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Figure 2 Ascl2 Depletion Does Not Drive the Bulk of Gene Expression Alterations in mDKO EPCs Show full caption (A) mRNA-seq data reveal deregulated maternally controlled imprinted genes in DHet EPCs (red), whereas a paternally controlled imprinted gene is unchanged (Igf2, blue). (B) RT-qPCR data from Ascl2 WT and mKO E7.5 EPCs shows that some key genes deregulated in mDKO EPCs are driven by Ascl2 downregulation (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; t test). Error bars represent SD. (C) Differential expression analysis from mRNA-seq of Ascl2 mKO EPCs reveals that only a minority of Dnmt3 mDKO DE genes are explained by Ascl2 repression. (D) Log2 fold change in expression between WT and Ascl2 or Dnmt3 mutant EPCs for each grouping of genes defined in (C). Ascl2-dependent genes display similar expression changes in Dnmt3 mDKO EPCs, whereas Dnmt3 unique DE genes are unchanged in Ascl2 mKO EPCs. See also Figure S2

Oocyte methylation controls several ICRs that are essential for maternal regulation of imprinted genes, which are important for both embryo and trophoblast development. As previously suggested (), maternal effects on trophoblast development may therefore be a result of loss of specific imprinted genes. To test this hypothesis, we first identified known methylation-dependent imprinted genes within our mDKO DE gene list. Of 79 imprinted genes (paternal and maternal) in our mRNA-seq data, 59 were robustly expressed in EPCs from at least one of the genotypes. However, only five were consistently altered in all mDKO genotypes: Zrsr1, Cd81, Ascl2, Phlda2, and Cdkn1c ( Figure 2 A ). Zrsr1 (also known as U2af1-rs1) was upregulated in mDKO EPCs but is unlikely to be involved in the phenotype of mDKO conceptuses, as mice with paternal disomy of chromosome 11 (where Zrsr1 lies) are viable (). Cd81, Cdkn1c, Phlda2, and Ascl2, which are all part of the same imprinting cluster, were all robustly downregulated in mDKO EPCs, as lack of oocyte methylation leads to activation of the non-coding transcript Kcnq1ot1 on the maternal allele, which is known to drive silencing of genes in its vicinity (). Cd81 KO mice are viable (), and both Cdkn1c and Phlda2 KO placentas are enlarged and show an expansion of the spongiotrophoblast layer, which is a very different phenotype from that observed in mDKOs (). However, trophoblast from maternal KO of Ascl2 (also known as Mash2) has a severe phenotype mainly characterized by a lack of spongiotrophoblast formation, which leads to embryonic lethality at around E10 (). Given the similarity in phenotype timing to the mDKO trophoblast, as well as the downregulation of spongiotrophoblast marker Tpbpa observed in both models, we decided to test whether Ascl2 downregulation was driving the transcriptional changes seen in mDKO EPCs. For this purpose, we used an Ascl2-lacZ knockin mouse line () to generate mKO conceptuses of Ascl2. We first performed histological analysis at E9.5, which revealed that Ascl2 mKOs had a reduced or absent labyrinthine layer despite having completed chorio-allantoic fusion, and had an enlarged TGC layer ( Figure S2 A), as previously described (). However, unlike Dnmt3a mKO trophoblast, the TGC layer expansion did not involve a significant increase in extracellular space ( Figure S2 B), inferring that TGC cell adhesion is largely intact in Ascl2 mKO mutants. We then isolated E7.5 EPCs from Ascl2 WT and mKO conceptuses for RT-qPCR analyses. We confirmed that Ascl2 repression leads to Tpbpa downregulation, but also found drastic downregulation of Cdx2 and Pcdh12, similar to that seen in Dnmt3 mDKO EPCs ( Figure 2 B). We then extended this analysis by performing mRNA-seq on control and Ascl2 mKO EPCs. We found that, while 43 genes were commonly deregulated between Ascl2 mKO and Dnmt3 mDKO EPCs, there were 94 DE genes that were unique to the Dnmt3 mDKOs ( Figure 2 C). Surprisingly, we also found 216 genes seemingly only deregulated in Ascl2 mKOs. However, when we analyzed the expression of these genes in Dnmt3 mDKO EPCs, we found that they displayed expression changes very similar to those seen in Ascl2 mKOs ( Figure 2 D), but had not passed our stringent criteria for differential expression calling. Importantly, genes deregulated only in the Dnmt3 mDKO EPCs did not display substantial changes in expression in Ascl2 mKOs ( Figure 2 D), demonstrating that these are indeed Ascl2-independent effects. Our data suggest that the majority of transcriptional alterations in mDKO trophoblast are independent of imprinting of a key regulator of placental development. While we cannot completely rule out that the combined loss of imprinting at other loci may drive the gene expression changes seen in mDKOs, it is likely that maternally derived methylation marks outside of ICRs play a major role in trophoblast gene regulation.