The transcriptional repressor Blimp1/PRDM1 regulates the maternal decidual response in mice

Abstract

The transcriptional repressor Blimp1 controls cell fate decisions in the developing embryo and adult tissues. Here we describe Blimp1 expression and functional requirements within maternal uterine tissues during pregnancy. Expression is robustly up-regulated at early post-implantation stages in the primary decidual zone (PDZ) surrounding the embryo. Conditional inactivation results in defective formation of the PDZ barrier and abnormal trophectoderm invasion. RNA-Seq analysis demonstrates down-regulated expression of genes involved in cell adhesion and markers of decidualisation. In contrast, genes controlling immune responses including IFNγ are up-regulated. ChIP-Seq experiments identify candidate targets unique to the decidua as well as those shared across diverse cell types including a highly conserved peak at the Csf-1 gene promoter. Interestingly Blimp1 inactivation results in up-regulated Csf1 expression and macrophage recruitment into maternal decidual tissues. These results identify Blimp1 as a critical regulator of tissue remodelling and maternal tolerance during early stages of pregnancy.

Fig. 1. Induction of Blimp1 protein expression during pregnancy and PR-Cre-driven deletion in decidual tissues.

a Blimp1 IHC in virgin, E3.5, E4.5, E5.5 and E6.5 wild-type uteri. In virgins, Blimp1 was detected in a small number of cells underlying the luminal epithelium. Upon fertilisation, Blimp1 was induced in uterine stroma (E3.5) followed by strong upregulation at the site of implantation in both the decidualising stroma and luminal epithelium (E4.5-E6.5). Results are representative of triplicate staining experiments performed using independent samples. b Western blot analysis confirmed strong induction of Blimp1 protein in the uterus during pregnancy. Source data are provided as a Source Data file. Duplicate experiments were performed with comparable results. c IHC confirms loss of Blimp1 protein in decidual stromal cells in Blimp1 mutant mice at E6.5. Data from 5 tissue sections from triplicate decidual samples per genotype. Source data are provided as a Source Data file. Bars represent mean. Two-tailed unpaired Student’s t-test ***p = 1.36 × 10⁻⁴. LE = luminal epithelium, GE = glandular epithelium, B = blastocyst, E = embryo, ExE = extra-embryonic ectoderm, Ep = epiblast, VE = visceral endoderm, M = mesometrial, AM = antimesometrial. Scale bar = 100 μm.

Fig. 2. Blimp1 mutants are receptive to implantation but display gross decidual and embryonic defects.

a Sites of decidualisation at E6.5 appear grossly similar between wild-type and Blimp1 mutant uteri, as visualised by Chicago Blue dye injection (n = 3 per group). b Chicago Blue content is reduced in decidua but not the ovaries of mutants (0.271 ± 0.013 μg/decidua) in comparison to wild type (0.348 ± 0.008 μg/decidua) mice. Data from 3 Blimp1 mutant mice (19 deciduae and 6 ovaries) and 3 wild-type mice (27 deciduae and 6 ovaries) are shown. Bars represent mean. Two-tailed unpaired Student’s t-test ***p = 3.49 × 10⁻⁶. Source data are provided as a Source Data file. c Alkaline phosphatase staining of wild-type and Blimp1 mutant E5.5 decidua shows a reduction in intensity and the spread of alkaline phosphatase staining in Blimp1 mutant decidua indicative of impaired decidualisation (n = 3 decidua per genotype). d H&E staining of E6.5 deciduae. Embryos in Blimp1 mutant mice are highly abnormal and display mis-localised implantation (n = 4 decidua per genotype). e Cell density analysis of DAPI-stained nuclei identifies reduced cell density in the PDZ of Blimp1 mutants. Data from 12 tissue sections from four samples per genotype are shown. Bars represent mean. Two-tailed unpaired Student’s t-test ***p = 1.08 × 10⁻¹². Source data are provided as a Source Data file. PDZ = primary decidual zone, M = mesometrial, AM = antimesometrial, E = embryo. Scale bars = a 4 mm or c–d 100 μm.

Fig. 3. Loss of the primary decidual zone barrier in Blimp1 mutants.

a IHC identifies invasion of Eomes positive trophoblasts into the PDZ in Blimp1 mutant decidua (n = 5 decidua per genotype). b IF staining of Oct4 and Eomes in isolated E5.5 embryos from wild-type and Blimp1 mutant mice counterstained with DAPI. Embryos isolated from Blimp1 mutant deciduae display normal epiblast morphology but distinct disruption of the extraembryonic ectoderm (n = 5 embryos per genotype). c Immunofluorescence staining of RFP (to identify embryonic cells) and K8 (trophoblasts) in wild type; mTmG and Blimp1 mutant; mTmG decidua (E5.5) counterstained with DAPI. White dotted lines outline embryos. White arrows in Blimp1 mutant;mTmG images indicate migrating trophoblast cells. RFP-labelled embryos from Blimp1 mutant; mTmG mice are highly disrupted and invade into the PDZ (n = 5 per genotype). d IF staining of E-cad (embryo) and ZO-1 in E5.5 mutant and wild-type decidua counterstained with DAPI. Reduced ZO-1 staining indicates impaired tight junction formation in the PDZ of mutants (n = 5 per genotype). White dotted lines outline the PDZ. e Reduced ZO-1 positive area forming a ‘complete barrier’ in the PDZ of E5.5 mutants demonstrates impaired barrier formation. Data from 12 tissue sections from four samples per genotype are shown. Bars represent mean. Two-tailed unpaired Student’s t-test ***p = 2.01 × 10⁻¹². Source data are provided as a Source Data file. f TEM of E6.5 PDZ confirms loss of a complete barrier in mutants (n = 3 per genotype). Top panel shows sections of resin embedded samples counterstained with toluidine blue for general morphology before TEM was undertaken. Yellow arrows indicate tight junctions at cell–cell borders. Asterisks mark intercellular spaces. E = embryo, M = mesometrial, AM = antimesometrial, a.u. = arbitrary units. Scale bars = 100 μm unless otherwise indicated.

Fig. 4. Transcriptional changes in E5.5 and E6.5 Blimp1 mutant decidua.

a RNA-Seq analysis of Blimp1 mutant and wild-type decidua at E5.5 and E6.5. Principal component analysis based on the expression of all genes (n = 34,245) clearly separates samples according to stage and Blimp1 genotype. The most pronounced transcriptional differences between the genotypes are observed at E6.5. b Over-represented gene categories among ≥2-fold up-(n = 703) and down-(n = 458) regulated genes in E6.5 mutants based on GO Biological process with affinity propagation as analysed using Webgestalt 2019 with Benjamini-Hochberg multiple testing correction. c Differential expression of Prl genes and Timp3 in E6.5 mutants. Prl genes that are selectively (Prl8a2) or predominantly expressed (Prl3c1 and Prl6a1) in decidual cells at early stages of gestation are decreased. In contrast, P-TGC restricted Prl genes (Prl3d1, 3d2 and 3d3, and Prl7a1 at this stage of gestation) are upregulated. The PDZ-associated Timp3 gene is downregulated. d GSEA comparing DESeq2 FDR-ranked genes in E6.5 mutants with uterine progesterone and oestrogen target genes^,. Downregulated gene expression in E6.5 Blimp1 mutants significantly correlates with both progesterone- and oestrogen-target genes. **FDR p = 0.024. ***FDR p = 0.000. e Multiple IFNγ-inducible genes are upregulated in E6.5 Blimp1 mutant decidua. Ifnγ is increased in Blimp1 mutant decidua but due to low level of expression was excluded from RNA-Seq analysis based on the FPKM ≥ 1 minimum level filter. f QPCR analysis confirms significant upregulation of Ifnγ transcripts in E6.5 Blimp1 mutant decidua. Data from four samples per genotype are shown. Bars represent mean. Two-tailed unpaired Student’s t-test *p = 0.0289. Source data are provided as a Source Data file.

Fig. 5. ChIP-Seq analysis of E6.5 decidua identifies potential Blimp1 target genes.

a Venn diagram overlaps of genome-wide Blimp1 binding sites identified by ChIP-Seq analysis of; E6.5 NEG decidua (n = 5808); multiple other NEG mouse tissues (n = 22,641); and embryonic small intestine from BEG mice (n = 2685) identifies decidual-specific and common Blimp1 binding sites. b Over-represented gene categories associated with common (n = 1129) and decidual-specific Blimp1 ChIP peak subsets (n = 2962) within 100 kb of gene TSSs based on PANTHER pathway as analysed using GREAT V3.0 bionomial test with multiple testing correction. The number of genes associated with each gene category are indicated. c MA plot of E6.5 Blimp1 mutant vs wild-type decidual RNA-Seq profiles with up- (green) and down-(red) regulated E6.5 NEG Blimp1 target genes indicated. Candidate Blimp1 target genes with potential links to mutant decidual abnormalities are highlighted. d Over-represented gene categories among upregulated Blimp1 target genes based on GO Biological process with affinity propagation as analysed using Webgestalt 2019 with Benjamini-Hochberg multiple testing correction. The number of genes associated with each gene category are indicated.

Fig. 6. Blimp1 mutant decidua displays increased Csf1 and macrophage invasion into the PDZ.

a ChIP-Seq analysis identifies significant Blimp1 binding at the promoter of the Csf1 gene in mouse decidua. A canonical Blimp1 binding motif underlies the ChIP-seq peak. b QPCR analysis confirms upregulated Csf1 expression in Blimp1 mutant decidua at both E5.5 and E6.5. Data from 4 (E5.5) and 5 (E6.5) samples per genotype. Bars represent mean. Two-tailed unpaired Student’s t-test *p = 0.0123 for E5.5 and 0.0138 for E6.5. Source data are provided as a Source Data file. c IF staining of F4/80 and MHC Class II in E6.5 decidua with DAPI counterstain identifies infiltration of macrophages into central decidual regions in mutants. In contrast, staining in wild types is largely restricted to the surrounding myometrium (n = 3 decidua per genotype). White dotted lines mark the boundary between decidual stroma and myometrium. d IF staining of c-fms in E6.5 decidua with DAPI counterstain. C-fms is strongly expressed in the maternal decidua directly adjacent to the embryo in both mutant and wild types. Images are representative of triplicate staining experiments. E = embryo. Scale bars = 100 μm.