Extended Data Fig. 1: Validation of vitamin C depletion and the downregulation of TET1-dependent germline genes in a mouse model of gestational vitamin C deficiency.
From: Maternal vitamin C regulates reprogramming of DNA methylation and germline development
a, Expression of vitamin C transporters (SVCT1, SVCT2 and GLUT8, encoded by Slc23a1, Slc23a2 and Glut8, respectively) in developing female (F) germ cells. Data are from Seisenberger et al.3. b, Kinetics of vitamin C depletion from the serum of pregnant Gulo−/− mice after withdrawal from their drinking water. Pregnant female mice were removed from vitamin C supplementation at E3.5 and circulating blood serum was tested over the time course indicated. It takes five days of withdrawal for the circulating vitamin C levels to be <25%, and seven days to be essentially undetectable. Line plot connects the average blood serum values of three biological replicates. c, Vitamin C levels measured by mass spectrometry in E13.5 embryonic tissue with and without gestational vitamin C removal. E13.5 female head or liver were normalized by tissue weight. Samples with non-detectable levels of vitamin C were set to zero. n = 5 biological replicates per condition. d, The reduction in E13.5 female germ cells numbers after vitamin C deficiency is confirmed using both Oct4–eGFP and SSEA1 positivity. n = 6 matched biological replicates per condition. e, Gestational vitamin C deficiency does not affect average somatic cell number. n = 13 control and n = 10 vitamin-C-depleted biological replicates. f, Vitamin-C-deficient E13.5 female germ cells express lower levels of key germline genes, as measured by qRT–PCR. n = 3 control and n = 4 vitamin-C-depleted biological replicates. The variability in Stra8 expression in controls as assessed by qRT–PCR at E13.5 is because it is just being induced at this stage; RNA-seq and immunofluorescence data further document the significant reduction in Stra8 RNA and protein levels in vitamin-C-deficient PGCs (Extended Data Fig. 3b, c). g, Expression of select germline genes are induced in a TET1/2-dependent manner after the addition of vitamin to cultured mouse ES cells11. Gene expression was measured by qRT–PCR, plotted as mean of technical triplicates. Data are from Blaschke et al.11. h, Decreased expression of select germline genes in Tet1−/− E13.5 germ cells as measured by RNA-seq. Data are from Yamaguchi et al.8, and denote averages of three controls and two Tet1−/− samples. i, Select germline genes induced by vitamin C in cultured ES cells (c) and downregulated in Tet1−/− germ cells (d) are also downregulated in vitamin-C-deficient E13.5 germ cells. Gene expression was measured by RNA-seq. n = 6 biological replicates. j, Gestational vitamin C deficiency does not affect transcription in the somatic cells of E13.5 female gonads as shown by unsupervised clustering of RNA-seq analysis. Clustering performed on n = 6 biological replicates per condition. Data are single values per stage (a), mean values (b, h), mean ± s.e.m. (c, i) or mean ± s.d. (d–g). P values determined by two-tailed t-test with Welch’s correction (c, d, e), two-tailed Student’s t-test (f).
