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1 Development 144: doi:1.1242/dev.1473: Supplementary information Supplementary Figures A (f) FRT LoxP B All Males Females I Ovary 1 (+) 77 bps (f) 78 bps (-) >13 bps (-) 2 4 (-) 424 bps M +/f +/- H 2 O f + - genotype +/- total p-value # % # % # % αkdm4a αkdm4a C All D Males E Females Percent survival 7 n.s. p=.78 (n=14) +/- (n=16) (n=16) Percent survival 7 n.s. p=.98 (n=8) +/- (n=1) (n=1) Percent survival 7 n.s. p=.96 (n=6) +/- (n=6) (n=6) time (weeks) time (weeks) time (weeks) F Males G Females 4 8 weeks 2 weeks 3 weeks 3 8 weeks 2 weeks 3 weeks H weight (g) +/β-gal p=.7 p=.38 +/- +/- genotype +/- weight (g) 3 2 +/- +/- +/- genotype 3. dpc 8. dpc 1. dpc 12. dpc 14. dpc
2 Development 144: doi:1.1242/dev.1473: Supplementary information Fig S1. Characterization of Kdm4a mice (A) Genotyping strategy used to detect wild type (+), LoxP flanked (f) and KO (-) alleles of Kdm4a. Positions of primers and their respective product sizes are indicated. (B) Kdm4a +/- mice were inter-crossed and the number of pups at time of weaning are reported. P values were calculated using a Chi-square test. Viability is shown for all, male and female animals. (C-E) Survival rates of wild type () and Kdm4a heterozygous (+/-) and KO () animals. Survival is shown for all (C), male (D) and female (E) animals. Log-rank (Mantel-Cox) test was used to determine significance. (F-G) Boxplot depicting average weights of Kdm4a, Kdm4a +/- and Kdm4a male (F) and female (G) animals at the indicated ages. Unpaired t-test was used to determine statistical significance. (H) Beta-galactosidase staining of embryos at the indicated stages expressing the LacZ reporter gene under control of the endogenous Kdm4a promoter (+/ Beta-Gal). Wild type embryos () were used as control. (I) Immunohistochemistry on paraffin embedded sections of control and Kdm4a ovary stained for Kdm4a. Staining is seen in the oocyte as highlighted with asterisk () as well as the surrounding granulosa cells of the ovarian follicle.
3 Development 144: doi:1.1242/dev.1473: Supplementary information Haematoxylin and eosin Oviduct Ovary Uterus BlV Lu En MuF Lu GC Myo TC Myo BlV TC Lu MuF En Lu GC Fig S2. Reproductive tissue histology in Kdm4a females Representative haematoxylin and eosin stained sections of ovary, oviduct and uterus of wild type () and Kdm4a females (n=3 tested /group). GC- Granulosa cell; TC Theca Cell; Endometrium; Myo Myometrium CL Corpus luteum; MuF Mucosal Fold; BlV Blood Vessel; Lu Lumen; En
4 ru s te ) (n =3 =3 ) Processed Mask (n Mask va ry te ru s 3) 3) (n = 3) (n = t( n= du c vi U O O Ki67 - Oviduct Ki67 - Uterus % Ki67 positive cells Ki67 - Ovary Processed Mask U IHC du ct =3 ) (n B Mask % Caspase 3 positive cells Caspase3 - Ovary IHC vi O va ry O Caspase3 - Oviduct A Caspase3 - Uterus Development 144: doi:1.1242/dev.1473: Supplementary information Ki67 (proliferation) Cleaved caspase-3 (Apoptosis) 1
5 Development 144: doi:1.1242/dev.1473: Supplementary information Fig S3. Proliferative and apoptosis levels in Kdm4a female reproductive tissues appear normal (A-B) Sections of ovary oviduct and uterus of wild type () and Kdm4a females were stained for Ki67 (A) and cleaved Caspase 3 (B). The result of image processing in ImageJ is depicted and the average percentages of positive cells (n=3 animals) are presented with SD. Unpaired t-test was used to determine statistical significance. Processed mask represents visualization of positive staining cells across the tissue section derived by equal colour threshold adjustment on unprocessed mask of wildtype and knockout samples
6 Development 144: doi:1.1242/dev.1473: Supplementary information A Flushed from oviduct at 3. dpc B p+/m+ p-/m+ p+/m- % ESC lines / plated embryos 1 7 Male Female n p<.1 +/- +/ Fig S4. Kdm4a females produce poor quality blastocysts in vivo (A) Representative images of embryos at 3. dpc flushed from Kdm4a, Kdm4a +/- and Kdm4a females that were crossed with males as indicated in the top right of each image. Delayed embryos (no sign of cavitation) are indicated with an asterisk. (B) 3. dpc embryos from wild type (), heterozygous (+/-) and Kdm4a () females were plated and the percentage of embryonic stem cell lines that were successfully generated is indicated. n indicated number of embryos plated in mesc medium to generate lines.
7 Development 144: doi:1.1242/dev.1473: Supplementary information Ovarian steroidogenesis pathway (2. dpc), n=11, n= p value p<. p<.1 Fold change / Rplp 2 1 Jmjd2a Lhr Prlr Ar Fshr Pgr Esr1 Esr2 Star Cyp17a1 Cyp1 1a1 Cyp19a1 2alphaHSD Hsd17b1 17b2 Hsd17b7 Hsd17b12 Hsd3 C Hsd cnd2 Cebp -beta Gdf-9 p<.1 Fig S. Gene expression of ovarian steroidogenesis pathway genes in Kdm4a females at 2. dpc Boxplot depicting average expression levels of the indicated genes in wild type () and Kdm4a ovary. Gene expression was determined through RT-qPCR from 11 animals per group. Statistical significance was determined using unpaired t-test and significance was reported for each gene if p<. (), p<.1 () and p<.1 ().
8 Development 144: doi:1.1242/dev.1473: Supplementary information A Genes downregulated in Kdm4a ovary Genes upregulated in Kdm4a ovary GO biological process count GO biological process count Sex differentiation Development of primary sexual characteristics Male gamete generation Cellular process involved in reproduction Cellular response to endogenous stimulus Response to hormone Development process involved in reproduction Reproduction Response to endogenous stimulus Cell development Phagocytic vesicle Cation transmembrane transporter activity Inorganic ion transmembrane transport Inorganic cation transmem. transporter activity Innate immune response Extracellular space Immune response Positive regulation of organismal process Defense response Immune system process B C Genes upregulated in Kdm4a uterus GO biological process Nuclear chromosome segregation Sister chromatid segregation Chromosome segregation Organelle fission Mitotic nuclear division Cell division Extracellular space Mitotic cell cycle Cell cycle process Cell cycle count GE enriched 2. dpc glandular epithelium (GE) genes vs 2. dpc Kdm4a knockout uterus Kdm4a _down GE enriched Kdm4a _up Fig S6. Investigation of ovarian and uterine target genes in Kdm4a (A) Gene ontology (GO) analysis on down and up regulated genes for processes affected by loss of Kdm4a in the 2.dpc ovary as determined from RNA Seq (n=3/group). (B) Overlap of published uterine glandular epithelium (GE) enriched genes at 2.dpc with genes that were downregulated or upregulated in the Kdm4a uterus. (C) Gene ontology (GO) analysis on upregulated genes for processes affected by loss of Kdm4a in the 2.dpc uterus as determined from RNA Seq.
9 Development 144: doi:1.1242/dev.1473: Supplementary information A Embryo transfer - foetus genotyping Ladder 1a 1b 1c 1d 1e 1f 2a 2b 2c 2d 2e 2f 2g 2h 2i 3a Ladder 3b 3c 3d 3e 3f 3g 3h 4a 4b 4c 4d 4e 4f a b c Ladder d e f g h i j k 6a 6b 7a 7b 7c wildtype allele knockout allele Number - Recipient female ID Alphabet - Foetus ID - Dead foetuses B Embryo transfer - Total foetus distribution Total embryos ( 17.dpc ) Cumulative distribution 12 m+/p- (Control) m-/p- (Mutant) 1 Embryo numbers % Mutant : Control Expected Total Recipient ID (n= 4) Fig S7. Bi-lateral embryo transfer of Kdm4a maternal zygotic mutants (A) Genotyping of individual embryos from recipient females following C-section at 17. dpc. The wildtype and knockout alleles are highlighted and dead/deformed foetuses obtained are highlighted with a red asterisk. (B) Individual numbers and cumulative distribution of total Kdm4a maternal zygotic mutants and control embryos from each recipient female. The red line at % represents the expected distribution based on the 1:1 mix of embryos transplanted into each oviduct.
10 Development 144: doi:1.1242/dev.1473: Supplementary information Table S1 Click here to Download Table S1 Table S2 Click here to Download Table S2 Table S3 Click here to Download Table S3
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