Supplemental Figure S1. (A) Relative Expression of the Top 30 Highest Expressed Mirnas

Supplemental Figure S1.(A) Relative expression of the top 30 highest expressed miRNAs at early post-implantation stages. (B) In situ hybridization for individual miRNAs located in each of the highly expressed clusters.

Supplemental Figure S2.(A) Change in morphology observed in Dicerfx/fx ESCs after their differentiation into EpiSCs. (B) A switch from naïve (Klf4, Rex1) to primed (Fgf5) pluripotency markers is seen after Dicerfx/fxESC differentiation into EpiSCs. Average from two ESC and EpiSC samples +/- SEM is shown. (C) Methodology of Dicer deletion experiments in EpiSCs. (D) Reduction in relative expression of highly expressed miRNAs at day 5 after tamoxifen induction. Average of three experiments +/- SEM is shown. (E) Cleaved Caspase 3 staining showing an increase in apoptosis in Dicer deleted EpiSCs at day 5 after tamoxifen treatment. (F) In situ hybridization for miR-467b.

Supplemental Figure S3. (A)Treatment for 48h with the pan-caspase inhibitor Z-VAD-FMK restores Dicer-/- EpiSCs’ survival. Average of 3 experiments +/- SEM is shown. (B) Cytochrome C release occurs in Dicer deleted EpiSCs. Total ERK was used as loading control for the cytosolic fraction. (C) Microarray data showing relative expression of pro- and anti-apoptotic factors in Dicer deleted compared to Dicerfx/fx EpiSCs. (D, E) Western blots for pro- (D) and anti-apoptotic (E) factors in Dicer deleted and Dicerfx/fx EpiSCs 5 days after tamoxifen treatment.

Supplemental Figure S4.(A) Time course of BIM expression in EpiSCs upon tamoxifen induction of Dicer deletion. Average of three experiments +/- SEM is shown. (B) Reduction in BIM protein levels after transfection of Dicer deleted EpiSCs with a Bim siRNA. Cells were transfected with a Bim siRNA or a control siRNA at day 2 and analyzed at day 4 after Dicer deletion. Average from three experiments +/-SEM is shown.

Supplemental Table S1.Raw Ct values from microRNA qPCR analysis conducted in 5.5dpc, 6.5dpc, 7.5dpc and 8.5dpc embryos. Average Ct value and standard deviation from three replicates are shown.

Supplemental Materials and Methods

AnnexinV and DiOC6 staining and FACS analysis

2x105 cells were re-suspended in Annexin V binding buffer (0.1%BSA in 10mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH7.4), stained with APC-conjugated AnnexinV (Molecular Probes) and 0.1mg/ml Propidium Iodide (Sigma) and analyzed by flow cytometry. The graphs represent cells positive for AnnexinV and negative for propidium iodide. 2x105 cells were re-suspended in PBS containing 40nM DiOC6 (Sigma), incubated for 15 min at 37oC and analyzed by flow cytometry. A BD LSR2 cytometer and the FlowJo software (BD) were used for data acquisition and analysis respectively.

Cell culture and manipulation

Dicerfx/fx ESCs were maintained in GMEM media (Gibco) containing 10% FCS plus LIF (1500U) on gelatin-coated dishes. Dicerfx/fx EpiSCs were differentiated from Dicerfx/fx ESCs as described elsewhere (Guo et al. 2009). Briefly, Dicerfx/fx ESCs were plated on dishes coated with human fibronectin (Millipore) in N2B27 media containing 20ug/ml Activin and 12ng/ml bFGF. Cells were passaged by mechanical disruption as previously described (Brons et al. 2007) until passage 12, where a stable EpiSCs line could be maintained. Dicerfx/fxEpiSCs were grown on FCS coated dishes in N2B27 media containing 20ug/ml Activin (R&D Systems) and 12ng/ml bFGF (R&D Systems). To induce Dicer deletion Dicerfx/fxEpiSCs were cultured in the presence of 0.3 M 4-OH-Tamoxifen (Sigma) for three days and left untreated from the third day onwards (Fig. S2C). EpiSCs were treated with 100M Z-VAD-FMK (R&D Systems) or DMSO for from day 3 to day 5 after the induction of Dicer deletion.

SiPORT transfection reagent (Ambion) was used according to manufacturers’ instructions to transfect individual microRNA mimics (Ambion) at a final concentration of 70nM. The miR-pool contains 70nM of each miRNA mimic (miR-20a, miR-92a, miR-302a and miR-19b). Those family members higher expressed in the early embryo were selected as representative for each miRNA seed family. Control samples were transfected with 70nM Pre-miR Negative Control #1 (Ambion). HiPerFect transfection reagent (Qiagen) was used according to manufacturers’ instructions to transfect Bim siRNA (Mm_Bcl2l11_2 FlexiTube siRNA, Qiagen) at a final concentration of 75nM. Three other Bim siRNAs (Mm_Bcl2l11_1 FlexiTube siRNA, Mm_Bcl2l11_3 FlexiTube siRNA and Mm_Bcl2l11_4 FlexiTube siRNA, Qiagen) were tested and showed lower level of Bim inhibition (data not shown).

EpiSC immunocytochemistry

EpiSCs were fixed for 10 min at room temperature in 4% paraformaldehyde/PBS, permeabilised for 5 min in 0.4% Triton X-100/PBS and incubated in 10% BSA/0.1% Triton X-100/PBS (Sigma) for 1h at room temperature. Cells were incubated overnight with antibodies against Cleaved CASPASE 3 (1/100, Cell Signalling), BIM-14A8 (1/100, Millipore) or TOM20 (1/100, Santa Cruz) in 1% BSA/0.1%Triton X-100/PBS. Cells were incubated with the appropriate Alexa-Fluor conjugated secondary antibody (Invitrogen) for 1h at room temperature and mounted in Vectashield Mounting media with Dapi (Vector). Images were acquired with a Zeiss confocal microscope and processed with ImageJ(Schneider et al. 2012).

Cytochrome C release assay and western blot

For the separation of cytosolic and membrane fractions cells were washed once with PBS and lysed on the plate with Digitonin Buffer (20mM HEPES/KOH pH7.5, 100mM sucrose, 2.5mM MgCl2, 100 mM KCl, 1mM DTT, 0.025% digitonin, 1mM PMSF) for 10 minutes on ice. After collection of the supernatant (cytosolic fraction) Triton X-100 extraction buffer (Ramsby and Makowski 2011)was added to the plates and these were incubated for 30 min on ice to obtain the supernatant containing the membrane fraction. Proteins were separated by 12% SDS-PAGE (Bis-Tris CriterionXT pre-cast gels, Bio-Rad) and transferred to PVDF membranes (Thermo Scientific). Membranes were incubated overnight in 5%BSA/TBST with antibodies against:-TUBULIN (1/2000, Cell Signalling), DICER (1/1000, Santa Cruz), BIM/BOD (1/1000, Enzo), Phospho-ERK1/2 (1/10000, Sigma), Phospho-SMAD2 (1/1000, Calbiochem), Phospho-AKT (Cell Signalling, 1/1000), Phospho-STAT3 (1/1000, Cell Signalling), Phospho-JNK (Cell Signalling, 1/1000), Phospho-p38 (1/1000, Cell Signalling), Phopho-eIF2 (1/1000, Cell Signalling), b-ACTIN (Cell Signalling, 1/1000), PCNA (1/2000, Santa Cruz), PUMA (1/1000, Cell Signalling), BID (1/1000, Cell Signalling), CASPASE2 (0.8ug/ml, R&D Systems), BCL2 (1/500, BioLegend), MCL1-S19 (1/500, Santa Cruz), A1 (1/500, R&D Systems), BCL-XL (1/1000, Santa Cruz), CYTOCHROME C (1/1000, BD Pharmigen), Total ERK1/2 (1/20000, Sigma). Western blot quantification was carried out using the ImageJ software (Schneider et al. 2012).

Microarray analysis and mRNA qPCR primers

For the microarray analysis, RNA from three independent experiments was extracted as previously described. Labeling and hybridization to Affimetrix Mouse Gene 1.0ST microarrays were performed at UCL Genomics at the Institute of Child Health. Normalization and statistical analysis were performed using GeneSpring software. The Babelomics data analysis platform (Medina et al. 2010)was used to analyze the enrichment for miRNA targets amongst those genes significantly up or down-regulated upon Dicer deletion compared to all genes expressed in Dicerfx/fxEpiSCs. A two-tailed Fisher exact test was used to determine significance (adjusted p<0.05).

The following pairs of primers were used for mRNA qPCR analysis:

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