A. Quantitative RT-PCR analysis of endogenous Wdr74 mRNA during preimplantation development. B. RT-PCR with Wdr74 intron-spanning primers confirms relative abundance of transcripts observed by qRT-PCR. C–H. Microinjected and cultured embryos photographed at 36, 60, and 84 hours post fertilization. Control dsGFP-injected embryos show normal development and form blastocysts by 84 hpf (C–E). dsWdr74 injected embryos develop normally to the morula stage (F–G) but fail to make blastocysts (H). I. Quantification of percent 2 cell embryos that develop to the blastocyst stage by 84 hpf (# blastocysts/# 2-cell ×100). J. qRT-PCR of Wdr74 transcripts indicates robust RNAi mediated knockdown due to microinjection of dsWdr74. K. Immunofluorescence of Wdr74 in morula stage dsGFP embryos shows nuclear localization; which is drastically reduced in dsWdr74 embryos of the same stage (L). hpf, hours post fertilization. Results of student T-test shown, error bars represent standard deviation. All data shown normalized to embryo equivalents; MII, Metaphase II oocyte. Scale bar in F representative for C–H. K′ and L′ show DAPI signal (DNA) from the same embryos shown in K and L, respectively.

A–B. Morphological evaluation of dsWdr74-injected and dsWdr74+dsTrp53 co-injected embryos at 84 hpf. dsWdr74 embryos do not develop past the morula stage (A). Reduction of Trp53 permits differentiation of Wdr74-deficient blastocysts (B). C. Percent of 2-cell embryos reaching the blastocyst stage by 84 hpf in dsGFP, dsWdr74 and dsWdr74+dsTrp53 co-injected embryos. D. qRT-PCR confirms knockdown of Wdr74 and Trp53 as expected. Results of student T-test shown, error bars represent standard deviation. All data shown normalized to embryo equivalents.

A–B. Hoechst 33322 (blue) and Pyronin Y (red) stains bind to DNA and RNA, respectively, in dsGFP (A) and dsWdr74 (B) embryos. Note the dramatic decrease in Pyronin Y fluorescence in the absence of Wdr74. C–D. Bioanalyzer electropherograms from total RNA extracted from dsGFP (C) and dsWdr74 embryos (D) show that 18s and 28s ribosomal RNAs are nearly absent in Wdr74-deficient embryos. E. Relative levels of microRNAs miR-125 and miR-721 and RNA polymerase III derived transcripts, Mvp, Rnu6, Snord110 and Snord65 in dsGFP and dsWdr74 morula. Results of student T-test shown, error bars represent standard deviation. All data shown normalized to embryo equivalents.

A–B. E-cadherin (Cdh1) localization by immunofluorescence marks blastomere cell-cell adhesion as expected in dsGFP morula (A). E-Cadherin is appropriately localized but present at reduced in dsWdr74 morula (B). C. qRT-PCR assays show reduced RNA polymerase II derived transcripts of Pouf51, Tead4, Actβ, GapdH, Bax, and Cdh1 but Trp53 shows an increase in transcripts in Wdr74-deficinet embryos. D. The average number of cells in dsGFP and dsWdr74 morula is not significantly different. E–F. Localization of Trp53 by immunofluorescence shows a marked increase of Trp53 protein in dsWdr74 embryos (compare F to E), consistent with the increase in Trp53 mRNA. Results of student T-test shown, error bars represent standard deviation. All data shown normalized to embryo equivalents. N.S., not significant. Scale bar in B and F representative for A–B and E–F, respectively.

A–H. Immunofluorescence localizes Oct4 and Cdx2 to the inner cell mass and trophectoderm, respectively, in dsGFP embryos (A–E). In dsWdr74/dsTrp53 rescued blastocysts (2 shown, F–J and K–O), Cdx2 is expressed and Oct4 is reduced (but present) in some TE-like cells (arrows in F–O). Scale bar in K representative for all panels. DIC, differential interference contrast microscopy.