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Characterization of Genes Required for Preimplantation Embryo Development
Preimplantation embryo development in the mouse is a time of rapid cellular morphological and molecular changes leading to embryo implantation for the generation of offspring. The Mager lab studies these events occuring between fertilization and implantation in order to better understand the initial events which set the stage for all future aspects of development. The result of this research impacts many scientific disciplines including in-vitro based means of embryo culture, establishment of epigenetic marks, differentiation and cellular reprogramming and can be used in translational research for the improvement of in-vitro culture techniques and develop novel therapies such as cell replacement in the case of macular degeneration (Bin, L., 2009).
Through the use of in-vitro embryo culture, RNA interference (RNAi) approaches and daily observations, gene function required in preimplantation embryo development can be determined. In the initial published body of work evaluating gene knockdown using our RNAi approach (Maserati M 2011), WDR74 was characterized in preimplantation embryo development. We now understand that WDR74 is implicated in RNA production and/or stability as gene knockdown at the 1 cell stage significantly depletes mRNA within the embryo by the morula stage. Furthermore, double knockdown of Trp53 and Wdr74 results in a partial rescue of blastocyst formation suggesting p53 mediated apoptosis in the failure to make a blastocyst phenotype.
The initial characterization of 4 RNA processing genes (SF3b14, SF3b1/SAP155, Rpl7l1 and Rrp7a) required for blastocyst formation was later evaluated. The results of this work has been submitted for publication and will be published soon in the journal Zygote. SF3b14 and SF3b1, identified as being part of the splicesome complex, disproportionally contributes to gene transcription of those genes containing more than 1 exon verifying a role in RNA splicing. Rpl7l1, identified by GO terms as a possible ribosomal gene, was found to be present in the cytoplasm and, surprisingly, in the nucleus. It is surmised this gene influences polymerase 2 activity as Rpl7l1 gene knockdown embryos demonstrate reduced active polymerase 2 activity at the morula stage. Rrp7a was identified as being critical in blastocyst formation and is present in the cytoplasm while excluded from the nucleus. Based on location and GO terms, this suggests a role in translation. Taken together, these 4 genes act in 3 different ways impacting RNA production, splicing or translation promoting blastocyst formation in the mouse.
The final gene evaluated in this work was Bcl-6 corepressor (Bcor). As opposed to our previous work with RNA processing factors, this gene knockdown does not result in a failure to make a blastocyst. Bcor knockdown increases the rate of physiologically normal blastocysts in both murine and bovine models. Although further characterization must be done, temporary Bcor gene knockdown might be a useful improvement of in-vitro embryo culture systems including murine, bovine, equine and possibly even human.
This manuscript is divided into 4 chapters, the first of which is a review of preimplantation embryo development. This covers selected and relevant events between fertilization and just before implantation of the embryo into the uterus. I mainly focus on events after fertilization and the necessary changes required for zygotic genome transcription and lineage specification.
The second chapter characterizes WDR74, a gene we identified as critical in the formation of a blastocyst in a reverse genetic screen. As state before, we assess WDR74 function with the developing embryo and conclude the protein plays a role in RNA production and/or stability of RNA transcripts. We also test to rescue blastocyst formation in WDR74 knockdown embryos in an attempt to further evaluate WDR74 function.
We continue the characterization of genes whose temporary reduction causes the failure of blastocyst formation in the third chapter. Here we report on four additional RNA processing genes in a body of work which has been published in the journal Zygote. Since these genes contained similar GO terms, we assumed they may all function in a similar way so they were assayed together as a group. As function of these genes were unknown, we determined protein localization within the cell, function in RNA splicing, alternative splicing and to determine if the failure to make a blastocyst is due to lineage specification.
In the final chapter, BCOR gene expression is characterized in preimplantation embryo development as in the former 2 chapters. However, the result of this gene knockdown does not lead to the failure to make a blastocyst, rather this improves the number of blastocysts formed during the correct physiological time; the same time that blastocysts form invivo. Undoubtedly, this could lead to possible commercial applications which are reviewed along with the preliminary data we have been able to collect thus far. Specifically, the continuation of the BCOR gene knockdown research in preimplantation embryo development is pitched in the form of academic and international business collaboration with InvitroBrasil for the production of cloned bovine, equine and ICSI in equine
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
A journey through horse cloning
Interest in equine somatic cell nuclear transfer technology has increased significantly since the first equid clones were produced in 2003. This is demonstrated by the multiple commercial equine cloning companies having produced numerous cloned equids to date; worldwide, more than 370 cloned horses have been produced in at least six different countries. Equine cloning can be performed using several different approaches, each with different rates of success. In this review we cover the history and applications of equine cloning and summarise the major scientific advances in the development of this technology in horses. We explain the advantages and disadvantages of different procedures to produce cloned equine embryos and describe the current status of equine clone commercialisation, along with observations of differences in regional breed association registration regulations.Fil: Gambini, Andres. Universidad de Buenos Aires. Facultad de AgronomÃa. Pabellón de Zootecnica. Laboratorio de BiotecnologÃa Animal; Argentina. Consejo Nacional de Investigaciones CientÃficas y Técnicas; ArgentinaFil: Maserati, Marc. In Vitro Clonagem Animal; Brasi
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
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
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
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
Bovine blastocyst image - blq101
Image of an in vitro produced bovine blastocyst