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Fig. S1. Comparison of Cdx2 and Oct4 expression. Cdx2 (green) and Oct4 (red) expression was determined at different embryonic stages by double immunostaining. In comparison to the faint signal at the 8-cell stage, stronger nuclear staining of Cdx2 protein was initially present in one to three blastomeres at the 12-cell stage and then gradually extended to all blastomeres on the outer part of the embryos at a later stage. Oct4 was evenly distributed in all blastomeres until the blastocyst stage.
Fig. S2. Efficient reduction of Cdx2 mRNA and protein levels results in a hatching block phenotype similar to Cdx2 knockout. (A) Evaluation of different Cdx2 siRNA duplexes for knockdown efficiency after microinjection of siCdx2 into zygotes. Three conventional 19-nt Cdx2 siRNA duplexes and three 25-nt Stealth siRNAs were injected into zygotes, which were cultured for 96-98 hours until the morula and blastocyst stages and then tested for Cdx2 expression by real-time RT-PCR with the corresponding scramble controls (siControl and StControl); untreated embryos were used as an additional control. With a unique sequence and high efficacy, siRNA3 was selected for subsequent experiments. (B) Observation of Cdx2 silencing at the morula stage by immunocytochemistry. These confocal images show that nuclear Cdx2 could not be detected even when the green fluorescent background staining of siCdx2-treated morulae was brightened. (C) Validation of Cdx2 silencing at the protein level by western blot analysis with duplicates. Each lane was loaded with total protein extracts from 40 E4.0 blastocysts treated with siControl and siCdx2. A monoclonal antibody detects Cdx2 protein near the 37 kDa marker band in the siControl group, but not in the siCdx2 group, indicating elimination of Cdx2 protein. (D) Highlights of a time-lapse movie. At 2.5 dpc, siControl (on the right side of the white line) and siCdx2 (on the left side of the line) treated OG2-GFP 8-cell embryos were placed in a chamber and examined by confocal microscopy. The recording time of each picture is marked in hours (h). Even though all siCdx2-treated embryos showed a delay in blastocyst expansion and failed to hatch, they did not show any phenotype of compaction or cell polarization. Compaction at the 8-cell stage (6.5 hours) and initiation of blastocyst formation (20-23 hours) was observed in Cdx2-deficient embryos, at the same time as for the control embryos.
Fig. S3. Effect of Cdx2 reduction on gene expression. (A) Relative gene expression levels of Cdx2-deficient 8-cell embryos. Cdx2 knockdown reduced expression of some of the crucial genes in TE lineage differentiation without overexpression of pluripotency-related genes at this stage. (B) Expression of ISP1, a novel murine tryptase with actions of a 'hatching enzyme', is not altered in Cdx2-deficient blastocysts. Confocal images of immunohistochemistry of ISP1 in siControl (left) and siCdx2 (right) treated blastocysts. (C) Ectopic expression of Oct4 and Nanog in the TE after microinjection of siCdx2 into MII oocytes Confocal images of immunohistochemistry of Oct4 (left) and Nanog (right). DNA counterstaining is with DRAQ5.
Fig. S4. Compaction of mouse embryos at 2.5 dpc after Tead4 knockdown by microinjection of siRNA into zygotes. Embryos compacted just like untreated embryos even though Tead4 was effectively knocked down with siTead4A and siTead4B. These are the same embryos as shown in Fig. 6B but 1 day earlier.
