Genetic gradual reduction of OGT activity unveils the essential role of O-GlcNAc in the mouse embryo
Fig 3
A mild reduction in OGT’s activity affects placental development in a sexually-dimorphic manner.
(A) Litter size for intercrosses of WT mice and for crosses between mice bearing the Ogt-Y851A mutation. P-values are from unpaired two-sided Student’s t-test, assuming unequal variance. N = 20 WT crosses, 8 OgtY851A/+ x OgtY851A/Y and 6 OgtY851A/Y851A x OgtY851A/Y crosses. (B) Breeding scheme used to produce placentae with the four different genotypes, which were analyzed via western blot and mRNA-Seq. (C) Western blot analysis of OGT, OGA and O-GlcNAc levels in the placenta isolated from E12.5 embryos of the four genotypes produced by the cross described in (B). LF: long form, SF: short form. (D) Normalized optical density for OGT (top), O-GlcNAc entire lane (middle) and ratio OGA long form vs. OGA short form from the western blot in (C). In (C,D), for each genotype, the two placentae belong to embryos from two different litters (the same two litters for all genotypes), except for the hemizygous males which both come from the same litter. (E,F) Separate DESeq2 analyses for female and male placentae, comparing (E) OgtY851A-homozygous versus heterozygous female placentae or (F) hemizygous versus WT male ones. All genes with adj. p-value < 0.05, any log2FC are colored, and their number is indicated. Genes standing out (and with abs(log2FC) ≥ 0.2) are labeled. (G) Scatter plot representing the average DESeq2-normalized counts of upregulated and downregulated DEGs found in OgtY851A/Y (versus WT male) placentae, in single placentae of all four genotypes analyzed. (H) GSEA of gene expression change in hemizygous OgtY851A/Y versus WT male placentae. The first 10 GO terms for the three gene ontologies based on Normalized Enrichment Score (NES) are shown. Terms are ordered based on gene ratio. The size of dots is proportional to the number of total genes of a GO term. Gene ratio = fraction of total genes of the GO term which are concordantly changing in mutant embryos. (I) Enrichment analysis of placental clusters’ marker genes [63] expression changes in OgtY851A/Y versus WT male placentae. All enriched clusters (adj. p-value < 0.05) are shown, ordered by gene ratio. The size of dots is proportional to the number of markers of each placental cluster. Gene ratio = fraction of total markers which are concordantly changing in mutant embryos. JZP: junctional zone precursors; LaTP: labyrinth trophoblast progenitors; SynT-I: syncytiotrophoblast layer I; SynT-II: syncytiotrophoblast layer II; sTGC: sinusoidal trophoblast giant cells; SpT: spongiotrophoblasts. In (E-I), N per genotype = 6 placentae coming from at least two different litters, except female OgtY851A/+ placentae for which one sample was excluded because outlier in unsupervised clustering.