title: Establishment of Functional Genomics Pipeline in Mouse Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.
abstract: The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of mouse embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics.
authors.affiliation: 1 Laboratory for In Vitro Histogenesis, RIKEN Center for Developmental Biology , Hyogo, Japan .
authors.forename: Nozomu
authors.initials: N
authors.lastname: Takata
authors.affiliation: 1 Laboratory for In Vitro Histogenesis, RIKEN Center for Developmental Biology , Hyogo, Japan .
authors.forename: Eriko
authors.initials: E
authors.lastname: Sakakura
authors.affiliation: 2 Large Scale Data Managing Unit, RIKEN Center for Life Science Technologies , Kanagawa, Japan .
authors.forename: Takeya
authors.initials: T
authors.lastname: Kasukawa
authors.affiliation: 3 Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University , Hiroshima, Japan .
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: 3 Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University , Hiroshima, Japan .
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: 4 Laboratory for Organogenesis and Neurogenesis, RIKEN Center for Developmental Biology , Hyogo, Japan .
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Sasai
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D051521:Fibroblast Growth Factor 5 / Q000172:deficiency* / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D016678:Genome; D023281:Genomics / Q000379:methods*; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D034622:RNA Interference
chemical_list: C496450:Fgf5 protein, mouse; D051521:Fibroblast Growth Factor 5