Double stranded RNA sensing is silenced during early embryonic development

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: minor_revision

Abstract
The type I interferon response is inactive during early mammalian development and becomes functional only after gastrulation. As a result, the totipotent and pluripotent embryonic stages remain susceptible to pathogens, including viruses. Here, we demonstrate that pluripotent mouse embryonic stem cells suppress the RIG-I-like receptor sensing pathway by silencing the expression of the double stranded RNA sensor MDA5. This silencing is necessary to avoid the recognition of double stranded RNAs of endogenous origin, which accumulate in mouse embryonic stem cells. Reintroducing MDA5 results in recognition of these endogenous double stranded RNAs and triggers the activation of the IFN response through IRF3. The production of interferon alters the differentiation ability of mouse embryonic stem cells, and affects the pluripotency gene expression programme, as shown by epigenetic, transcriptomic and proteomic analyses. Further, we show that zebrafish also repress MDA5 expression in early development and lack early-stage interferon activation, and that inducing double-stranded RNA-mediated signalling at this stage results in developmental defects. Altogether, we conclude that silencing the RIG-I-like receptor pathway during early development is important in preventing aberrant immune recognition of endogenous double stranded RNAs, safeguarding normal development.

Introduction
Type I interferons (IFNs) orchestrate the innate immune response against viruses. IFNs are produced by infected cells, typically upon sensing viral nucleic acids. Viral cytoplasmic DNA is sensed by the cyclic GMP-AMP synthase, cGAS and viral RNAs are sensed by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family of proteins1,2. This family has three members, RIG-I, melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics (LGP2). RIG-I recognises uncapped short double-stranded RNAs (dsRNAs), while MDA5 recognises long dsRNA molecules. Although LGP2 binds a variety of RNA moieties, the absence of CARD domains prevents downstream signalling. LGP2 regulates the activity of the other two members in the family, preventing RIG-I activation, while promoting MDA5 activity2. Upon the sensing of viral nucleic acids, several families of transcription factors are activated, including the interferon regulatory factors 3 and 7 (IRF3/7), which drive type-I IFN expression, and the nuclear factor-kappa B (NF-kB), which drives the production of pro-inflammatory cytokines, such as TNF-α3. While IRF3 is expressed in most cell types, IRF7 has lower basal expression levels, that only increase upon IFN activation4,5. Besides type I IFNs, IRF3 also promotes the expression of other immune-related genes, suggesting that it has a broader range of targets than IRF76–8. Once secreted, IFNs act in an autocrine and paracrine manner by binding to IFN receptors (IFNAR1/2) and inducing the expression of hundreds of interferon-stimulated genes (ISGs) via JAK-STAT signalling9. In addition to cytoplasmic sensors, the Toll-like receptors (TLRs), located on cell surfaces or endosomes, can also recognise viral nucleic acids and activate the type I IFN response10.

Healthy mammalian cells typically do not accumulate significant amounts of endogenous dsRNAs, as they have evolved mechanisms to prevent their buildup11,12. In contrast, certain pathological conditions, such as cancer or autoimmunity, are characterised by the activation of the IFN response upon sensing endogenous nucleic acids from nuclear or mitochondrial origins. This activation results from the malfunctioning of key RNA processing enzymes, as well as epigenetic regulators or nucleic acids sensors13,14.

There is increasing evidence that transposable elements (TEs), due to their repetitive nature and their multiple insertions in either sense or antisense orientation, constitute a natural source of dsRNA in cells15,16. Most somatic cells silence transposon expression to prevent the potential deleterious consequences of novel insertions. Interestingly, embryonic stem cells (ESCs) and the early stages of development exhibit unique patterns of transposon activity. In contrast to somatic cells, early embryonic development is characterised by sequential waves of expression from specific TE families, such as LINE-1 in the mouse 2-cell stage, and MERVL in preimplantation mouse embryos17. These patterns are partly a result of the profound epigenetic reprogramming that embryonic cells undergo during the first divisions to acquire a totipotent and pluripotent identity, which typically suppresses TE activity. As an adaptation, embryonic cells exploit TEs, co-opting their promoters (e.g. long-terminal repeats, LTRs) and derived RNAs to fulfil essential functions during development18–21.

ESCs and early development stages are also unique in aspects of immune defence. Both we and others have previously demonstrated that the IFN response is inactive in pluripotent ESCs and the early stages of mammalian development. When challenged with viruses or viral DNA/RNA mimics, both mouse and human ESCs are incapable of producing IFNs22–25. Similarly, mouse embryonic teratocarcinoma cells, which retain certain pluripotency traits, and human induced pluripotent stem cells also lack the capacity to produce IFNs25,26. The ability to synthesise IFNs is acquired after cells differentiate and lose their pluripotent state22–25. Supporting this, in vivo data show that in mice, the ability to produce IFNs is not acquired until after gastrulation, specifically at day 8 post fertilisation27. These observations lead to the hypothesis that the IFN response and pluripotency represent two incompatible cellular processes.

The critical question is why cells in early development, including ESCs, have sacrificed the type I IFN response if this renders them more susceptible to infections. While ESCs may use alternative antiviral pathways, we and others have shown that for specific viruses, ESCs are more permissive to infection than somatic cell types. This challenges the notion of a generalised intrinsic resistance of ESCs to viruses24,28–32. Within the inner cell mass, ESCs may be physically protected from viruses by the trophectoderm. In addition to the physical barrier, trophectoderm cells are immunologically competent and could stimulate the inner cell mass antiviral response by producing IFNs33–35. Although attenuated, ESCs can respond to exogenous IFNs, suggesting that maternal IFNs could also have protective properties during development24,25,36,37. For instance, constitutive expression of the type I IFN, IFN-ε, by the maternal reproductive system has been shown to provide protection against viral infection38.

To elucidate the mechanisms and rationale behind the absence of the type I IFN response in early development, we employed pluripotent mESCs and zebrafish as model systems. In both, we observed a silencing of the dsRNA-sensing pathway. Specifically, mESCs suppress the type I IFN response by silencing the expression of the dsRNA sensor MDA5. Unlike somatic cells, mESCs accumulate endogenous dsRNAs, that can induce the IFN response through MDA5 sensing. As a consequence, introducing MDA5 into mESCs activates the IFN response in the absence of infection. This inappropriate activation is highly detrimental, impairing ESCs ability to differentiate and maintain pluripotency, as shown by epigenetic, transcriptomic and proteomic analyses. The silencing of the dsRNA-sensing mechanisms appears to be a conserved evolutionary strategy amongst jawed vertebrates. Similarly, in zebrafish, MDA5 expression is low during the early stages of development, and activation of the IFN response results in obvious developmental defects.

We conclude that the gene expression program required for early development is inherently incompatible with an active IFN response. Expression of TEs is crucial for normal development but can lead to the formation of dsRNAs, potentially triggering the IFN response through MDA5 sensing. Our results indicate that IFNs disrupt normal development due to conflicts between IFN-associated transcription factors and the chromatin landscape in early development. Therefore, embryos must inactivate dsRNA sensing to suppress the IFN response, which, as a trade-off, increases their susceptibility to infections.

Results
The expression of the dsRNA sensor MDA5 is developmentally regulated
ESCs fail to make IFNs when challenged with dsRNA, but they acquire this ability after differentiation22–24,39–42. To identify which components of the IFN response may be responsible for the differential ability in producing IFNs, we compared the expression of genes involved in TLR signalling, RLR signalling and JAK/STAT signalling during stem cell differentiation. We used two different datasets, one of mouse ESCs differentiated to the three major embryonic lineages, ectoderm, mesoderm and endoderm43, and a second, where mouse ESCs were differentiated to neuronal stem cells and neurons44 (Fig. 1a–c and Supplementary Fig. 1A, B). Although varying between lineages, we observed a consistent increase in the levels of expression of Ifih1, the gene encoding for the dsRNA sensor MDA5, and Irf7, which encodes for the transcription factor IRF7. The expression of both genes was negligible in ESCs and increased after differentiation (Fig. 1a–c and Supplementary Fig. 1A, B). Both genes are involved in activating IFN production upon dsRNA stimulation, suggesting that the RLR pathway is silenced at multiple levels in ESCs. We confirmed these findings using in vitro differentiation assays of ESCs through induction of embryoid body formation followed by retinoic acid treatment45. After 20 days of differentiation, the expression of pluripotency factors Nanog and Pou5f1 was lost, while the expression of differentiation markers Sox17 and Foxa2 increased as expected (Fig. 1d, e). We observed that Ifih1 was the most differentially expressed gene involved in the RLR signalling pathway upon differentiation, displaying a significant increase (Fig. 1f). To evaluate potential mechanisms that drive silencing of Ifih1 in ESCs, we first tested the role of DNA methylation. ESCs were grown in the presence of 5-azacytidine, a hypomethylation-inducing reagent, but no increase in Ifih1 expression was found, while the known methylation-regulated gene Dazl was increased (Supplementary Fig. 1C). We next tested the contribution of polycomb-mediated silencing. RING1B, a component of the polycomb repressive complex 1 (PRC1), was found to bind the promotor region of Ifih1 (Supplementary Fig. 1D). Both RING1B and homolog RING1A are functionally redundant and required for PRC1 function46. Despite binding, RING1B degron-depletion in Ring1A–/– ESCs did not result in a significant upregulation of Ifih1 expression, suggesting it is not directly repressed by PRC147,48 (Supplementary Fig. 1E).

We confirmed that upon differentiation, and increased Ifih1 expression, mESCs acquire the ability to respond to the dsRNA mimic, poly(I:C), as measured by RT-qPCR of IFNs (Ifnb1), Tnf as well as of the ISG Cxcl10 (Fig. 1g). As sensing type I IFN is integral to the overall IFN response, we also compared the response of ESCs and differentiated cells to exogenous IFN-β. ESCs displayed an attenuated response to exogenous IFN-β, which increased upon differentiation, as shown by RT-qPCR analyses of a panel of ISGs (Fig. 1h). Despite some responsiveness to exogenous IFNs, Ifih1 was not induced compared to other ISGs (Fig. 1i). All these together confirmed that ESCs acquire the IFN response during differentiation, and this coincides with a significant upregulation of the dsRNA sensor MDA5 (Ifih1).

Introduction of MDA5 in ESCs leads to developmental disruption
MDA5 is expressed at very low levels in ESCs, therefore, we decided to explore the consequences of expressing MDA5 in these cells by generating ESC lines constitutively expressing MDA5. Although MDA5 expression was driven by a promoter known to be stable in mESCs (EF1a), after several passages, cells silenced the exogenous copy of MDA5 in an irreversible manner. To address this issue, we generated doxycycline-inducible ESCs lines expressing a FLAG-tagged form of MDA5. Two clones were selected for further characterisation, and MDA5 expression after doxycycline treatment was confirmed both by western-blot and RT-qPCR (Fig. 2a, b, respectively). Despite MDA5 expression reaching levels similar to those obtained after differentiation of cells (Supplementary Fig. 2A), we still observed silencing of MDA5 after 24 h, suggesting an adverse effect of long-term expression of MDA5. To further investigate the consequences MDA5 expression in ESCs, we performed a time course analysis by RNA-high-throughput sequencing (RNA-seq) after MDA5 expression (0, 4, 8, 24h after doxycycline induction) with two independent clonal cell lines compared to wild type ESCs (WT). Surprisingly, hundreds of genes were commonly differentially expressed in both MDA5-expressing cell lines compared to WT, with significant changes as early as 4h after induction (abs log2FC>0.4; pval≤ 0.05) (Supplementary Data 1). Gene ontology (GO) analysis of the shared differentially expressed genes showed that both upregulated and downregulated genes were associated with development, morphogenesis, and differentiation, confirming the profound rewiring of the developmental programme when MDA5 is expressed (Fig. 2c, Supplementary Fig. 2B and Supplementary Data 2). More specifically, the upregulated genes were associated with terms involving nervous system development. This observation was confirmed using the Mouse Gene Atlas to predict cell types based on differential gene expression data, and by analysing the expression of specific neuronal markers by RNA-seq and RT-qPCR, suggesting that MDA5 induction in ESCs results in a ‘neuronal-like’ gene expression profile (Fig. 2d, e and Supplementary Fig. 2C). Conversely, downregulated genes were associated with genes typically expressed in ESCs and embryonic fibroblasts, suggesting that MDA5 induces the loss of the ESC features (Fig. 2f, g). We conclude that the expression of MDA5 causes a profound disruption of the gene expression programme of ESCs, leading to an expression profile associated with differentiating cells.

MDA5 expression results in the loss of pluripotency factors and activation of immune pathways in ESCs
Considering the alterations in gene expression observed upon MDA5 induction, we hypothesise that these could be driven by epigenetic changes, including chromatin accessibility and/or differential activity of transcription factors. To test this possibility, we first assessed changes in the chromatin architecture upon MDA5 induction. We performed an Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-seq) and confirmed that MDA5 induces changes in chromatin accessibility, with 171 sites being more accessible, and a total of 382 losing accessibility after only 24h of MDA5 induction (Fig. 3a, Supplementary Fig. 3A–C and Supplementary Data 3). Gene Ontology analysis revealed that regions with more accessibility were associated with nervous system function, similar to the RNA high-throughput sequencing analyses, but also deleterious pathways such as apoptosis. On the other hand, regions losing accessibility were associated with signalling pathways regulating the pluripotency of stem cells, including TGF-β, Apelin and p53 (Fig. 3b). Interestingly, regions changing accessibility were enriched for transcription factor binding sites of pluripotency factors, including Oct4, Nanog, and several Sox and Klf family members (Supplementary Fig. 3B). These findings were confirmed at the transcriptional level using the RNA-seq dataset. Using the loss-of-function (LOF) tool from the ShinyGO package49, we found that the gene expression changes associated with MDA5 induction were predicted to be driven by the loss of transcription factors involved in pluripotency maintenance, such as Nanog, Oct4 (Pou5f1), and some members of the Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), including Suz12, Eed and Rnf2 (Fig. 3c, for full list see Supplementary Fig. 3D). PRCs repress the expression of lineage-specific markers during pluripotency and silence the expression of pluripotency factors upon differentiation50,51. In agreement with this prediction, we confirmed that the expression of the pluripotency factors Nanog, Klf4 and Pou5f1 decreased upon MDA5 induction. In contrast, Sox2 expression increased (Fig. 3d, e). Sox2 has been shown to increase during neuroectodermal differentiation, which agrees with the neuronal-like gene expression phenotype that ESCs acquire after MDA5 induction (Fig. 2d, e)52,53. Altogether, our combined analyses suggest that the function and expression of transcription factors essential for early development are affected by MDA5.

To provide further evidence of transcription factor dysregulation at the protein level and expand on the mechanism, we performed Chromatin-Enrichment for Proteomics followed by Mass Spectrometry (ChEP-MS). This approach provides a global overview of the chromatin composition and how these changes under different conditions54,55. The chromatin-associated proteome underwent substantial changes after 8h of MDA5 induction, with comparable numbers of proteins showing increased or decreased presence (absLog2FC>1, p<0.05). Similar patterns were also observed after 24 and 48h of MDA5 induction (Fig. 3f and Supplementary Data 4). KEGG pathway analyses revealed that enriched proteins in chromatin were associated with immune and pro-inflammatory signalling pathways after 24h of MDA5 induction, while at 48h, pathways involved in shutting down pro-inflammatory and IFN responses were activated56,57 (Fig. 3g). On the other hand, factors involved in Notch, Hippo and p53 signalling, which are important for normal development, were depleted from the chromatin fraction after MDA5 induction (Fig. 3g). These results were reminiscent of the pathways obtained by ATAC-seq (Fig. 3b). We also exploited these datasets to understand the dynamics of key pluripotency maintenance factors. OCT4 and members of the Myc and Klf families, as well as PRC components, were less enriched in the chromatin fraction after inducing MDA5. In contrast, factors involved in de novo DNA methylation were chromatin-enriched upon MDA5 induction (Fig. 3h). To investigate if some of the changes observed in chromatin enrichment were driven by changes in gene expression, we compared a late ChEP-MS data point with an early RNA-seq timepoint. This comparison revealed a significant positive correlation (p=0.003, ρ=0.46) between RNA and protein levels, suggesting that the observed changes are not solely due to alterations in the subcellular localisation of chromatin factors, but also reflect shifts in the gene expression profile of these cells (Fig. 3i and Supplementary Fig. 3E, F). Interestingly, the group of shared genes with changes in the RNA and protein level was associated with innate immune signalling, including the ISG DDX58 (Fig. 3j). We predict that expression of MDA5 in ESCs results in innate immune activation, and a concomitant loss of pluripotency, accompanied by epigenetic and transcriptional reprogramming.

Expression of MDA5 results in type I IFN activation upon recognition of endogenous dsRNAs
Both the high-throughput proteomic and RNA analyses suggested that MDA5 expression results in immune activation in ESCs in the absence of infection. To confirm this finding, we tested the expression levels of the type I IFN Ifnb1, Tnf, and the ISGs Isg15 and Ifitm1 in MDA5-induced cells using RT-qPCR. MDA5 induction resulted in the expression of type I IFNs and ISGs (Fig. 4a). On protein level, we observed production of IFN-β by ELISA in cells expressing inducible or constitutive forms of MDA5 (Fig. 4b). To test the importance of dsRNA recognition in activating the IFN response, we generated ESCs that express an MDA5 mutant form lacking the ability to recognise dsRNA. The C-terminal domain (CTD, residues 898–1020) of MDA5 is proposed to play a key role in the initial binding and oligomerisation on dsRNA58,59. Based on these findings an MDA5 variant lacking the CTD was generated (ΔCTD). Using in vitro pull-down assays, we confirmed that full-length MDA5 binds dsRNA (poly(I:C)), but not ssRNA (poly(C)), while the ΔCTD mutant failed to bind both dsRNA and ssRNA (Fig. 4c). We generated ESC lines expressing the inducible ΔCTD mutant and found that expression of this truncated MDA5 form was not repressed as observed for full-length MDA5 (Fig. 4d). In addition, this mutant failed to activate the type I IFN response and consequent ISG expression in comparison with full-length MDA5 (Fig. 4b, e), suggesting that MDA5 requires binding to endogenous dsRNA and oligomerisation to initiate the IFN response. Based on these results, we tested whether ESCs accumulate endogenous dsRNA by performing flow cytometry with antibodies against dsRNA (J2). As a control for staining specificity, we pre-treated cells with RNAse III, a dsRNA-specific endonuclease, before probing with J2 antibodies. We confirmed that dsRNA accumulated in ESCs and this signal was RNAse III sensitive (Fig. 5a, b, and Supplementary Fig. 4A). On the other hand, the somatic IFN-competent microglial cell line, BV2, did not accumulate any RNAse III-sensitive dsRNA (Fig. 4a, b). To further confirm the presence of dsRNA in ESCs we performed immunofluorescence analyses in ESCs and in vitro differentiated cells. While differentiated cells did not display any dsRNA-positive cells, ESCs did, confirming that dsRNA is accumulated during pluripotency (Fig. 5c–e). We next aimed at identifying the transcripts present in the dsRNA population of ESCs using immunoprecipitation (IP) with J2 antibody followed by RNA high-throughput sequencing. We performed enrichment analyses by comparing the transcripts enriched in the dsRNA IPs (‘enriched dsRNA’) over input, versus all non-enriched transcripts (‘non-enriched control’). This comparison revealed that dsRNAs were enriched in transcripts from protein-coding genes (Fig. 5f). Next, we calculated the minimum free energy (MFE) of these enriched transcripts and found a significant decrease compared to all non-enriched ones, confirming the presence of more structured conformations, possibly dsRNA (Fig. 5g). Both sets of transcripts displayed similar GC content, indicating that the lower MFE is not merely the consequence of a higher GC content (median %GC=49.1 in non-enriched control, 48.9% in enriched dsRNAs) (Fig. 5h). Interestingly, the dsRNA-enriched transcripts were longer than the controls (2866 vs 1894 median nucleotides) (Fig. 5i). The main contributors for the lower MFE in dsRNA-enriched genes were the 3′ UTR (untranslated regions) and coding sequence (CDS), and a significant increase in the proportion of intronic reads was found in the dsRNA-enriched transcripts (Fig. 5j, k). In agreement with the proposed roles for TEs in contributing to dsRNA formation in mammalian cells, and their enrichment in both 3′ UTR and intronic regions14, we found that the enriched dsRNAs contain significantly more TEs per gene, suggesting that TE-sequences contribute to dsRNA formation in ESCs (Fig. 5l). TE-enriched classes were both DNA and retrotransposons, from the SINE (B1, B2 and B4) and LTR (ERV-MaLR) class (Fig. 5m and Supplementary Fig. 5A).

These findings indicate that ESCs provide a permissive environment for dsRNA accumulation. However, this permissiveness is broken upon introduction of the dsRNA sensor MDA5, which activates the type I IFN response after dsRNA recognition.

Who interferes with pluripotency: IFN production or IFN signalling?
Our results led us to hypothesise that IFN production is responsible for the dysregulation of pluripotency in ESCs. To test this possibility, critical genes for IFN production were depleted, and tested if MDA5 was no longer capable of perturbing pluripotency markers expression (Fig. 6a, b and Supplementary Fig. 6A). Only when IRF3 and MAVS were depleted, and no IFN response was activated, pluripotency genes remained unchanged after MDA5 induction. On the other hand, depletion of IRF7 mimicked control cells, still showing decreased expression of pluripotency genes with MDA5 (Fig. 6c). This suggests that ultimately the activation of IRF3 and possibly the production of IFN is responsible for the loss in the expression of pluripotency genes.

To further confirm that pluripotency dysregulation is specific to MDA5 and IFN production, we measured the expression of pluripotency genes in MDA5-ΔCTD cells and MAVS overexpressing cells, which do not activate IFN production on its own. Neither of these resulted in perturbation of pluripotency genes nor activation of neuronal marker expression (Fig. 6d–f and Supplementary Fig. 2C). It therefore seems that the production of IFN is responsible for perturbing the pluripotency-associated gene expression programme. Finally, to test whether exogenous type I IFN stimulation could lead to a similar perturbation, ESCs were incubated with different amounts of IFN-β. This resulted in increased expression of ISGs such as Oas1, Stat1 and Isg15 by RT-qPCR, however, no significant changes in the expression of the pluripotency factors Nanog, Klf4 or Pou5f1 was observed (Fig. 6g). To further corroborate that IFN production rather than IFN signalling is responsible for the developmental dysregulation, we compared the expression of pluripotency genes in cells expressing MDA5 in the presence of Ruxolitinib. This compound is a well-studied JAK-STAT inhibitor that blocks intracellular IFN signalling60. As expected, no ISGs were induced in the presence of Ruxolitinib, despite a significant decrease in the expression of pluripotency genes (Fig. 6h). All these results together revealed that IRF3-mediated production of IFN, and not the sensing and signalling by exogenous IFN, is responsible for perturbing the pluripotency-associated gene expression programme.

Suppression of the dsRNA sensing pathway is conserved in other vertebrates
Both mouse and human ESCs fail to produce IFNs when challenged with dsRNA, suggesting that the suppression of this pathway is conserved across mammals32. Jawed vertebrates, including fish and birds, also use the type I IFN response as the primary mechanism to defend against viruses. In zebrafish, the adaptive immune system is not well developed until 4 weeks post fertilisation, making it a useful model to study the innate antiviral response in the early stages of development61–63. Besides this, zebrafish are a powerful in vivo tool to assess the long-term effects of an active IFN response in early development on body plan formation. First, we confirmed that the expression of most RLR-signalling genes, including RIG-I, MDA5 and MAVS were also developmentally regulated in zebrafish (Fig. 7a)64.

Next, we investigated whether this developmentally regulated expression correlated with the ability of zebrafish to respond to dsRNA. To this end, 1-cell embryos were injected with the dsRNA analogue poly(I:C), and IFN production was measured by in situ hybridisation with probes against the ISG Trim25. Only after 24h did we observe an increase in Trim25 staining (Fig. 7b) which was confirmed by analysing the expression of both Trim25 and Isg20 by RT-qPCR (Fig. 7c). DsRNA stimulation resulted in deleterious phenotypes in ~50% of the injected embryos. Some embryos exhibited impaired head development (class 1), while others showed other defects including issues with trunk development (class 2) (Fig. 7d, e). These results suggest that activating the response to dsRNA in early zebrafish development can also have negative consequences.

We also investigated whether the dsRNA-induced phenotype was independent from IFN signalling and ISG production, as we previously observed in ESCs. To this end, dsRNA-injected zebrafish were treated with 25μM of Ruxolitinib. The treatment resulted in decreased induction of Trim25 (Fig. 7f). However, a similar number of fish displayed the dsRNA-induced phenotype with or without Ruxolitinib treatment (Fig. 7g). These results suggest that the dsRNA-induced developmental phenotype in zebrafish is independent of IFN signalling, similarly to what we observed in ESCs (Fig. 6g, h). Finally, we also tested the consequences of IFN production in the differentiation ability of mESCs, as a model of in vitro development. IFN activation resulted in failure of mESCs to efficiently silence the expression of the major pluripotency factors during the first hours of differentiation, and premature expression of differentiating markers, such as Hand1 (Supplementary Fig. 7A).

All these together suggest that the ability to synthesise IFNs in zebrafish is developmentally regulated and, like mice, activation of the dsRNA-mediated responses during early embryonic stages can have severe developmental consequences. We hypothesise that silencing of the dsRNA-sensing pathway is conserved across jawed vertebrates.

Methods
Ethics statement
All experiments performed with zebrafish at the University of Granada comply with national and European Community regulations for the use of animals in experimentation and were approved by the ethical committees of the University of Granada and the Junta de Andalucía.

Mouse and zebrafish datasets and analysis
Sequencing data from mouse ESC differentiation studies were obtained from the Geo Expression Omnibus data repository44, GSE127741) and ArrayExpress data collection43, E-MTAB-4904). FastQ files were extracted from the repositories and quality assessed using FASTQC (v0.11.8). Cutadapt (v3.4) was used to trim the sequences using the command [cutadapt -a AGATCGGAAGAG -A AGATCGGAAGAG -j 0 -m 50 -o tr.$fq1 -p tr.$fq2 $fq1 $fq2 > tr.$sample.log]. Sequences were not collapsed within each sample, and duplicates were retained. Alignment of reads (--very-sensitive -p 24 --no-mixed --no-discordant --no-unal -x $db −1 tr.$fq1 −2 tr.$fq2 2» $bt2log) to the REL104 reference set was performed using hisat2 (v2.2.1). Alignment rates were typically 90% of input reads per sample. Alignments were stored in sorted, indexed BAM files. Raw tag counts per gene in each sample were extracted from the BAM files using the gtf file in combination with featureCounts function of Rsubread (featureCounts(files=bamfiles, annot.ext=‘Mus_musculus.104.gtf.gz’, isGTFAnnotationFile=TRUE, countMultiMappingReads=TRUE, allowMultiOverlap=TRUE, isPairedEnd=TRUE, nthreads=24, minOverlap=10)), and were normalised as part of the DESeq2-based groupwise comparisons: no conversions (e.g. reads per kb of gene, reads per million, etc.) were performed. The zebrafish dataset was obtained from the Geo Expression Omnibus data repository64, GSE106430). Raw reads were aligned to the complete assembly of zebrafish genome GRCz11 and quantified according to NCBI RefSeq D. rerio Annotation Release 106 (June 2, 2017) using STAR v2.7.11b85 and quantified according to NCBI RefSeq D. rerio Annotation Release 106 (June 2, 2017) using featureCounts function of Subread v2.0.8.

Cells and differentiation protocols
The mouse ESC line v6.5 was obtained from ThermoFisher (MES1402) and cultured in Dulbecco’s modified Eagle Medium (DMEM, ThermoFisher) supplemented with 15% heat-inactivated foetal calf serum (ThermoFisher), 1X Minimal essential amino acids (ThermoFisher), 2mM L-glutamine, 103 µ/ml of LIF (Stemcell Technologies) and 50µM 2-mercaptoethanol (ThermoFisher). ESCs were grown on plates coated with 0.1% gelatine, detached using 0.05% Trypsin (ThermoFisher). The mouse microglial BV-2 cells were cultured in Dulbecco’s modified Eagle Medium (DMEM, ThermoFisher) supplemented with 10% heat-inactivated foetal calf serum (ThermoFisher) and 2mM L-glutamine. Cells were incubated at 5% CO2 at 37°C.

Mouse ESCs were differentiated using hanging droplets and retinoic acid to induce embryoid body formation and differentiation as described before45. In brief, ESCs were washed and resuspended in culture media without LIF. Individual drops of 20µl cell suspension are pipetted on non-culture treated Petri dish lids and cultured upside down for 48h. During that time embryoid bodies (EBs) will form and are collected into non-culture treated plates for another 48h before adding retinoic acid to the media (250nM). After 7 days of further incubation, the EBs are moved to gelatine-coated cell culture treated plates where they will attach and are ready for subsequent experiments.

Differentiated cells were collected and RNA was extracted using Tri-reagent (Sigma-Aldrich) and processed for real time PCR as described below.

For all doxycycline (dox) treatments, 250ng/ml concentration was used. For poly(I:C) treatment, differentiated cells were collected and seeded in 24 well plates, transfected with 2.5µg poly(I:C) using lipofectamine 2000 and incubated for 6h before collecting the cells in Tri-reagent. For exogenous IFN-β treatment, differentiated cells were collected and seeded in 24 well plates and incubated with 10 or 100pg/ml of mouse IFN-β (Bio-techne, 8234-MB-010) for 4h before collecting the cells in Tri-reagent. Ruxolitinib (Invivogen, tlrl-rux-3) treatment was performed at 5μM concentration. To assess the impact of the IFN response during differentiation a modified differentiation protocol was used. V6.5 and MDA5-overexpressing cells were pre-treated with doxycycline for 8h before a single-cell suspension of 5×105 cells/ml was prepared in medium without LIF and in the presence or absence of doxycycline. Twenty µl drops were pipetted on the inside of the lid of a 10 cm petri dish and incubated upside-down at 37°C, 5% CO2 for 24h. The embryoid bodies were consequently washed from the lids, pelleted by centrifugation and RNA extracted using Tri-reagent.

RNA extraction and real time PCR
Total RNA was extracted using Tri-reagent (MilliporeSigma), and cDNA was synthesised using M-MLV (Promega) or the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in accordance with the manufacturer’s instructions. qPCR reactions were performed using GoTaq qPCR master mix (Promega) or qPCR BIO sygreen (PCR Biosystems) using previously published primers (Supplementary Data 5) on a QuantStudio 5 (ThermoFisher) or Azure Cielo 6 (Azure biosystems). Data was analysed using Quantstudio Design & Analysis software or Azure Cielo Manager software. Differences were analysed by single factor ANOVA to calculate significant differences amongst comparisons, followed by an F-test for variance and appropriate two-tailed t-test *p-val≤0.05, **p-val≤0.01, ***p-val≤0.001.

Western blot assay
Whole-cell extracts were collected in RIPA buffer containing protease inhibitors followed by sonication and centrifugation for clarification of extracts. Extracts were quantified using the BCA assay, mixed with LDS sample buffer (Invitrogen) and Sample reducing agent (Invitrogen) and run on 4–12% Bis-Tris precast gels (ThermoFisher). Proteins were transferred to nitrocellulose membrane using semi-dry (iBlot2, ThermoFisher) or wet transfer (mini blot module, ThermoFisher) according to the manufacturer’s recommendations. Membranes were blocked for 1h at room temperature in PBS-T (0.1% Tween-20) and 5% milk powder before overnight incubation at 4°C with primary antibody. Antibodies used were anti-mouse HRP (7076, CST), anti-Rabbit HRP (7074, CST), anti-mouse 680RD (925-68070, LICORbio), anti-mouse 800CW (926-32210, LICORbio), anti-rabbit 680RD (925-68071, LICORbio), anti-rabbit 800CW (926-32211, LICORbio) anti-FLAG (M2, Merck), anti-tubulin (CP06, Calbiochem), anti-GAPDH (CB1001, Merck), anti-Nanog (ebioMLC-51, Invitrogen), anti-KLF4 (4038, CST), anti-IRF3 (12A4A35, BioLegend), anti-IRF7 (MA5-52511, Invitrogen) and anti-MAVS (sc365334, Santa Cruz). Proteins bands were visualised using ECL (Pierce) on a Bio-Rad ChemiDoc imaging system or Li-cor imaging system (LICORbio), depending on the secondary antibody used. Protein bands were quantified using ImageJ (v1.53q) software and expression levels calculated normalised to tubulin.

J2 flow cytometry and Immunofluorescence
Cells were dissociated using 0.05% Trypsin, washed in PBS and resuspended in FACS buffer (PBS with 1% FBS). Cells were pelleted and resuspended in Fixation buffer (420801, BioLegend) and incubated for 15’ at 4°C, washed twice with Intracellular Staining Permeabilisation Wash Buffer (421002, BioLegend) and stained overnight with the anti-dsRNA antibody J2 (English & Scientific Consulting) in Intracellular Staining Permeabilisation Wash Buffer. After washing, cells were incubated with anti-mouse Alexa Fluor 647 (ThermoFisher) for 1h at room temperature and washed three times with Intracellular Staining Permeabilisation Wash Buffer before finally resuspending the cells in FACS buffer. Cells were analysed using a MACS Quant analyser 10 (Miltenyi), and data were processed using FlowJo software (Treestar).

For immunofluorescence, cells were seeded on gelatine coated culture slides (Corning Falcon), washed twice with PBS with 2% FBS, fixed in fixation buffer (420801, BioLegend) and incubated for 15’ at 4°C. The fixed cells were washed three times with Intracellular Staining Permeabilisation Wash Buffer (421002, BioLegend) and stained overnight with the anti-dsRNA antibody J2. Cells were washed three times with Intracellular Staining Permeabilisation Wash Buffer and stained with anti-mouse FITC (406001, BioLegend) and incubated at RT for 1h. After washing three times with Intracellular Staining Permeabilisation Wash Buffer, cells were stained with DAPI in antifade mounting media (Vectashield, Vector laboratories) and visualised using a fluorescence microscope (Axio, Carl Zeiss).

Stable cell lines
Plasmids containing the sequences of mouse MDA5 (GE-healthcare, MMM1013-202762875) and 3xFLAG (pcDNA3.1-3xFLAG) were used to amplify Gibson assembly fragments to construct the MDA5 sequence with a 3xFLAG tag at the N-terminal end. A plasmid containing the MAVS sequence (GE-healthcare, MMM1013-202764911) was used as template to directly amplify the MAVS open reading frame with specific restriction sites and a N-terminal FLAG tag (for oligonucleotides, see Supplementary Data 5). The amplified and digested fragments were ligated into the pLenti-GIII-EF1a plasmid for constitutive expression and/or purification, and the pCW57-MCS1-P2A-MCS2 (89180, Addgene) plasmid, in which were placed the hPGK promoter with the EF1a promoter for optimal expression in mESCs. For the ΔCTD mutant of MDA5, a reverse primer containing a stop codon and specific restriction site was used to amplify a fragment that was ligated into the same pLenti-GIII-EF1a and pCW57-MCS1-P2A-MCS2 plasmids. Verified plasmids containing the genes of interest were transfected in mESCs using Lipofectamine 2000 and selected with the appropriate antibiotic. Clonal cell lines were isolated, expanded and tested for expression by qRT-PCR and Western blot. ShRNA sequences targeting IRF3, IRF7 and MAVS were designed using the Broad institute design tool (https://portals.broadinstitute.org/gpp/public/gene/search) and ligated into the pLKO.1 plasmid (24150, Addgene). After sequencing to verify integrity, the plasmids were transfected into mESCs using Lipofectamine 2000 and selected with the appropriate antibiotic. Clonal lines were isolated, expanded and tested for knockdown of the target gene by RT-qPCR (Supplementary Data 5).

MDA5 purification and poly(I:C) pulldown
HEK293T cells were transfected with the pLenti-GIII-EF1a plasmids expressing FLAG-MDA5 and FLAG-ΔCTD using Lipofectamine 2000, following manufacturer’s instructions. Cells were collected after 48h and resuspended in lysis buffer (20mM HEPES-KOH pH 7.9, 100mM KCl, 0.2mM EDTA, 0.5mM DTT, 0.2mM PMSF, 5% glycerol, supplemented with Complete Protease inhibitors), followed by sonication. Lysates were incubated with pre-washed FLAG Dynabeads (Sigma-Aldrich, M8823) overnight. Beads were washed five times with lysis buffer, supplemented with 1M KCl, with an additional final wash with TBS (50mM Tris pH 7.4, 150mM NaCl). Purified MDA5 proteins were eluted using FLAG peptide (Sigma-Aldrich, F4799) to a 100µg/ml final concentration in TBS buffer, following manufacturer’s instructions. Eluted proteins were quantified by Coomassie staining and nanodrop. For poly(I:C) pulldowns, poly(C)-agarose beads (Sigma-Aldrich, P9827) were washed twice with 50mM Tris pH 7.0, 200mM NaCl, and resuspended in 50mM Tris-pH 7.0, 50mM NaCl. Poly(I) (Sigma-Aldrich, P4154) was dissolved in 50mM Tris-pH 7.0, 150mM NaCl to a concentration of 2mg/ml. One part of poly(C) beads and two parts of poly(I) solution were mixed and rocked for 1h at 4°C to form double-stranded RNA (dsRNA). Next, beads were pelleted, washed and resuspended in 50mM Tris-pH 7.0, 150mM NaCl. Poly(C) beads only were used as a control (single-stranded RNA, ssRNA). For pulldowns, equilibrated dsRNA-beads were resuspended in binding buffer (50mM Tris-pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40). A volume of beads slurry was combined with an equal volume of MDA5 protein (300µg of WT or ΔCTD), plus protease/phosphatase inhibitors and 25 U RNAsin/ml. Reactions were incubated at 4°C with gentle rotation for 1h, washed three times with binding buffer and resuspended in SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) loading buffer for downstream analyses by western blot.

Total RNA high-throughput sequencing and analysis
Total RNA was extracted from cells using Tri-reagent and the quality assessed on the Agilent 2100 Electrophoresis Bioanalyser Instrument (Agilent, G2939AA). RNA was quantified using the Qubit 2.0 Fluorometer (ThermoFisher, Q32866). DNA contamination was quantified using the Qubit dsDNA HS assay kit and confirmed to be <6% (ThermoFisher, Q32854). Libraries for total RNA sequencing were prepared using the NEBNext Ultra 2 Directional RNA library prep kit (Illumina, E7760) and the NEBNext rRNA Depletion kit (Human/Mouse/Rat) (Illumina, E6310) according to the provided protocol. Sequencing was performed on the NextSeq 2000 platform (Illumina, 20038897) using NextSeq 2000 P3 Reagents (200 Cycles, 2x100bp) (20040559). Raw sequences from the two sequencing runs were combined prior to being quality assessed using FASTQC (v0.11.8), and html format outputs generated. Cutadapt (v3.4) was used to trim the sequences with the following command: cutadapt -a AGATCGGAAGAG -A AGATCGGAAGAG -j 0 -m 50 -o tr.$fq1 -p tr.$fq2 $fq1 $fq2 > tr.$sample.log. Sequences were not collapsed within each sample, and duplicates were retained. The murine genome (release 104, “REL104”) and gtf formatted annotation was obtained by ftp from ensembl (ftp.ensembl.org/pub/release-104/fasta/mus_musculus). Alignment of reads (--very-sensitive -p 24 --no-mixed --no-discordant --no-unal -x $db −1 tr.$fq1 −2 tr.$fq2 2» $bt2log) to the REL104 reference set was performed using hisat2 (v2.2.1. Alignments were stored in sorted, indexed BAM files. Raw tag counts per gene in each sample were extracted from the BAM files using the gtf file in combination with featureCounts function of Rsubread (featureCounts(files = bamfiles, annot.ext = ‘Mus_musculus.104.gtf.gz’, isGTFAnnotationFile = TRUE, countMultiMappingReads = TRUE, allowMultiOverlap = TRUE, isPairedEnd = TRUE, nthreads=24, minOverlap=10)), and were normalised as part of the DESeq2-based groupwise comparisons: no conversions were performed. Pairwise comparisons of sample groups were performed using the DESeq2 Bioconductor package, with alpha set at 1; as each sample group had 3 replicates, all groups were used. logFC values were shrunk using the ashr model in lfsShrink() and a significance threshold of p<0.01 (adjusted) was applied. Gene ontology (biological process) analysis was performed using the ClueGo plugin in the Cytoscape package86. Cell type predictions were performed using the Mouse Gene Atlas within the Enrichr package (https://maayanlab.cloud/Enrichr/)87. Transcription factor predictions were done using the LOF dataset within the ShinyGO (0.81) package49.

Chromatin enrichment for proteomics (ChEP)
ChEP was conducted based on previously established protocols with slight modifications54,88. Briefly, 1×10⁷ cells were crosslinked using 1% formaldehyde at room temperature (RT) for 10min, followed by quenching with 0.25M glycine. The cells were lysed in 1ml ice-cold cell lysis buffer (25mM Tris pH 7.4, 0.1% Triton X-100, 85mM KCl, and 1× Roche protease inhibitor) and centrifuged at 2300×g for 5min at 4°C to isolate the nuclei. After discarding the cytoplasmic fraction, the pellet was resuspended in 500μl SDS buffer (50mM Tris pH 7.4, 10mM EDTA, 4% SDS, and 1× Roche protease inhibitor) and incubated at RT for 10min. The solution was diluted to 2ml using urea buffer (10mM Tris pH 7.4, 1mM EDTA, and 8M urea) and centrifuged at 16,100×g for 30min at RT, repeating the step once. Subsequently, the pellet was resuspended with 2ml SDS buffer and centrifuged at 16,100×g for 30min at RT. The resulting protein pellet was dissolved in 250μl storage buffer (10mM Tris pH 7.4, 1mM EDTA, 25mM NaCl, 10% glycerol, and 1× Roche protease inhibitor) and sonicated using a Bioruptor (Diagenode) for five cycles (30s on/off) to enhance solubility. Protein concentration was quantified via a Qubit protein assay (Invitrogen). For mass spectrometry (MS) analysis, 100μg of protein extract was de-crosslinked at 95°C for 30min. Each time point included two biological replicates.

For proteomic analyses, de-crosslinked chromatin protein extracts (100μg) were precipitated by adding 10 volumes of ice-cold 100% acetone and incubated at −20 °C for 15min. The samples were centrifuged at 16,000×g for 10min in a pre-cooled centrifuge. The resulting protein pellets were washed with 500μl ice-cold acetone, centrifuged, and the acetone was discarded. The pellets were resuspended in 100μl urea buffer (8M urea, 100mM Tris-HCl, pH 8.0, 10mM DTT) and incubated at room temperature for 20min. Subsequently, 10μl iodoacetamide was added, and the mixture was incubated in the dark for 15min at room temperature. For protein purification and on-bead digestion89, 5μl of pre-washed carboxyl magnetic beads (Thermo Scientific) and 154μl binding buffer (100% acetonitrile, ACN) were added, followed by incubation for 18min at room temperature. The beads were washed twice with 180μl of 70% ethanol and once with 200μl of 100% ACN. Proteins were eluted and digested on beads in 100μl of ammonium bicarbonate buffer (ABC) containing 3μl trypsin (Promega) at 37°C for 2h. After removing the beads, the solution was incubated at 37°C overnight to complete digestion. The digested peptides were fractionated using strong anion exchange into three fractions90: flow-through (FT), pH 8 elution, and pH 2 elution. The pH 8 and pH 2 fractions were combined. Finally, peptides were enriched and desalted using Stage-Tip91 desalting before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a Orbitrap Exploris 480 instrument.

Proteomic data analysis was carried out following previously described protocols with minor modifications88. Raw mass spectrometry data were processed using MaxQuant software92 (v2.4.2) with searches performed against the mouse UniProt database (downloaded June 2017). Default parameters were used, with LFQ, iBAQ, and the “match between runs” feature enabled. Protein identification was validated against a decoy database generated within MaxQuant. Biological replicates were grouped to identify differentially expressed proteins. Data were filtered for three valid values in at least one group. Missing values were imputed using default settings in Perseus93 (v2.0.11) based on the assumption that they were not detected because they were under or close to the detection limit. Differentially enriched proteins were determined using a two-tailed Student’s t-test (P<0.05) with a fold change threshold of >2. Differential proteins at 8, 24, and 48h were excluded from the differential proteins at 0h. Downstream analyses, including data visualisation, were performed using R software (version 4.4.1).

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and analysis
The ATAC (mitochondrial DNA-remove) protocol was adapted from previously established methods with modifications94,95. A total of 10,000 cells were collected, washed with 1mL of ice-cold PBS containing EDTA-free protease inhibitor, and centrifuged at 500×g for 5min at 4°C. The supernatant was completely removed, and the cell pellet was resuspended in 20μL of transposase reaction mixture, consisting of 1× Tagment DNA buffer, 1μL Tn5 enzyme, and 0.02% digitonin. The transposition reaction was carried out at 37°C for 30min with continuous agitation. Following transposition, DNA was purified and the ATAC-seq library was constructed using the KAPA Hyper Prep Kit (KAPABiosystems, 07962363001) and barcoded with NEXTflex DNA barcodes (Integrated DNA Technologies). Paired-end sequencing of the library was performed on an Illumina NextSeq 500 platform.

Sequence reads were processed using the seq2science pipeline (v1.2.2)96. Paired-end reads were trimmed with fastp v0.23.2 with default options. Genome assembly GRCm39 was downloaded with genomepy 0.16.1. Reads were aligned with bwa-mem2 v2.2.1 with options ‘-M’. Afterwards, duplicate reads were marked with Picard MarkDuplicates v3.0.0. Before peak calling, paired-end info from reads was removed with seq2science so that both mates in a pair get used. Bam files were randomly sampled down to the reads number as the same as the lowest reads sample. Peaks were called with macs2 v2.2.7 with options ‘--shift 75 --extsize 200 –nolambda’. Differential peaks were calculated by Diffbind (v3.12.0). Peak tracks and heatmaps were generated by Deeptools (v3.5.5). Peaks were annotated by ChIPseeker v1.38.0. GO analysis was conducted by clusterProfiler v4.10.1.

Double-stranded RNA immunoprecipitations and analyses
Double stranded RNA was isolated using the dsRNA specific antibody J2 (English & Scientific Consulting). For each replicate, cells from one confluent 10 cm plate were collected, washed twice with cold PBS and lysed in 1ml IP-buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100) supplemented with protease inhibitors (S8820, Sigma-Aldrich). For each 1ml of lysate, 10μl DNAse (M6101, Promega) and 2μl RNAse inhibitor (N2111, Promega) was added and incubated on ice for 30min after which the lysate was spun at 13k rpm, 4°C for 15’ and the supernatant collected. To couple J2 antibodies to magnetic protein G beads, 50μl of protein G slurry (88848, Thermo Scientific) was washed three times with cold IP buffer, resuspend in 100μl IP buffer with 5μg of J2 antibody and mixed at 4°C while rotating for 3h. After incubation, the beads were washed three times with IP buffer, mixed with the lysate and incubated at 4°C for 4h while rotating. Beads were washed three times 5min at 4°C while rotating and tubes were changed once to prevent RNA carryover. After the last wash, the beads were resuspended in 250μl of 0.3M NaAc (pH 5.2) and RNA was extracted using Trireagent LS according to the manufacturer’s protocol. Purified RNA was sent for sequencing and processed according to the protocol described in the ‘Total RNA high-throughput sequencing and analysis’ section. Sequencing reads were processed, aligned and counted according to the protocol described in the ‘Total RNA high-throughput sequencing and analysis’ section. Normalisation (TMM method) and differential expression analysis between J2 IP and RNAseq gene counts were performed using the glmQLFit function from edgeR v. 4.2.2 R package97,98. Transcripts were classified as enriched dsRNAs when J2 IP vs RNAseq log2FC>0.75 and false discovery rate <0.05. The non-enriched transcripts were used as ‘non-enriched control’. Sequences of processed transcripts (exons only) were obtained from genome reference GRCm39 using Ensembl genome annotation (release 104). Transcript lengths and lengths of 5′ UTR, CDS and 3′ UTR were also obtained from Ensembl release 104. Minimum free energy values were calculated via RNAfold from the ViennaRNA package v. 2.7.0 with default parameters99. Transposable elements (TE) annotation was obtained from the Hammel Lab (https://labshare.cshl.edu/shares/mhammelllab/www-data/TEtranscripts/TE_GTF/). Overlap between TE and exon coordinates was performed with findOverlaps function from the GenomicRanges v. 1.56.2 R package. Differences between RNA population features were calculated with one-tail Wilcoxon Test as implemented in wilcox_test function from rstatix v0.7.2 package.

5’ azacytidine treatment
WT mESCs (v6.5) were grown in the presence of different concentrations of 5’ azacytidine (A2385, Sigma-Aldrich) for 48h to allow for several replication cycles and depletion of DNA methylation. Cells were collected, RNA extracted and analysed as described above.

Zebrafish experiments
Zebrafish wild-type strains AB/Tübingen/TAB (AB/Tu/TAB) were gifts from the zebrafish facilities at LARCEL (Málaga, Spain) and MPI-CBG (Dresden, Germany). The fish were maintained and bred under standard conditions100. Wild-type zebrafish embryos were obtained by natural mating of AB/Tu and TAB zebrafish of mixed ages (5–18 months). Pairs were randomly selected from 20 males and 20 females and zebrafish embryos were staged using the morphological criteria described before101. After fertilisation, eggs were collected and one-cell stage embryos were microinjected with 2nL of 1μg/μL poly(I:C) in PBS71. Control embryos were injected with the same volume of PBS. For Ruxolitinib experiments, embryos were first injected with poly(I:C) as described above and then incubated with 25μM Ruxolitinib diluted in E3 medium 1-h post injection (as in ref. 102). Control embryos were incubated in E3 containing an equivalent concentration of DMSO. Phenotypes were monitored at 24h post fertilization (hpf).

For whole-mount in situ hybridisation, a partial region of Trim25 was cloned into pGEM-T using specific primers (Supplementary Data 5), linearised with NcoI and in vitro transcribed using SP6 (P1085, Promega) to generate an antisense trim25 probe, which was labelled with digoxigenin. Zebrafish embryos were fixed, hybridised, and stained using NBT/BCIP103, imaged and analysed using ImageJ. Total RNA was isolated from 50 to 60 embryos using Trizol (Invitrogen) according to the manufacturer’s protocol. One microgram of RNA was DNase I treated and purified via phenol/chloroform extraction. cDNA was synthesised using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and used for qPCR (GoTaq qPCR Mix, Promega). Embryos of the same age were used to compare the differences in developmental defects due to their treatments. Embryos were classified based on severity of the development defects, where embryos were assigned class 1 defects if embryos exhibited failure to develop the head and class 2 defects if there were defects in trunk development.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The type I interferon (IFN) system is critical in vertebrate somatic cells to provide protection against viruses. One key intracellular innate receptor, MDA5, senses the presence within the cytosol of double-stranded RNA (dsRNA) of viral or endogenous source and triggers the IFN system. However, stem cells are endowed with an attenuated IFN response and therefore rely on alternative antiviral mechanisms for their protection. How and why the IFN system is attenuated in stem cells is not well understood.\nIn this study, the authors show that the silencing of MDA5 expression in mouse embryonic stem cells (mESCs) is crucial to prevent aberrant IFN response activation and this is essential to maintain pluripotency. First, the authors found that mESCs do not express crucial proteins from the RIG-I-like receptors (RLRs) pathway and found that MDA5 and IRF7 both become gradually expressed upon differentiation. They then use an inducible MDA5-expressing system to address the impact of MDA5 expression on mESCs. They show, by RNA-seq, that expression of MDA5 causes a development disruption with upregulation of genes involved in nervous system development while genes involved in maintaining mESCs features (e.g. pluripotency factors) were downregulated. They further find that these changes at the expression levels are accompanied by alterations of chromatin accessibility and chromatin-associated proteins in regions controlling nervous system functions, pluripotency and immune responses. The authors then show that mESCs express endogenous dsRNA in contrast to differentiated cells and that the expression of MDA5 induces the upregulation of several interferon-stimulated genes (ISGs). Further analyses show that the MDA5-mediated activation of the IRF3 pathway and not the IFN-mediated signalling pathway impact on the pluripotency programme of the cells. Finally, the importance of their findings was further shown by using a zebrafish model, in which the premature activation of the IFN response resulted in development defects.\nThis study is well-written and the findings are important and very interesting for the field of innate immunity and stem cell biology as it provides further key insights into the incompatibility between the IFN system and pluripotency. While this a comprehensive and thorough study, some findings need to be strengthened by further experimental evidence in order to fully support the conclusions of this study. The authors should therefore address the following comments:\nMajor comments:\nFigure 4a: the authors nicely show that the induction of Flag-tagged MDA5 in mESCs results in the activation of a type I IFN response based on the upregulation of ifnb gene as well as three ISGs (tnf, isg15 and ifitm1). While the upregulation of these 3 ISGs is compelling at the transcription level, it is unclear how robust is this response and whether the levels of ISG mRNAs observed upon MDA5 expression lead to readily detectable levels of the corresponding proteins as observed in viral infections or in autoinflammatory contexts. Given that the activation of the IFN response upon induction of MDA5 in mESCs constitutes a central point of the study, the authors should test whether the activation of the IFN response upon MDA5 expression leads to upregulation of ISGs at the protein level by western blot and/or ELISA/Multiplex assay kit (e.g. Meso Scale Discovery biomarker assay). As a general comment, this study monitors changes of gene expression by measuring mRNA levels, the authors should verify whether these changes occur at the protein level as well by analysing the level of some ISGs-encoded proteins and pluripotency factors.\nFigure 4c: the authors aimed to address whether the observed upregulation of ISGs upon the expression of MDA5 is caused by its well-established ability to sense endogenous dsRNA and consequently induce an IFN response. For this the authors engineered mESCs stable cell lines expressing a MDA5 mutant lacking its C-terminal domain known to be the region that recognises target RNAs. They showed that the induction of a truncated mutant of MDA5 did not lead to an upregulation of ISGs and also didn’t cause changes in the levels of pluripotency factors (Nanog, Klf4, Pou5f1 in Figure 5b). Given that the induction of the full-length MDA5 protein was shown to induce an IFN response, the authors conclude that MDA5’s ability to sense dsRNA is required to then initiate the signalling cascade and induce an IFN response. Yet, it is key to ensure that the expression level of the truncation mutant is comparable to the full-length to then compare their effects on the IFN pathway and the pluripotency state of the cells. To rule that the truncation mutant is simply too lowly expressed compared to full-length MDA5, the authors should provide a western blot comparing the expression levels of FLAG-MDA5 delta CTD versus FLAG-MDA5 WT at different time point post doxycycline treatment (0, 8, 24, 48 hrs post dox) and assess that the mESCs clones used express similar levels of FLAG-tagged MDA5. If it is indeed the case, the induction of ifih1, ifnb1, isg15 and ifitm1 as well as the pluripotency factors should be monitored by qRT-PCR and/or western blot within the same experiment to compare any changes at each time point caused by the mutant versus the full-length version of MDA5.\nFigure 5a and b: To further address whether the observed upregulation of ISGs and the alteration of pluripotency upon MDA5 expression in mESCs is caused by the IFN induction pathway, the authors depleted essential components of the pathway using shRNA-mediated silencing in their inducible FLAG-MDA5 mESCs clone. They found that the upregulations of ifnb1 and tnf as well as the downregulation of pluripotency factors were abolished upon silencing of IRF3 and MAVS, but not by knockdown of IRF7. These results suggest that the pathway leading to the production of IFN is necessary for the alterations observed in mESCs upon MDA5 expression. However, the authors showed in fiugrue5a that the induction of ifih1 (encoding MDA5) expression is clearly attenuated especially in mESCs expressing shRNA targeting IRF3 where the induction upon doxycycline treatment appears to be of 2-fold versus 6 fold in cells expressing shRNA control (figure 5a, top left panel with Ifih1 level of expression assessed by qRT-PCR). Can the authors rule out that the loss of ISGs upregulation and pluripotency factors downregulation are not caused by a diminished induction of MDA5 in mESCs expressing shRNA against either IRF3 or MAVS? To circumvent this cofounding effect, the authors could select a mESCs clone expressing shIRF3 and efficiently knocking down IRF3 but in which the level of MDA5 is induced at a similar level than in cells expressing the shRNA control.\nFigure 5d: the authors nicely show that activation of the IFN signalling by treatment of mESCs with recombinant IFN-b doesn’t lead to pluripotency dysregulation. Given all the changes the authors observed at the chromatin level upon MDA5 induction, the authors should strengthen their findings by analysing the importance of the IFN signalling in the same cellular context analysed throughout their study, e.g. in mEScs in which MDA5 expression is induced. For this, the authors inhibit the type I IFN receptor IFNAR with, for instance, the inhibitor Ruxolitinib and test whether the observed changes in ISGs as well as in the pluripotency factors do still occur upon MDA5 induction.\nMinor comments:\n- The current title “Double stranded RNA sensing drives interferon silencing in early\ndevelopment” is misleading. The study doesn’t show that the sensing of endogenous dsRNA by MDA5 and the subsequent activation of the IFN response causes the silencing of the pathway. The authors should change their title to reflect more accurately their main findings.\n- Line 78-80: “There is increasing evidence that transposable elements (TEs)…constitute a natural source of dsRNA in cells (6-8). Please mention the correct references here as the cited studies (Brownell et al., 2014; Xu et al. 2014); Smith et al., 2012) show the direct binding of IRF3 transcription factor to the promoter of some specific ISGs but do not look at the production of dsRNA from TEs.\n- Line 158-161: The authors investigated the mechanism responsible for the silencing of Ifih1 gene in mESCs and tested the contribution of the Polycomb repressive complex 1 (PRC1). For this, the authors depleted one component of PRC1, RING1B, in a Ring1A -/- ESCs. The authors should clarify in their manuscript the rationale behind using Ring1A-/- cells to deplete RING1B and mention that both RING1A and RIG1B are functionally redundant Polycomb proteins and that the inactivation of the PRC1 requires the depletion of both proteins. Additionally, in extended data figure 1, the authors should show that RINGB1 depletion is indeed degron-depleted by, for instance, western blot to support their conclusion that PRC1 is not required for Ifih1 silencing.\n- Please ensure that legends on the x-axis are present for each graphs (e.g legends on the x-axis in figures 1d, 4a, 4c are missing): and that the same labelling of x- and y-axis is used for similar analyses throughout the manuscript.\n- Please ensure that the figures legends are clear and provide all the necessary information to understand the data shown. For example, the figure legends of Figure 5b and c doesn’t specify the cells and the treatment (dox) used.\n- Figure 1c: It is unclear how the statistical analyses show a significant difference in ifnb1 and cxcl10 gene expression upon treatment of mESCs with poly (I:C) while there is no upregulation shown in the graphs. The authors should perhaps consider using a log scale on the y-axis to show the differences occurring in mESC gene expression upon poly (I:C) treatment.\n- Please display the size reference (molecular weight in kDalton) on all the western blot analyses shown in this study.\n- Line 713, figure legend of Figure 2c: the authors mean “two Ifih1-inducible” clones not genes, please clarify the figure legend.\n- It is unclear what the extended figure 3c shows, please provide more information in the figure legend. In the figure legend, it is mentioned that the second clone doesn’t harbour the same deletion. If it is an example of inaccessible region found after ATAC-seq analysis, why the authors chose a region that is not found as inaccessible in the second MDA5-expressing clone of this study? What do the authors mean by “second clone”, is it a the second MDA-5 expressing clone or a WT mESCs (v6.5 mESCs)? Please clarify accordingly in the corresponding legend. Shouldn’t the control be an ATAC-seq analysis on MDA5 expressing clone before treatment with doxycycline?\n- PLease provide more clarification in the text and the figure legends of the data shown shown in figure 3h and 3i. In figure 3h, can the authors explain why they found an enrichment of DDX58 (encoding RIG-I) bound to chromatin upon MDA5 expression? To the knowledge of this reviewer, RIG-I is localised in the cytosol and is not associated to the chromatin. Is figure 3I showing the group of genes for which there is a positive correlation between early RNA-seq data and late ChEP-MS data? Many/most of the ISGs are not binding chromatin, why would an upregulation of innate immune genes correlate with these same proteins enriched with chromatin? Is the group of innate immune genes that are upregulated and whose encoded proteins is enriched to chromatin upon MDA5 induction (genes that are part of the \"innate immune system\" and cytokine signalling in immune system\" in figure 3I) mainly consisting of transcription factors/repressors known to be involved in the control of immune genes? Please provide more details about the genes/proteins shown in figure 3I.\n- Figure 4b: a faint band of MDA5 is detectable in the ssRNA IP, while a much robust band is observed upon dsRNA IP, which is consistent with the known ability of MDA5 to bind dsRNA. Yet, it is currently unclear if the blot shown in figure 4b is a single membrane or if there are two blots, one for the ssRNA pulldowns and the other for the dsRNA pulldowns. Can the authors clarify please? To assess that MDA5 preferentially bind dsRNA and that this binding is indeed lost when the C-terminal domain of MDA5 is lacking, the pulldowns of ssRNA and dsRNA with the two versions of MDA5 should be loaded on the same blot to then have the same exposure time and allow proper comparison.\n- Figure 4f. The authors used as “Non-enriched control” the whole transcriptome obtained by RNA-seq. Can the authors explain why they didn’t use an IgG control for this experiment to remove from their analysis any non-specific binding of RNA to the beads and antibody? Could the labelling be changed to “whole transcriptome” vs “anti-dsRNA-IP”? Finally, does “protein coding” RNAs include intronic regions?\n- Please clarify the extended figure 6a: WT and MDA5-expressing clones were differentiated into embryoid bodies for 24 hours. Was the doxycycline treatment to induce MDA5 also performed for 24 hours and therefore were both the differentiation induction and doxycycline treatments induced at the same time? While the expression of MDA5 leads to the upregulation of differentiation markers (Sox2 and Hand1), can the authors explain why it also leads to upregulation of pluripotency markers (Nanog and Klf4) while they showed earlier in their study that MDA5 expression results in a decrease of pluripotency factors?\n- The concentration of doxycycline used in the study is not mentioned in material and methods or in figure legends.\n- Line 115: typo, change “supress” by “suppress”.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The manuscript by Macias et al. aims to dissect the mechanism in which the disable on type I interferon production in mESCs is due to the lack of dsRNA sensor MDA5. Ectopically expression of MDA5 sensitizes the mESC for interferon signaling activation which results in the damage of their stemness. Moreover, the high level of endogenous dsRNAs in mESC, which is normally absent in somatic cells, are responsible for triggering MDA5-mediated type I interferon pathway activation, leading to the upregulation of both interferon and ISGs. It has been shown that cells at early developmental stage including ESC and iPSC display some uniqueness on innate immune responses and modulation, including the resistance on viral infection with intrinsic ISG upregulation and others. The complexity of the cell destinies along differentiation and their different responses to IFN leave many open questions in this field. This manuscript adds some information for understanding the complicated scenario further, but the data is a little bit preliminary which hardly support a strong piece of theory add up to the existing understanding.\nHere are a few of my comments in specific:\n1. Figure 2, especially Figure 2c-d claimed that MDA5 overexpression inducted a “neuronal-like” gene expression profile of ESC. As the treatment for cell clone screening easily affect the differential potential of ESCs, this experiment lacks necessary control (such as a MDA5 mutant or truncation) to prove that the expression profile change is indeed driven by MDA5, not a non-specific consequence of ectopic expression of proteins per se.\n2. In Figure 3, the author showed that expression of MDA5 in ESCs results in innate immune responses, mainly rely on GO analysis from sequencing data. qPCR for signature genes of activated pathways, and the biochemistry approach for detecting the protein level of key players are also required to support the conclusion.\n3. In Figure 4m, the author claimed that enriched dsRNAs harbour significantly more TEs per gene. As TEs with inverted sequences are capable to form dsRNA duplex, to further check which type of TEs enriched in ESCs specifically is suggested.\n4. In Figure 5d, exogeneous type I IFN stimulation led to upregulation of multiple ISGs such as Oas1, Stat1 and Isg15, and the author showed that sensing of exogenous IFN was unrelated to pluripotency perturbation. Since MDA5 also acting downstream of IFN signaling as an ISG, it will be worth to check if its expression could be induced by IFN treatment, the upregulation of which should impact the pluripotency according to the ectopic expression result.", "reviewer_4": "The manuscript by Witteveldt et al reports that the sensing of dsRNA by MDA5 during embryonic development is specifically silenced in order to allow for necessary gene expression patterns for proper development and differentiation. Specifically, the authors found that MDA5 is silenced early in embryogenesis, and that expression of MDA5 at this early stage leads to dysregulation of key gene expression patterns, driven by IRF3. Authors claim that MDA5 specifically requires silencing because ESCs inherently have high dsRNA that activates MDA5. They extend their findings to a zebrafish model, where they show similar inhibition of dsRNA sensing early in development.\nThis manuscript presents an interesting new discovery that MDA5 is specifically inhibited early in embryogenesis, and proposes that gene transcription driven by IRF3, rather than simply a toxic effect of interferon-driven gene expression, drives the deleterious effects. This novel finding is in theory quite attractive, but the manuscript has several points that need to be addressed to support the conclusions being made.\n1. The biological model used for many of the experiments consists of an inducible MDA5 expression system. My concern with this model is that overexpression of MDA5 in many cells induces non-specific activation of the receptor. I imagine that the goal of this model is to express a biologically relevant amount of MDA5, that would be similar to the expression of MDA5 in the same cell type but at a later point of differentiation, e.g. ESCs at Day 6 of differentiation vs Day 0. Have the authors compared, either by WB or by RT-qPCR, the expression of MDA5 in their overexpressed early ESC to expression of later-stage differentiated cells, to see if their levels of MDA5 expression are comparable? A WB to show expression at protein levels would be particularly useful. Otherwise, it may be hard to draw conclusions about a natural state of ESCs being particularly prone to MDA5 activation due to, as they claim, high dsRNA levels, vs nonspecific effects from high MDA5 overexpression.\n2. In Fig 2c, authors report on transcriptional changes resulting from MDA5 expression in ESCs. However, it is surprising that there were no obvious IFN/ISG expression patterns reported. One would expect that activation of MDA5 would lead to these sorts of transcriptional changes. Could author look at these specifically?\n3. In Fig 3h, the authors do report an “innate immune signalling” pattern associated with MDA5 expression. Why was this not apparent in earlier datasets? Is this conclusion drawn from highly processed data points a valid analytical approach?\n4. Authors state that in Extended Data 4 “ISG induction was also confirmed on the RNAseq data obtained from the two independent MDA5 overexpressing clones”, but this is not apparent. Can authors better indicate which of these genes are known ISGs? Furthermore, are the genes upregulated by PCR in Fig 4a not significantly differentially expressed in the RNAseq data? If so, why not?\n5. I am unsure about the claim the authors make that a delta-CTD MDA5 results in no dsRNA binding. In fact, one of the papers cited by the authors (Peisley et al 2011 PNAS) specifically reports that the CTD is involved in cooperative filament assembly (i.e. MDA5 oligomerisation for activation) and does NOT affect dsRNA binding capacity. Therefore, use of a delta-CTD MDA5 is not really differentiating from an MDA5 that cannot bind to endogenous dsRNA, but rather one that has loss-of-function in downstream signalling. The data presented in their PolyIC pulldown is not well explained, and does not go far enough as to convince differently than what was previously reported. Rather, use of an MDA5 mutant that does not have dsRNA-binding capacity would be more appropriate. These have been reported by others:\nhttps://pmc.ncbi.nlm.nih.gov/articles/PMC5502429/\nhttps://pubmed.ncbi.nlm.nih.gov/30449722/\n6. The authors claim that ESC have inherently high dsRNA loads, which is the driver of MDA5 activation and thus why these cells specifically require silencing of MDA5. However, the J2 flow cytometry on which this point strongly relies requires better controls. The comparison of the ESC is done to an unrelated cell type (a microglia). As the authors state in their manuscript, there is high variability in dsRNA levels between cell types. Since the authors claim that ESCs in particular have high dsRNA, and that this is concordant with low MDA5, and that as ESCs differentiate this MDA5 expression increases, then is there a concordant drop in dsRNA load in these more differentiated cells? Performing the J2 staining on control cells that are closer to the ESC would be more a convincing comparison. Furthermore, the authors could consider doing J2 confocal microscopy as an additional experiment.\n7. In Fig 4i, could the authors further analyse the data to report on e.g. introns vs exons, or 3/5’UTRs? TEs are often found in these regions, and increased pulldown of e.g. intron-containing mRNA, or extended 3’UTR would support their theory.\n8. The data reported in Fig 4g-m is quite interesting, but requires better description of the analysis methods.\n9. For Fig 5a, WBs are required to demonstrate knockdown of the target proteins.\n10. The data in Fig 6 is a critical part of the author’s proposal that activation of IRF3 is the driver for the gene dysregulation effects in ESCs. The treatment with IFNb is one way to test their theory, however, is it possible that a 4 hr treatment with IFNb may not be long enough to show an effect on the tested pluripotency genes? It is not clear in the legends how this compares to the length of dox treatment and the timing of MDA5 upregulation/activation. A more definitive approach could be to treat these MDA5-expressing cells with IFNAR-blocking antibodies to allow the activation of IRF3 and expression of genes, but block the IFN activity. Alternatively, authors could use IFNAR2-KO cells.\n11. The zebrafish model presented in Fig 7 is an excellent development by the authors, but it could be further explored in order to strengthen their claims. For example, authors could include treatments similar to what was done for cells in Fig 6, e.g. exogenous IFNb treatment compared to dsRNA stimulation, and inclusion of an IFNAR2-KO or of IFNAR-blocking antibodies. The experiments as they stand cannot really disentangle the previously well-established damaging effects of interferon during early development vs the effects resulting from activation of MDA5."}, "author_response": "We thank the reviewer for their constructive comments. The suggested experiments have strengthened the conclusions of our study.\nMajor comments:\n1. Figure 4a: the authors nicely show that the induction of Flag-tagged MDA5 in mESCs results in the activation of a type I IFN response based on the upregulation of ifnb gene as well as three ISGs (tnf, isg15 and ifitm1). While the upregulation of these 3 ISGs is compelling at the transcription level, it is unclear how robust is this response and whether the levels of ISG mRNAs observed upon MDA5 expression lead to readily detectable levels of the corresponding proteins as observed in viral infections or in autoinflammatory contexts. Given that the activation of the IFN response upon induction of MDA5 in mESCs constitutes a central point of the study, the authors should test whether the activation of the IFN response upon MDA5 expression leads to upregulation of ISGs at the protein level by western blot and/or ELISA/Multiplex assay kit (e.g. Meso Scale Discovery biomarker assay). As a general comment, this study monitors changes of gene expression by measuring mRNA levels, the authors should verify whether these changes occur at the protein level as well by analysing the level of some ISGs-encoded proteins and pluripotency factors.\nWe have now tested IFN- protein production by ELISA with both inducible and constitutively expressed MDA5 in ESCs (new Fig 4B). Inducible MDA5 results in IFN- protein production, slightly over the detection limit (~ 8.5-9 pg/ml). Constitutively expressed MDA5 results in a more pronounced production of IFN- (~13 pg/ml). Although the constitutively expressed form leads to a more obvious immune phenotype, these cells cannot be indefinitely propagated, as they eventually silence MDA5 expression after few passages. We have also compared constitutively expressed MDA5 with the CTD mutant. In agreement with our previous results, overexpression of the CTD mutant of MDA5 does not result in IFN- protein production (new Fig 4B, lower panel).\nWe have also tried to assess the expression of some ISGs by western blot, including RIG-I and OASL. RIG-I increase was detected by mass spectrometry analyses (Fig 3i). However, due to problems with the sensitivity of the antibodies, the generally low expression of ISGs, the short-lived nature of MDA5 expression and the attenuated response of ESCs to IFNs (see Fig 1h), these were, unfortunately, undetectable by western blot.\nWe have also measured the protein levels of key pluripotency markers, including Nanog and KLF4, both of which showed marked decreases in expression during induction of MDA5 (new Fig 3e).\n2. Figure 4c: the authors aimed to address whether the observed upregulation of ISGs upon the expression of MDA5 is caused by its well-established ability to sense endogenous dsRNA and consequently induce an IFN response. For this the authors engineered mESCs stable cell lines expressing a MDA5 mutant lacking its C-terminal domain known to be the region that recognises target RNAs. They showed that the induction of a truncated mutant of MDA5 did not lead to an upregulation of ISGs and also didn’t cause changes in the levels of pluripotency factors (Nanog, Klf4, Pou5f1 in Figure 5b). Given that the induction of the full-length MDA5 protein was shown to induce an IFN response, the authors conclude that MDA5’s ability to sense dsRNA is required to then initiate the signalling cascade and induce an IFN response. Yet, it is key to ensure that the expression level of the truncation mutant is comparable to the full-length to then compare their effects on the IFN pathway and the pluripotency state of the cells. To rule that the truncation mutant is simply too lowly expressed compared to full-length MDA5, the authors should provide a western blot comparing the expression levels of FLAG-MDA5 delta CTD versus FLAG-MDA5 WT at different time point post doxycycline treatment (0, 8, 24, 48 hrs post dox) and assess that the mESCs clones used express similar levels of FLAG-tagged MDA5. If it is indeed the case, the induction of ifih1, ifnb1, isg15 and ifitm1 as well as the pluripotency factors should be monitored by qRT-PCR and/or western blot within the same experiment to compare any changes at each time point caused by the mutant versus the full-length version of MDA5.\nWe have now compared the expression levels of WT and CTD MDA5, (new Figure 4d and 4e, respectively) and observed comparable levels of expression on both protein and transcript levels. Using the same samples, we have further confirmed that only WT MDA5 is capable of inducing IFN and ISG expression (new Figure 4e), but not the CTD mutant. In agreement, only WT MDA5 perturbs the expression of pluripotency factors and differentiation markers (new Figure 6d and Suppl. Figure 2b)\n3. Figure 5a and b: To further address whether the observed upregulation of ISGs and the alteration of pluripotency upon MDA5 expression in mESCs is caused by the IFN induction pathway, the authors depleted essential components of the pathway using shRNA-mediated silencing in their inducible FLAG-MDA5 mESCs clone. They found that the upregulations of ifnb1 and tnf as well as the downregulation of pluripotency factors were abolished upon silencing of IRF3 and MAVS, but not by knockdown of IRF7. These results suggest that the pathway leading to the production of IFN is necessary for the alterations observed in mESCs upon MDA5 expression. However, the authors showed in fiugrue5a that the induction of ifih1 (encoding MDA5) expression is clearly attenuated especially in mESCs expressing shRNA targeting IRF3 where the induction upon doxycycline treatment appears to be of 2-fold versus 6 fold in cells expressing shRNA control (figure 5a, top left panel with Ifih1 level of expression assessed by qRT-PCR). Can the authors rule out that the loss of ISGs upregulation and pluripotency factors downregulation are not caused by a diminished induction of MDA5 in mESCs expressing shRNA against either IRF3 or MAVS? To circumvent this cofounding effect, the authors could select a mESCs clone expressing shIRF3 and efficiently knocking down IRF3 but in which the level of MDA5 is induced at a similar level than in cells expressing the shRNA control.\nWe have now compared the induction of FLAG-MDA5 in all shRNA lines by western blot and confirm that there are no apparent differences in protein expression levels (new Fig. 6b). In addition, we are including new western blot analyses to confirm successful depletion of IRF3, IR7 and MAVS (new Fig. 6a).\n4. Figure 5d: the authors nicely show that activation of the IFN signalling by treatment of mESCs with recombinant IFN-b doesn’t lead to pluripotency dysregulation. Given all the changes the authors observed at the chromatin level upon MDA5 induction, the authors should strengthen their findings by analysing the importance of the IFN signalling in the same cellular context analysed throughout their study, e.g. in mEScs in which MDA5 expression is induced. For this, the authors inhibit the type I IFN receptor IFNAR with, for instance, the inhibitor Ruxolitinib and test whether the observed changes in ISGs as well as in the pluripotency factors do still occur upon MDA5 induction.\nAs suggested by the reviewer, we have blocked IFN signalling using Ruxolitinib. Despite blocking IFN signalling, the expression of pluripotency genes is still significantly down regulated upon MDA5 induction (new Fig. 6h). We have also performed a similar experiment in zebrafish (new Fig 7f-g). Both experiments suggest that IFN signalling is not required to observe the developmental phenotypes.\nMinor comments:\n- The current title “Double stranded RNA sensing drives interferon silencing in early development” is misleading. The study doesn’t show that the sensing of endogenous dsRNA by MDA5 and the subsequent activation of the IFN response causes the silencing of the pathway. The authors should change their title to reflect more accurately their main findings.\nWe agree with the reviewer and we have therefore changed the title to: ‘Silencing of double stranded RNA sensing is required for early embryonic development’\n- Line 78-80: “There is increasing evidence that transposable elements (TEs)…constitute a natural source of dsRNA in cells (6-8). Please mention the correct references here as the cited studies (Brownell et al., 2014; Xu et al. 2014); Smith et al., 2012) show the direct binding of IRF3 transcription factor to the promoter of some specific ISGs but do not look at the production of dsRNA from TEs.\nThere references have now been corrected to: Chiappinelli (Cell 2015; https://pmc.ncbi.nlm.nih.gov/articles/PMC4556003/), Roulois (Cell 2015; https://pmc.ncbi.nlm.nih.gov/articles/PMC4556003/), Mehdipour (Nature 2020; https://pmc.ncbi.nlm.nih.gov/articles/PMC4556003/).\n- Line 158-161: The authors investigated the mechanism responsible for the silencing of Ifih1 gene in mESCs and tested the contribution of the Polycomb repressive complex 1 (PRC1). For this, the authors depleted one component of PRC1, RING1B, in a Ring1A -/- ESCs. The authors should clarify in their manuscript the rationale behind using Ring1A-/- cells to deplete RING1B and mention that both RING1A and RIG1B are functionally redundant Polycomb proteins and that the inactivation of the PRC1 requires the depletion of both proteins. Additionally, in extended data figure 1, the authors should show that RINGB1 depletion is indeed degron-depleted by, for instance, western blot to support their conclusion that PRC1 is not required for Ifih1 silencing.\nThe PRC1 complex is a E3 ubiquitin-ligase that monoUb H2A at K119. Although depleting the catalytic subunit of the complex RING1B is enough to observe depletion of ubH2A, the double knock-out of RING1B and its homolog RING1A is required to totally erase this mark (https://www.sciencedirect.com/science/article/pii/S1534580704003673?via%3Dihub). The functional redundancy of these two genes has now been highlighted in the text. The RING1B-degron dataset is published and it has been extensively used. For more details see: (https://www.nature.com/articles/s41556-024-01493-w ; https://www.nature.com/articles/s41594-021-00661-y ; https://www.sciencedirect.com/science/article/pii/S2211124719317140?via%3Dihub)\n- Please ensure that legends on the x-axis are present for each graphs (e.g legends on the x-axis in figures 1d, 4a, 4c are missing): and that the same labelling of x- and y-axis is used for similar analyses throughout the manuscript.\nCorrected. Please note that the legends in 1d, 4a and 4c were not missing. These data are not represented as fold-change (FC) but normalised to Gapdh or Actb.\n- Please ensure that the figures legends are clear and provide all the necessary information to understand the data shown. For example, the figure legends of Figure 5b and c doesn’t specify the cells and the treatment (dox) used.\nThese have now been corrected. The concentration of doxycycline used is 250ng/ml. This information has been added to material and methods.\n- Figure 1c: It is unclear how the statistical analyses show a significant difference in ifnb1 and cxcl10 gene expression upon treatment of mESCs with poly (I:C) while there is no upregulation shown in the graphs. The authors should perhaps consider using a log scale on the y-axis to show the differences occurring in mESC gene expression upon poly (I:C) treatment.\nCorrected. These data have now been represented using a log scale (new Fig 1G).\n- Please display the size reference (molecular weight in kDalton) on all the western blot analyses shown in this study.\nMolecular weight sizes have now been included in all blots.\n- Line 713, figure legend of Figure 2c: the authors mean “two Ifih1-inducible” clones not genes, please clarify the figure legend.\nThis has now been corrected\n- It is unclear what the extended figure 3c shows, please provide more information in the figure legend. In the figure legend, it is mentioned that the second clone doesn’t harbour the same deletion. If it is an example of inaccessible region found after ATAC-seq analysis, why the authors chose a region that is not found as inaccessible in the second MDA5-expressing clone of this study? What do the authors mean by “second clone”, is it a the second MDA-5 expressing clone or a WT mESCs (v6.5 mESCs)? Please clarify accordingly in the corresponding legend. Shouldn’t the control be an ATAC-seq analysis on MDA5 expressing clone before treatment with doxycycline?\nApologies for the confusion. After performing ATAC-seq analyses, we realised that the MDA5 clone (1) has a small genomic deletion, probably caused by insertion of the doxycycline-inducible form of MDA5. Also, our analysis suggests that the MDA5 clone (2), which was also used for the time course RNA-seq analyses, does not contain this deletion. All these together suggest that the defects observed in the developmental programme upon MDA5 induction are not indirectly caused by the microdeletion, since both clones behave similarly. These observations have now been included in the figure legend.\nWith regards the ATAC control, we have analysed the ATAC-seq data using both normalisation to WT (v6.5) ESCs and non-dox MDA5 ESCs. We noticed some leaky expression of MDA5 in the absence of doxycycline, making the changes induced upon MDA5 expression less obvious.\n- Please provide more clarification in the text and the figure legends of the data shown shown in figure 3h and 3i. In figure 3h, can the authors explain why they found an enrichment of DDX58 (encoding RIG-I) bound to chromatin upon MDA5 expression? To the knowledge of this reviewer, RIG-I is localised in the cytosol and is not associated to the chromatin. Is figure 3I showing the group of genes for which there is a positive correlation between early RNA-seq data and late ChEP-MS data? Many/most of the ISGs are not binding chromatin, why would an upregulation of innate immune genes correlate with these same proteins enriched with chromatin? Is the group of innate immune genes that are upregulated and whose encoded proteins is enriched to chromatin upon MDA5 induction (genes that are part of the \"innate immune system\" and cytokine signalling in immune system\" in figure 3I) mainly consisting of transcription factors/repressors known to be involved in the control of immune genes? Please provide more details about the genes/proteins shown in figure 3I.\nThe ChEP-MS protocol performs an enrichment for chromatin-bound proteins to facilitate their detection. However, this protocol will still detect abundant non-chromatin bound proteins. In support of this, the manuscript that originally developed this protocol detected 3,522 proteins after chromatin enrichment, which agrees with this protocol being an enrichment step rather than being a pure ‘only chromatin-bound’ fraction (https://pmc.ncbi.nlm.nih.gov/articles/PMC4300392/). This protocol has also been used to compare chromatin-enriched factors in naïve vs primed hPSCs. A total of 4,576 proteins were detected after enrichment (https://www.nature.com/articles/s41556-022-00932-w). We hypothesise that the induction of RIG-I is enough to detect it after chromatin-enrichment, but it is not necessarily directly associated with chromatin. The same rationale would apply for other regulators of the IFN response that are not known to associate with chromatin, such as Vimentin (see Fig 3i-j). However, it is important to note that RIG-I has been shown to be able to localise in the nucleus in some settings (PMID: 35598407).\nInnate immune genes that correlate at the RNA and protein level (Fig 3i-j) are Hck, Myo1c, Dusp6, Sugt1, Anxa2, Ddx58, Pnp2 and Pnp (annotated in Reactome, the R-MMU-168249 Innate Immune system). Using GO Biological pathways, we obtained similar genes: Hck, Ddx58, Epha2, Myo1c, Vim, Foxp1, Trim35, Anxa3, Anxa2, Ass1, Cdkn1c and Pnp2.\nTo confirm that the innate immune activation is also observed in the RNA-seq datasets, we have included the infection/immune pathways obtained by KEGG analyses (Suppl Fig 3e).\n- Figure 4b: a faint band of MDA5 is detectable in the ssRNA IP, while a much robust band is observed upon dsRNA IP, which is consistent with the known ability of MDA5 to bind dsRNA. Yet, it is currently unclear if the blot shown in figure 4b is a single membrane or if there are two blots, one for the ssRNA pulldowns and the other for the dsRNA pulldowns. Can the authors clarify please? To assess that MDA5 preferentially bind dsRNA and that this binding is indeed lost when the C-terminal domain of MDA5 is lacking, the pulldowns of ssRNA and dsRNA with the two versions of MDA5 should be loaded on the same blot to then have the same exposure time and allow proper comparison.\nThis is a single membrane. The uncropped blot has now been included in Suppl. Fig 8. Apologies for forgetting to include the uncropped version in the initial submission.\n- Figure 4f. The authors used as “Non-enriched control” the whole transcriptome obtained by RNA-seq. Can the authors explain why they didn’t use an IgG control for this experiment to remove from their analysis any non-specific binding of RNA to the beads and antibody? Could the labelling be changed to “whole transcriptome” vs “anti-dsRNA-IP”? Finally, does “protein coding” RNAs include intronic regions?\nOur initial experiments included IgG as a negative control, however no RNA was recovered and library preparation failed. Therefore, we decided to use the input to calculate the enrichment for each transcript in the dsRNA IP. This approach has been successfully used in the past by Tebaldi’s team for dsRNA identification (https://www.sciencedirect.com/science/article/pii/S2666166721000733 and https://pubmed.ncbi.nlm.nih.gov/32497523/). The enrichment over input approach identifies two groups of transcripts; first, those enriched in the dsRNA IP, and second, those not enriched. These are the two subgroups that have been consistently used in Figure 5 (non-enriched control vs enriched dsRNA).\nSince we have used transcripts, these did not contain introns. However, we agree with the reviewer that this is an important distinction we should address and have now reanalysed the datasets and quantified the number of intronic reads in input vs IP. We have identified that the dsRNA IPs contain, proportionally, more reads mapping to introns that input, suggesting that introns are also prone to generate dsRNA.\n- Please clarify the extended figure 6a: WT and MDA5-expressing clones were differentiated into embryoid bodies for 24 hours. Was the doxycycline treatment to induce MDA5 also performed for 24 hours and therefore were both the differentiation induction and doxycycline treatments induced at the same time? While the expression of MDA5 leads to the upregulation of differentiation markers (Sox2 and Hand1), can the authors explain why it also leads to upregulation of pluripotency markers (Nanog and Klf4) while they showed earlier in their study that MDA5 expression results in a decrease of pluripotency factors?\nESCs, with or without MDA5, were differentiated for 24 in the presence of doxycycline, we have clarified this in the legend (Suppl. Fig 7). There are two different defects caused by MDA5 induction. First, MDA5 in ESCs causes the downregulation in the steady-state levels of pluripotency factors (Figure 3D-E). However, when ESCs are forced to differentiate (Suppl. Fig. 7), MDA5 impairs the normal silencing of the pluripotency factors, suggesting that it also interferes with differentiation. Therefore, we do not expect to observe a downregulation in the pluripotency factors in any of the conditions of the Suppl. Fig. 7 upon MDA5 induction, as none represents normally grown ESCs with MDA5.\n- The concentration of doxycycline used in the study is not mentioned in material and methods or in figure legends.\nThis has now been included in material and methods.\n- Line 115: typo, change “supress” by “suppress”.\nCorrected\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Macias et al. aims to dissect the mechanism in which the disable on type I interferon production in mESCs is due to the lack of dsRNA sensor MDA5. Ectopically expression of MDA5 sensitizes the mESC for interferon signaling activation which results in the damage of their stemness. Moreover, the high level of endogenous dsRNAs in mESC, which is normally absent in somatic cells, are responsible for triggering MDA5-mediated type I interferon pathway activation, leading to the upregulation of both interferon and ISGs. It has been shown that cells at early developmental stage including ESC and iPSC display some uniqueness on innate immune responses and modulation, including the resistance on viral infection with intrinsic ISG upregulation and others. The complexity of the cell destinies along differentiation and their different responses to IFN leave many open questions in this field. This manuscript adds some information for understanding the complicated scenario further, but the data is a little bit preliminary which hardly support a strong piece of theory add up to the existing understanding.\nHere are a few of my comments in specific:\n1. Figure 2, especially Figure 2c-d claimed that MDA5 overexpression inducted a “neuronal-like” gene expression profile of ESC. As the treatment for cell clone screening easily affect the differential potential of ESCs, this experiment lacks necessary control (such as a MDA5 mutant or truncation) to prove that the expression profile change is indeed driven by MDA5, not a non-specific consequence of ectopic expression of proteins per se.\nWe have now included the log2FC expression of several neuronal markers in both MDA5 clones using RNA-seq (new Figure 2e). We have also confirmed the induction of these neuronal markers by RT-qPCR, and confirmed that only WT MDA5, but not the CTD mutant, can induce their expression (new Suppl. Fig 2b).\n2. In Figure 3, the author showed that expression of MDA5 in ESCs results in innate immune responses, mainly rely on GO analysis from sequencing data. qPCR for signature genes of activated pathways, and the biochemistry approach for detecting the protein level of key players are also required to support the conclusion.\nWe have now included a new panel of ISGs induced by MDA5 in both individual clones by RNA-seq (new Suppl. Fig 4a). We have also confirmed their induction both at the transcript and protein level. For transcripts, we have performed RT-qPCR for a panel of ISGs during a time course of MDA5 induction (new Fig 4e). As a control, we have also confirmed that the CTDMDA5 mutant fails to induce these ISGs, as expected (new Fig 4e). At the protein level, we have confirmed that both inducible and constitutively expressed MDA5 in ESCs results in IFN- production by ELISA. Please see also response to reviewer 1, point 1.\n3. In Figure 4m, the author claimed that enriched dsRNAs harbour significantly more TEs per gene. As TEs with inverted sequences are capable to form dsRNA duplex, to further check which type of TEs enriched in ESCs specifically is suggested.\nWe have now performed a more detailed analysis and confirmed that retrotransposons are enriched in the dsRNA IP. These belong to the SINEs and LTRs class (new Fig 5m). More specifically, these belonged to the B1 (Alu), B2 and B4 families as well as ERVL-MaLR (new Suppl. Fig 5). Importantly, B1 (Alu) and B2 are the most enriched and also known to be inverted repeats.\n4. In Figure 5d, exogeneous type I IFN stimulation led to upregulation of multiple ISGs such as Oas1, Stat1 and Isg15, and the author showed that sensing of exogenous IFN was unrelated to pluripotency perturbation. Since MDA5 also acting downstream of IFN signaling as an ISG, it will be worth to check if its expression could be induced by IFN treatment, the upregulation of which should impact the pluripotency according to the ectopic expression result.\nWe have now confirmed that exogenous IFN cannot induce endogenous MDA5 expression (new Fig 1e). We conclude that MDA5 is silenced in ESCs and IFN stimulation is not enough to derepress its silencing.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Witteveldt et al reports that the sensing of dsRNA by MDA5 during embryonic development is specifically silenced in order to allow for necessary gene expression patterns for proper development and differentiation. Specifically, the authors found that MDA5 is silenced early in embryogenesis, and that expression of MDA5 at this early stage leads to dysregulation of key gene expression patterns, driven by IRF3. Authors claim that MDA5 specifically requires silencing because ESCs inherently have high dsRNA that activates MDA5. They extend their findings to a zebrafish model, where they show similar inhibition of dsRNA sensing early in development.\nThis manuscript presents an interesting new discovery that MDA5 is specifically inhibited early in embryogenesis, and proposes that gene transcription driven by IRF3, rather than simply a toxic effect of interferon-driven gene expression, drives the deleterious effects. This novel finding is in theory quite attractive, but the manuscript has several points that need to be addressed to support the conclusions being made.\n1. The biological model used for many of the experiments consists of an inducible MDA5 expression system. My concern with this model is that overexpression of MDA5 in many cells induces non-specific activation of the receptor. I imagine that the goal of this model is to express a biologically relevant amount of MDA5, that would be similar to the expression of MDA5 in the same cell type but at a later point of differentiation, e.g. ESCs at Day 6 of differentiation vs Day 0. Have the authors compared, either by WB or by RT-qPCR, the expression of MDA5 in their overexpressed early ESC to expression of later-stage differentiated cells, to see if their levels of MDA5 expression are comparable? A WB to show expression at protein levels would be particularly useful. Otherwise, it may be hard to draw conclusions about a natural state of ESCs being particularly prone to MDA5 activation due to, as they claim, high dsRNA levels, vs nonspecific effects from high MDA5 overexpression.\nAll our results lead us to hypothesise that overexpression of MDA5 induces the IFN response due to dsRNA recognition. First, overexpression of an MDA5 mutant that fails to bind dsRNA and oligomerise does not result in IFN activation, despite expression levels being similar to WT MDA5 (new Fig, 4d-e). Second, overexpression of the adaptor protein of MDA5, MAVS, that also aggregates its CARD domains for signalling transduction, does not result in IFN activation.\nWe agree with the reviewer that overexpression of MDA5 in other cell lines can also lead to signalling. However, we believe this signalling is specific, and caused by varying degrees of accumulation of dsRNAs in different cell types. Most established cell lines in laboratories are derived from tumours. Tumours tend to have alterations in epigenetic silencing, and increased expression of transposable elements, which could possibly lead to dsRNA formation (https://pubmed.ncbi.nlm.nih.gov/33888553/). For instance, HeLa or PA-1 cells have detectable levels of dsRNA as shown by immunofluorescence or flow cytometry (https://pubmed.ncbi.nlm.nih.gov/30046113/ and https://www.biorxiv.org/content/10.1101/2025.05.28.656609v1.full).\nWe have now compared the absolute expression levels of MDA5 in in vitro differentiated ESCs vs the induced MDA5 clones (1) and (2) after 8 hours of doxycycline by RT-qPCR. These results lead us to conclude that in our system there are no aberrantly high levels of overexpression of MDA5.\nMDA5 (Ifih1) transcript levels were compared after normalisation to Gapdh. Samples analysed are normal ESCs (ESCs) and in vitro differentiated ESCs (diff), compared to clones (1) and (2) of MDA5 expressing cells after 8 hours of doxycycline addition.\n2. In Fig 2c, authors report on transcriptional changes resulting from MDA5 expression in ESCs. However, it is surprising that there were no obvious IFN/ISG expression patterns reported. One would expect that activation of MDA5 would lead to these sorts of transcriptional changes. Could author look at these specifically? 3. In Fig 3h, the authors do report an “innate immune signalling” pattern associated with MDA5 expression. Why was this not apparent in earlier datasets? Is this conclusion drawn from highly processed data points a valid analytical approach? 4. Authors state that in Extended Data 4 “ISG induction was also confirmed on the RNAseq data obtained from the two independent MDA5 overexpressing clones”, but this is not apparent. Can authors better indicate which of these genes are known ISGs? Furthermore, are the genes upregulated by PCR in Fig 4a not significantly differentially expressed in the RNAseq data? If so, why not\nApologies if this part was not reported clearly. To resolve any confusion, we now have included graphs to represent the enrichment for infection/immune pathways obtained with the RNA-seq timecourse dataset (new Suppl Fig 3e). We have also included a new panel of ISGs induced by MDA5 in both individual clones by RNA-seq (new Suppl Fig 3f).\nAt the protein level, we have confirmed that both inducible and constitutively expressed MDA5 in ESCs results in IFN- production by ELISA (new Fig 4b). Please see also response to reviewer 1, point 1 and reviewer 2, point 2.\nIn addition, we have explored the potential for the changes in gene expression induced by MDA5 to be caused by type I IFN expression using the Interferome database https://interferome.org/interferome/home.jspx. From the 396 genes differentially expressed at t=4h, 269 are predicted to be regulated by type-I IFNs. From the 368 genes commonly dysregulated at t=8, 245 are predicted to be regulated by type I IFNs. Similarly, from the 248 genes dysregulated at t=24h, 152 are regulated by type I IFN.\nTo end, in our experience detecting the expression of IFN-and ISGs by RNA-seq in cell lines that are not IFN-proficient is challenging due to their generally low expression, which can lead to variability and non-significant enrichment. This is why we favour quantifying these genes by alternative more sensitive and reliable methods, such as RT-qPCR.\n5. I am unsure about the claim the authors make that a delta-CTD MDA5 results in no dsRNA binding. In fact, one of the papers cited by the authors (Peisley et al 2011 PNAS) specifically reports that the CTD is involved in cooperative filament assembly (i.e. MDA5 oligomerisation for activation) and does NOT affect dsRNA binding capacity. Therefore, use of a delta-CTD MDA5 is not really differentiating from an MDA5 that cannot bind to endogenous dsRNA, but rather one that has loss-of-function in downstream signalling. The data presented in their PolyIC pulldown is not well explained, and does not go far enough as to convince differently than what was previously reported. Rather, use of an MDA5 mutant that does not have dsRNA-binding capacity would be more appropriate. These have been reported by others:\nhttps://pmc.ncbi.nlm.nih.gov/articles/PMC5502429/\nhttps://pubmed.ncbi.nlm.nih.gov/30449722/\nWe would like to thank the reviewer for pointing out the complexity of the mode of binding of MDA5 to dsRNA. Both the paper mentioned by the reviewer, which uses cryo-EM of MDA5-dsRNA filaments (https://pubmed.ncbi.nlm.nih.gov/30449722/) as well as another paper published on the same year using a crystallography approach (https://pubmed.ncbi.nlm.nih.gov/23273991/) show that all domains of MDA5 (Hel1, Hel2i, Hel2 and CTD) interact with dsRNA. MDA5 forms a ring around the dsRNA molecule, and different regions from all domains are also required to oligomerise. Therefore, the ability to bind dsRNA and to oligomerise are intimately connected, and to our knowledge there are no known mutants that can provide a separation of function. For instance, the second paper mentioned by the reviewer (https://pubmed.ncbi.nlm.nih.gov/28606988/) describes a loss-of-function mutation in MDA5. The mutation is a single amino acid substitution in Hel1 (K365E), and the mutation is predicted to abolish dsRNA binding and oligomerisation. Quoting from the paper ‘Substitution with glutamic acid introduces a negative charge that is predicted to abolish the protein–nucleic acid interaction and MDA5 oligomerization (Fig. 2 C)… suggesting that the defect of this mutant lies in MDA5 dimerization or oligomerization upon binding or detecting RNA, although we did not test this further.\nUsing the same polyIC pulldown approach we used, this manuscript demonstrates that the point mutation abolished stable binding or oligomerisation to the dsRNA polyIC, thus resulting in defective interferon signalling.\nSimilarly, the CTD seems to be required for both dsRNA binding as well as oligomerisation. Although in isolation the CTD from MDA5 binds dsRNA less efficiently than the CTD from RIG-I and LGP2 (Figure 1C, https://pmc.ncbi.nlm.nih.gov/articles/PMC2719387/), removing the CTD from MDA5 decreases its binding affinity to dsRNA (Figure 1B and FigureS1B with helicase+CTD and Figure S1D with helicase only, https://pubmed.ncbi.nlm.nih.gov/22314235/). These results agree with most domains from MDA5 being required to initially bind dsRNA.\nSecond, despite the challenges of generating mutants that uncouple dsRNA binding from oligomerisation, it is important to note that oligomerisation can only happen on a dsRNA substrate, and not in the absence of RNA (https://pubmed.ncbi.nlm.nih.gov/30449722/, Figure S7C), suggesting that MDA5 can only trigger the IFN response upon dsRNA binding and oligomerisation (or filament formation). Finally, further confirming the critical role for oligomerisation in the discrimination of bona-fide MDA5 substrates, MDA5 has been shown to be capable of binding dsRNA, dsDNA and ssRNA in vitro, however, only binding to dsRNA is cooperative (multimeric), and capable of downstream signalling (Fig 1, https://pubmed.ncbi.nlm.nih.gov/22160685/).\nFor all these reasons, we believe that overexpression of the MDA5-CTD is a useful control to test if the negative impact of MDA5 on pluripotency requires dsRNA binding and oligomerisation. This control demonstrates that the defects observed are not an indirect consequence of overexpressing MDA5 in ESCs, but that this is mediated by dsRNA recognition, oligomerisation and signalling. We will be modifying the text to make this concept clearer.\nThe polyIC pulldown assay is used to test the ability of proteins to bind dsRNA (polyIC) vs ssRNA (polyC). This assay has been successfully used in the past to test MDA5 dsRNA binding ability (https://pubmed.ncbi.nlm.nih.gov/16116171/ or https://pubmed.ncbi.nlm.nih.gov/28606988/). This approach cannot discriminate if MDA5 is binding as a monomer or is oligomerising on the dsRNA. Considering the current literature, we predict that upon polyIC binding WT-MDA5 will be oligomerising, thus resulting in a strong pulldown with polyIC, while the CTD, which it is known to bind with less affinity to dsRNA and fails to oligomerise, will result in decreased pulled-down levels with polyIC, as we observe.\n6. The authors claim that ESC have inherently high dsRNA loads, which is the driver of MDA5 activation and thus why these cells specifically require silencing of MDA5. However, the J2 flow cytometry on which this point strongly relies requires better controls. The comparison of the ESC is done to an unrelated cell type (a microglia). As the authors state in their manuscript, there is high variability in dsRNA levels between cell types. Since the authors claim that ESCs in particular have high dsRNA, and that this is concordant with low MDA5, and that as ESCs differentiate this MDA5 expression increases, then is there a concordant drop in dsRNA load in these more differentiated cells? Performing the J2 staining on control cells that are closer to the ESC would be more a convincing comparison. Furthermore, the authors could consider doing J2 confocal microscopy as an additional experiment.\nTo address this point we have performed immunofluorescence with anti-dsRNA J2 antibody in ESC vs in vitro-differentiated ESCs. Quantification of dsRNA positive cells confirmed the results from the Flow Cytometry experiment that ESCs accumulates more endogenous dsRNA compared to IFN-competent cells (new Fig 5c-e).\n7. In Fig 4i, could the authors further analyse the data to report on e.g. introns vs exons, or 3/5’UTRs? TEs are often found in these regions, and increased pulldown of e.g. intron-containing mRNA, or extended 3’UTR would support their theory.\nWe have attempted to perform these analyses and we do not observed differences in enrichment between the different mRNA regions (5’UTR, CDS, 3’UTR). However, we had anticipated that this analysis would not be very informative. DsRNA IPs pull-down the whole transcript, which can contain both dsRNA and ssRNA regions. J2-based IPs do not provide positional information of which region is actually forming the dsRNA within the transcript. However, we have now calculated the density of intronic sequences and confirmed that dsRNA IPs are more enriched in intronic regions than input, confirming the suggestion of this reviewer.\n8. The data reported in Fig 4g-m is quite interesting, but requires better description of the analysis methods.\nMore details have been added, please see ‘double-stranded RNA immunoprecipitation and analyses’ section in material and methods.\n9. For Fig 5a, WBs are required to demonstrate knockdown of the target proteins.\nWe have now included WBs confirming the successful depletion of IRF3, IRF7 and MAVS (new Fig. 6a). We have also confirmed that all these depleted cell lines express similar levels of FLAG-MDA5 (new Fig. 6b).\n10. The data in Fig 6 is a critical part of the author’s proposal that activation of IRF3 is the driver for the gene dysregulation effects in ESCs. The treatment with IFNb is one way to test their theory, however, is it possible that a 4 hr treatment with IFNb may not be long enough to show an effect on the tested pluripotency genes? It is not clear in the legends how this compares to the length of dox treatment and the timing of MDA5 upregulation/activation. A more definitive approach could be to treat these MDA5-expressing cells with IFNAR-blocking antibodies to allow the activation of IRF3 and expression of genes, but block the IFN activity. Alternatively, authors could use IFNAR2-KO cells.\nWe have now tested this possibility using ruxolitinib, which is an inhibitor of IFN signalling. Despite blocking IFN signalling and ISG production, the expression of MDA5 still results in significant downregulation of the expression of pluripotency genes (new Fig. 6h). A similar approach has been used for zebrafish, see below.\n11. The zebrafish model presented in Fig 7 is an excellent development by the authors, but it could be further explored in order to strengthen their claims. For example, authors could include treatments similar to what was done for cells in Fig 6, e.g. exogenous IFNb treatment compared to dsRNA stimulation, and inclusion of an IFNAR2-KO or of IFNAR-blocking antibodies. The experiments as they stand cannot really disentangle the previously well-established damaging effects of interferon during early development vs the effects resulting from activation of MDA5.\nWe have tested if the dsRNA-induced phenotype is affected by blocking the IFN response with ruxolitinib. First of all, we tested if ruxolitinib is functional in zebrafish and used concentrations reported to drive differential susceptibility to viruses in zebrafish (25 uM; https://pubmed.ncbi.nlm.nih.gov/39861877/). We observed a reduction in the expression of the ISG Trim25 using ruxolitinib (new Figure 7f). This experiment suggest that blocking the IFN response using ruxolitinib does not rescue the dsRNA-induced phenotype (Fig 7g).\nWe also considered stimulating the fish with type I IFN, but unfortunately, zebrafish type I IFN is not commercially available to perform this type of assays."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All our comments have been thoroughly addressed with compelling experiments and additional supportive data, which have been incorporated into the manuscript.\nMinor comments:\n• -Figure 4b: Please display error bars as shown in other plots (e.g.figure 4a). Please also mention in the figure legend that the grey area depicts the threshold limit of the ELISA.\n• -Figures 4d, 4e: we thank the authors to confirm that the MDA5 full-length version (FLAG-MDA5 WT )and the C-terminal deletion mutant of MDA5 (FLAG-MDA5 delta CTD ) are expressed at similar levels by qRT-PCR (figure 4E). In figure 4D, the authors also confirm that the levels of protein of full-length MDA5 versus mutant at various times points are similar. These comparisons between protein levels can only be done if all samples are run in the same blots. In Supplementary figure 8.1d, we can see in the uncropped western blot that all the signals shown in Figure 4D are indeed derived from the same membrane and therefore the authors provided compelling evidence that the FLAG-MDA5 delta CTD and FLAG-MDA5 WT are indeed expressed at similar levels at both the RNA and protein level. The authors should specify in the figure legend that the two blots depicted in Figure 4D are signals from the same blot with the uncropped version shown in Supplementary Figure 8.1.\n• Please ensure for consistency that throughout the manuscript the axes’ labels are the same if the same analyses were performed. In Figure 7c, the y-axis is labelled as “expression levels (relative to gapdh)”, while in Figure 1g the y-axis is labelled with “normalised to gapdh” only.\n• Figure 6g: please specify which cells are used in the figure legend.\n• Figure 4d,6b,6e: the molecular weight references (in kDalton) are still missing on the western blots.\n• Figure 6f: perhaps add ” MAVS ESC” instead of “MAVS”in the legend present within the figure to be consistent with the labels present in figure 6d.\n• Figure 6h: the “w/o IFN” from the legend within the figure can be misleading as there is no IFN added in this experiment. This reviewer understands that the authors meant that there is no IFN signalling due to the presence of ruxolitinib but to avoid confusion please remove”w/o IFN” and leave “MDA (+dox +ruxo)” or perhaps add “MDA5 w/o IFN signalling (+dox +ruxo)”.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The manuscript by Witteveldt et al. reported that the sensing of dsRNA by MDA5 is silenced during embryogenesis. With quite a lot of new evidence being added in the revised version, the updated manuscript has fulfilled my requests regarding to the previous reviewing points.", "reviewer_4": "I have carefully read the authors' responses to all reviewer comments. I find that the authors have addressed all of my concerns, and I have no further comments to the manuscript in it's revised state."}, "author_response": "Point-by-point response to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nAll our comments have been thoroughly addressed with compelling experiments and additional supportive data, which have been incorporated into the manuscript.\nMinor comments:\n• -Figure 4b: Please display error bars as shown in other plots (e.g.figure 4a). Please also mention in the figure legend that the grey area depicts the threshold limit of the ELISA.\nError bars have been added and legend has been modified.\n• -Figures 4d, 4e: we thank the authors to confirm that the MDA5 full-length version (FLAG-MDA5 WT )and the C-terminal deletion mutant of MDA5 (FLAG-MDA5 delta CTD ) are expressed at similar levels by qRT-PCR (figure 4E). In figure 4D, the authors also confirm that the levels of protein of full-length MDA5 versus mutant at various times points are similar. These comparisons between protein levels can only be done if all samples are run in the same blots. In Supplementary figure 8.1d, we can see in the uncropped western blot that all the signals shown in Figure 4D are indeed derived from the same membrane and therefore the authors provided compelling evidence that the FLAG-MDA5 delta CTD and FLAG-MDA5 WT are indeed expressed at similar levels at both the RNA and protein level. The authors should specify in the figure legend that the two blots depicted in Figure 4D are signals from the same blot with the uncropped version shown in Supplementary Figure 8.1.\nLegend of figure 4d has been modified to reflect the reviewers suggestion.\n• Please ensure for consistency that throughout the manuscript the axes’ labels are the same if the same analyses were performed. In Figure 7c, the y-axis is labelled as “expression levels (relative to gapdh)”, while in Figure 1g the y-axis is labelled with “normalised to gapdh” only.\nAxis labels have been checked and modified when necessary to ensure a consistent nomenclature.\n• Figure 6g: please specify which cells are used in the figure legend.\nThe name of the cell line used in figure 6g has been added\n• Figure 4d,6b,6e: the molecular weight references (in kDalton) are still missing on the western blots.\nSize references have been added to the Western blots.\n• Figure 6f: perhaps add ” MAVS ESC” instead of “MAVS”in the legend present within the figure to be consistent with the labels present in figure 6d.\nNaming in Figure 6f has been modified\n• Figure 6h: the “w/o IFN” from the legend within the figure can be misleading as there is no IFN added in this experiment. This reviewer understands that the authors meant that there is no IFN signalling due to the presence of ruxolitinib but to avoid confusion please remove”w/o IFN” and leave “MDA (+dox +ruxo)” or perhaps add “MDA5 w/o IFN signalling (+dox +ruxo)”.\nWe have changed the name to the latter suggestion provided by the reviewer.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Witteveldt et al. reported that the sensing of dsRNA by MDA5 is silenced during embryogenesis. With quite a lot of new evidence being added in the revised version, the updated manuscript has fulfilled my requests regarding to the previous reviewing points.\nReviewer #4 (Remarks to the Author):\nI have carefully read the authors' responses to all reviewer comments. I find that the authors have addressed all of my concerns, and I have no further comments to the manuscript in it's revised state."}]

Decision letter