Loss of H3K27me3 imprinting in the Sfmbt2 miRNA cluster causes enlargement of cloned mouse placentas

Abstract

Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT.

Fig. 1. Placental weights and histology of IVF and SCNT from wild type or KO mice.

a Weights of term placentas derived from IVF or SCNT. The horizontal lines indicate the mean value. +, wild type; Δm, maternal KO; Δm/p, maternal or paternal KO; *P < 0.05, *****P < 0.0005, ******P < 0.0001 (Kruskal–Wallis test). N represents the number of biological replicates. Gab1 KO included six placentas cloned from Sertoli cells. Source data are provided as a Source data file. b Hematoxylin and eosin-stained tissue sections of E19.5 placentas from IVF and SCNT (wild type and Sfmbt2 maternal KO placentas). ST spongiotrophoblast layer, LB labyrinthine layer. Scale bar, 2 mm.

Fig. 2. Expression analysis of miRNAs in IVF- and SCNT-derived placentas at E11.5.

a Changes in the size of IVF- and SCNT-derived placentas during gestation. SCNT placentas were smaller than IVF placentas in the early stage but larger in the later stage. IVF and SCNT placentas at E11.5 were identical in size and structure, and they were considered to be appropriate for comparative transcriptome analysis, as shown in b. b Histology of IVF and SCNT placentas at E11.5. ST spongiotrophoblast layer, LB labyrinthine layer. Scale bar, 1 mm. c Venn diagram showing the numbers of miRNAs with differential expression levels in E11.5 SCNT placentas derived from cumulus and Sertoli cells. Twenty-one differentially expressed miRNAs were common to the two types of SCNT placentas. d List of the 21 common differentially expressed miRNAs. Rows in orange and blue indicate genes located on chromosome 2 (2qA1) and chromosome 12 (12qF1), respectively. Annotations were determined according to the information from Agilent SurePrint G3 mouse miRNA microarray miRBase rel.17 version. Mmu-miR-199a-5p and -3p are annotated on chromosome 9 in the current database. Also see Supplementary Data 1.

Fig. 3. Dysregulation of two clustered miRNAs in SCNT placentas.

a, b Scatterplot analysis of all miRNAs based on their expression levels in cumulus- a and Sertoli- b derived SCNT placentas compared with those detected in IVF placentas. Red and blue dots represent miRNAs from the Sfmbt2 and Mirg clusters, respectively. Dotted lines represent >1.3-fold changes. c Expression levels of Sfmbt2 and Mirg miRNAs. The Sfmbt2 miRNAs were highly expressed in SCNT placentas, regardless of the donor cell type. The Mirg miRNAs had lower expression levels in SCNT placentas and they exhibited donor cell type dependency. Also see Supplementary Figs. 2 and 3. N represents the number of biological replicates. The error bars represent the SEM. The Source data of Fig. 2c are provided as a Source data file.

Fig. 4. Localization of miR669f-3p, Sfmbt2 miRNA, in E11.5 placenta.

a In situ hybridization image of a normal (IVF-derived) placenta at E11.5. The areas corresponding to the magnified images in b and c are indicated by black squares. ST spongiotrophoblast layer, LB labyrinthine layer. Scale bar, 1 mm. b–e In situ hybridization using probes for miR669f-3p and negative-control probes. The nuclei in the cell layer of secondary trophoblast giant cells (TG) were clearly stained for miR669f-3p b. The nuclei of trophoblasts in the ST layer and immature trophoblasts in the LB layer d were also clearly stained. Their cytoplasm was faintly stained. Scale bar, 100 μm.

Fig. 5. Placental weights and histology of SCNT from miRNA KO mice.

a Weights of term placentas derived from IVF or SCNT. The horizontal lines indicate the mean value. +, wild type; Δm, maternal KO; ******P < 0.0001, ns, not significantly different (Kruskal–Wallis test). N represents the number of biological replicates. Weights of IVF and wild type SCNT placentas were same as Fig. 1a. b Areas of the ST and LB layers, and length of the boundary between ST and LB layers. *P < 0.05, *****P < 0.0005 (two-way ANOVA). N represents the number of biological replicates. The error bars represent the SEM. c Hematoxylin and eosin-stained tissue sections of E19.5 placentas from IVF and SCNT (wild type and two types of KO placentas). ST spongiotrophoblast layer, LB labyrinthine layer. Scale bar, 2 mm. The Source data of a and b are provided as a Source data file.

Fig. 6. Transcriptome analysis of IVF and SCNT placentas from wild type and miRNA KO.

a Principal component analysis of IVF, SCNT wild type, miRNA KO, and miRNA/Gab1 KO placentas at E11.5 and E19.5 (term). The numbers within brackets indicate the number of biological replicates. b Numbers of DEGs in SCNT (wild type and two types of KO) placentas vs IVF placentas. c Box plots showing the expression levels of Sfmbt2 pri-miRNAs (Mir467b, Mir297b, and Mir669a-3) and placenta-specific imprinted genes (Sfmbt2, Gab1, and Slc38a4) in IVF and SCNT placentas. Upper, lower, and center lines indicate minimum, maximum, and mean values. The mean expression level in all samples is indicated as 0.0. +, wild type; Δm, maternal KO. Also see Supplementary Figs. 4 and 5, and Supplementary Data 2–6. Source data are provided as a Source data file.

Fig. 7. Changes of canonical imprinted genes and predicted target genes in SCNT placentas.

a, b Fold changes in the expression levels of canonical imprinted genes a, and the predicted target genes of Sfmbt2 miRNAs b with different expression levels in IVF and SCNT placentas. The expression level in IVF placentas was set at 0. c Hematoxylin and eosin-stained tissue sections of E19.5 placentas from wild type and KO (Gkn2, Rgs4, and Fst) IVF. Scale bar, 1 mm. An increase in the number of glycogen cells was observed in the Fst KO placenta (arrowheads).