8 research outputs found
Wdr74 Is Required for Blastocyst Formation in the Mouse
Preimplantation is a dynamic developmental period during which a combination of maternal and zygotic factors program the early embryo resulting in lineage specification and implantation. A reverse genetic RNAi screen in mouse embryos identified the WD Repeat Domain 74 gene (Wdr74) as being required for these critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse
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Determining the Roles of Smyd3 and Wdr74 In Vivo
Preimplantation is a short yet critical time period for the embryo that includes dramatic changes in gene expression and developmental potential. During early cleavage, a combination of maternal and zygotic factors program the embryonic genome. These dynamic events result in critical differentiation steps including the first lineage specification (ICM and TE), which is required for implantation.
The first half of this thesis investigates the role of Smyd3 as a possible maternal-effect gene, which was identified through an mRNA-based expression assay. Using a Smyd3 conditional allele to remove the function of the protein, we were able to confirm successful allelic recombinations but were unable to confirm the absence of the protein. Due to a lack of a phenotype, two conclusions were made: 1) If we did disrupt Smyd3 function, the lack of a phenotype indicates that Smyd3 is not a maternal-effect gene, and is not required for embryonic viability or adult fertility. 2) It is possible that newly-annotated transcripts are responsible for the necessity of Smyd3, and that the conditional allele used did not disrupt the function of Smyd3.
The second half of this thesis uses a reverse genetic RNAi screen that identified Wdr74 as being required for the critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos, blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse
Wdr74 is required for blastocyst formation.
<p><b>A.</b> Quantitative RT-PCR analysis of endogenous <i>Wdr74</i> mRNA during preimplantation development. <b>B.</b> RT-PCR with <i>Wdr74</i> intron-spanning primers confirms relative abundance of transcripts observed by qRT-PCR. <b>C–H.</b> 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). <b>I.</b> Quantification of percent 2 cell embryos that develop to the blastocyst stage by 84 hpf (# blastocysts/# 2-cell ×100). <b>J.</b> qRT-PCR of Wdr74 transcripts indicates robust RNAi mediated knockdown due to microinjection of dsWdr74. <b>K.</b> 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.</p
Lineage specification in dsWdr74/dsTrp53 blastocysts.
<p><b>A–H.</b> 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.</p
Blocking <i>Trp53</i> permits blastocyst formation in Wdr74-deficient embryos.
<p><b>A–B.</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 <i>Trp53</i> permits differentiation of Wdr74-deficient blastocysts (B). <b>C.</b> Percent of 2-cell embryos reaching the blastocyst stage by 84 hpf in dsGFP, dsWdr74 and dsWdr74+dsTrp53 co-injected embryos. <b>D.</b> qRT-PCR confirms knockdown of <i>Wdr74</i> and <i>Trp53</i> as expected. Results of student T-test shown, error bars represent standard deviation. All data shown normalized to embryo equivalents.</p
Gene expression in dsWdr74 morula.
<p><b>A–B.</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). <b>C.</b> qRT-PCR assays show reduced RNA polymerase II derived transcripts of <i>Pouf51</i>, <i>Tead4</i>, <i>Actβ</i>, <i>GapdH</i>, <i>Bax</i>, and <i>Cdh1</i> but <i>Trp53</i> shows an increase in transcripts in Wdr74-deficinet embryos. <b>D.</b> The average number of cells in dsGFP and dsWdr74 morula is not significantly different. <b>E–F. </b>Localization of Trp53 by immunofluorescence shows a marked increase of Trp53 protein in dsWdr74 embryos (compare F to E), consistent with the increase in <i>Trp53</i> 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.</p