role during human pre-implantation development; for example , on the stage specific activation endogenous retroviruses ( Göke et al . , 2015; Grow et al . , 2015 ) . TEs have evidently been co-opted for the regulation of mammalian development as exemplified by the domestication of the retroviral env gene , which is essential for placental development ( Dupressoir et al . , 2012 ) . Here , we have shown that the expression of a subset of TEs are intimately linked to its nearest gene during early embryonic development . As we have shown in several cases , TEs could regulate gene expression through chimeric transcripts , which are misregulated in the absence of Stella . The impact of transcriptional inhibition of ERV programme and associated chimeric transcripts during MZT , and their subsequent effects on development merits further investigation . Intriguingly , we discovered that reducing MuERV-L mRNA/ protein levels at the 2-cell stage affects development . This could suggest that the presence of MuERV-L Gag protein has a functional role during early mouse development , similar to a recent report illustrating a role of endogenous LTR viral-like particles in human blastocyst development ( Grow et al . , 2015 ) . It is also possible that MuERV-L siRNA KD may downregulate a critical subset of chimeric transcripts that include MuERV-L sequence or , could feedback to target transcriptional repression to MuERV-L loci through small RNA pathways ( Castel and Martienssen , 2013 ) . Disentangling these possibilities warrants further investigation . In conclusion , we have revealed that Stella is involved in orchestrating the transcriptional changes and activation of endogenous retroviral ( ERV ) elements during maternal-to-zygotic transition . The appropriate expression of LTR-ERV driven zygotic genes and specific ERV elements ( MuERV-L ) in turn contribute towards normal early embryonic development in mice .
The maternal-to-zygotic transition ( MZT ) marks the period when the embryonic genome is activated and acquires control of development . Maternally inherited factors play a key role in this critical developmental process , which occurs at the 2-cell stage in mice . We investigated the function of the maternally inherited factor Stella ( encoded by Dppa3 ) using single-cell/embryo approaches . We show that loss of maternal Stella results in widespread transcriptional mis-regulation and a partial failure of MZT . Strikingly , activation of endogenous retroviruses ( ERVs ) is significantly impaired in Stella maternal/zygotic knockout embryos , which in turn leads to a failure to upregulate chimeric transcripts . Amongst ERVs , MuERV-L activation is particularly affected by the absence of Stella , and direct in vivo knockdown of MuERV-L impacts the developmental potential of the embryo . We propose that Stella is involved in ensuring activation of ERVs , which themselves play a potentially key role during early development , either directly or through influencing embryonic gene expression .
When a sperm cell fertilizes an egg cell , this creates a single-celled embryo called a zygote that will go on to divide repeatedly throughout development . The zygote inherits the contents of the egg including many important proteins that initially control how the embryo develops . In mice , the embryo takes over control of development once the zygote has divided to form a two-celled embryo . This transition of control destroys the maternally inherited proteins and selectively activates zygotic genes and some DNA sequences called transposable elements that evolved from virus DNA . One of the proteins inherited from the egg cell is called Stella . Embryos that lack Stella die within the first few cell divisions , which suggests that the protein is needed during the earliest stages of development . However , it is not clear what Stella's role in the early embryo is . Huang , Kim et al . decided to investigate how Stella might affect the genes that are switched on during development by using a technique called RNA-seq to study egg cells and early embryos from mice . Two-celled embryos that lacked Stella could not activate a number of genes that produce proteins that are critical for development . They also failed to activate a group of transposable elements called endogenous retroviruses . In particular , a lack of Stella significantly reduced the activity of an endogenous retrovirus called MuERV-L . Further experiments showed that MuERV-L is needed for normal embryonic development , and so suggests that transposable elements play important roles in this process . Future studies will aim to explore these roles in more detail . It will also be important to identify the genes that Stella targets in embryos , and to investigate the roles that similar maternally inherited proteins play in early embryonic development .