Figures and data
Female mice with an ancestral history of Khdc3 mutation manifest hepatic transcriptional dysregulation that is independent of genotype.
(a) Gene ontology scatterplot for dysregulated genes in Khdc3-null oocytes, based on RNA-Seq. The y-axis represents −log FDR (false discovery rate) and the x-axis represents log2 enrichment ratio. Dysregulation of genes important in lipid and carbohydrate metabolism are highlighted. (b) Murine expression of Khdc3 in various tissues, detected by qPCR (N=3). (c) Relative mRNA expression of Cyp17a1 in the livers of WT, WT*, KOKO, and KO* mice measured by qPCR. Each dot represents an individual mouse. (d) Relative mRNA expression of Cyp17a1 in the livers of WT* female mice generated from various male and female Khdc3-null grandparents measured by qPCR. (e) Volcano plots depicting differentially expressed genes identified by RNA-seq in the livers of WT vs. WT*, KO* and KOKO mice, respectively (N=4-6). Red dots represent upregulated genes in WT*, KO* and KOKO mice while blue dots represent genes that were downregulated in WT*, KO* and KOKO mice. The x-axis shows log2 fold change values, and the y-axis denotes −log10 p-values. (f) Dot plots of common dysregulated liver genes amongst the WT*, KO*, and KOKO mice compared to WT mice; ***p-adjusted < 1 x 10-5. (g) Dot plot of the significantly dysregulated genes in KO* livers (x-axis) versus WT* livers (y-axis), based on adjusted p-value. Red dots reveal significantly dysregulated genes in KO* mice, blue dots reveal significantly dysregulated genes in WT* mice, and grey dots represent commonly significantly dysregulated genes in both KO* and WT* mice. (h) Gene ontology of the most common dysregulated genes identified in the livers of both WT* and KO* revealed abnormalities in pathways critical for lipid and glucose metabolism.
WT* defects persist over multiple generations and can be passed through the maternal and paternal ancestral lines.
(a) Schematic of experimental mating to form WT**(P) mice. Khdc3-null mice are represented by black filled shapes, WT mice represented by white filled shapes, and Khdc3-heterozygote mice represented by half-filled black shapes. Volcano plot displaying dysregulated genes in the livers of WT**(P) mice compared to WT mice (N=4). Red dots represent upregulated genes and blue dots represent downregulated genes in the WT**(P) mice. (b) Schematic of experimental mating to form WT**(M) mice. Volcano plot displaying dysregulated genes in the livers of WT**(M) mice compared to WT mice (N=4). Red dots represent upregulated genes and blue dots represent downregulated genes in the WT**(M) mice. (c) Venn diagram representing the number of significantly dysregulated liver genes of WT**(P) and WT**(M) female mice compared to WT mice. (d) Pedigree schematic representing mating of WT*(P) male and female mice over successive generations to form WT******(P) mice. Dot plots represent relative liver mRNA expression of Cyp17a1 and 2610507I01Rik of the 2nd through 7th generation WT female mice descended from male Khdc3-null mice as measured by qPCR. (e) Pedigree schematic representing creation of a F2 outcross WT female from the mating of a female Khdc3-null female and WT male and subsequent F1 generation Khdc3-heterozygote female with WT male. Venn diagram depicting the overlap of commonly dysregulated genes in the livers of F2 outcross WT and WT**(M) female mice compared to WT mice (N=4-7). (f) Dot plots representing liver gene dysregulation amongst WT, WT*(P), WT*(M), and F2 outcross WT female mice. ***p < 1 x 10-5.
Wild type mice with ancestral history of Khdc3 mutation have dysregulation of multiple hepatically-metabolized molecule in the serum.
Dot plots revealing abnormal concentration of (a) bile acids, (b) xenobiotics, and (c) metabolic cofactors present in KOKO, WT**(P), and WT****(P) female mice compared to WT mice. (d) WT****(P) female mice exposed to a HFD had elevated serum concentration of lipid molecules compared to WT**** mice fed a conventional diet and WT mice fed a HFD. (e, f) Female WT****(P) female mice exposed to a HFD had decreased levels of multiple other hepatic metabolites, some of which were decreased in WT HFD female mice to a lesser magnitude *p < 0.05, **p < 0.005, ***p < 0.0005.
Oocytes of KOKO and WT**(P) mice have dysregulated expression of multiple miRNAs and tsRNAs.
(a) Dot plots representing dysfunctional expression of small RNA-processing genes of the livers of WT**(P) mice as measured in RNA-seq. (b) Volcano plots depicting differentially expressed miRNAs and tRNA fragments in KOKO oocytes compared to WT oocytes. Red dots represent upregulated small RNAs, and blue dots represent downregulated small RNAs (N=4). (c) Volcano plots depicting differentially expressed miRNAs and tRNA fragments in WT**(P) oocytes compared to WT oocytes. Red dots represent upregulated small RNAs, and blue dots represent downregulated small RNAs (N=4). (d) Scatterplot of the most significantly dysregulated small RNAs of WT**(P) oocytes (x-axis) versus KOKO oocytes (y-axis), based on p-value. Green dots reveal dysregulated miRNAs and tRNA fragments in WT**(P) oocytes, brown dots reveal dysregulated miRNAs and tRNA fragments in KOKO oocytes, and black dots represent commonly dysregulated small RNAs in both WT**(P) and KOKO oocytes.
Khdc3 expression in ovaries of WT, KO*, and WT* mice, from RNA-Seq.
