Restricted accessResearch articleFirst published online 2016-06
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
The epiblast (foremost embryonic epithelium) 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.
Get full access to this article
View all access options for this article.
References
1.
WellingM, and GeijsenN. Uncovering the true identity of naive pluripotent stem cells. Trends Cell Biol, 2013; 23:442–448.
2.
SasakiT, FasslerR, and HohenesterE. Laminin: the crux of basement membrane assembly. J Cell Biol, 2004; 164:959–963.
3.
TamPP, and LoebelDA. Gene function in mouse embryogenesis: get set for gastrulation. Nat Rev Genet, 2007; 8:368–381.
4.
TamPP, LoebelDA, and TanakaSS. Building the mouse gastrula: signals, asymmetry and lineages. Curr Opin Genet Dev, 2006; 16:419–425.
5.
NicholsJ, and SmithA. Pluripotency in the embryo and in culture. Cold Spring Harb Perspect Biol, 2012; 4:a008128.
6.
BrennanJ, LuCC, NorrisDP, et al.Nodal signalling in the epiblast patterns the early mouse embryo. Nature, 2001; 411:965–969.
7.
TaoH, SuzukiM, KiyonariH, et al.Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical–basal polarity. Proc Natl Acad Sci U S A, 2009; 106:14426–14431.
8.
LiWL, and DingS. Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming. Trends Pharmacol Sci, 2010; 31:36–45.
9.
Iwafuchi-DoiM, MatsudaK, MurakamiK, et al.Transcriptional regulatory networks in epiblast cells and during anterior neural plate development as modeled in epiblast stem cells. Development, 2012; 139:3926–3937.
10.
KoehlerKR, MikoszAM, MoloshAI, et al.Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature, 2013; 500:217–221.
11.
EirakuM, TakataN, IshibashiH, et al.Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature, 2011; 472:51–56.
12.
SasaiY, EirakuM, and SugaH. In vitro organogenesis in three dimensions: self-organising stem cells. Development, 2012; 139:4111–4121.
13.
SasaiY. Cytosystems dynamics in self-organization of tissue architecture. Nature, 2013; 493:318–326.
14.
SasaiY. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell, 2013; 12:520–530.
15.
LancasterMA, RennerM, MartinCA, et al.Cerebral organoids model human brain development and microcephaly. Nature, 2013; 501:373–379.
16.
MatanoM, DateS, ShimokawaM, et al.Modeling colorectal cancer using CRISPR–Cas9-mediated engineering of human intestinal organoids. Nat Med, 2015; 21:256–262.
17.
SanderJD, and JoungJK. CRISPR–Cas systems for editing, regulating and targeting genomes. Nat Biotechnol, 2014; 32:347–355.
18.
MarraffiniLA, and SontheimerEJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet, 2010; 11:181–190.
19.
HsuPD, LanderES, and ZhangF. Development and applications of CRISPR–Cas9 for genome engineering. Cell, 2014; 157:1262–1278.
20.
GajT, GersbachCA, and BarbasCF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol, 2013; 31:397–405.
21.
MusunuruK. Genome editing of human pluripotent stem cells to generate human cellular disease models. Dis Model Mech, 2013; 6:896–904.
22.
WatayaT, AndoS, MugurumaK, et al.Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation. Proc Natl Acad Sci U S A, 2008; 105:11796–11801.
23.
EirakuM, WatanabeK, Matsuo-TakasakiM, et al.Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell, 2008; 3:519–532.
24.
OhgushiM, MinaguchiM, and SasaiY. Rho-signaling-directed YAP/TAZ activity underlies the long-term survival and expansion of human embryonic stem cells. Cell Stem Cell, 2015; 17:448–461.
25.
SugaH, KadoshimaT, MinaguchiM, et al.Self-formation of functional adenohypophysis in three-dimensional culture. Nature, 2011; 480:57–62.
26.
KamiyaD, BannoS, SasaiN, et al.Intrinsic transition of embryonic stem-cell differentiation into neural progenitors. Nature, 2011; 470:503–509.
27.
UedaHR, MatsumotoA, KawamuraM, et al.Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J Biol Chem, 2002; 277:14048–14052.
28.
KasukawaT, MasumotoKH, NikaidoI, et al.Quantitative expression profile of distinct functional regions in the adult mouse brain. PLoS One, 2011; 6:e23228.
29.
IkeyaM, KawadaM, NakazawaY, et al.Gene disruption/knock-in analysis of mONT3: vector construction by employing both in vivo and in vitro recombinations. Int J Dev Biol, 2005; 49:807–823.
30.
HansenK, CoussensMJ, SagoJ, et al.Genome editing with CompoZr custom zinc finger nucleases (ZFNs). J Vis Exp, 2012; 64:e3304.
31.
LoveNR, ThuretR, ChenY, et al.pTransgenesis: a cross-species, modular transgenesis resource. Development, 2011; 138:5451–5458.
32.
OkabeM, IkawaM, KominamiK, et al.“Green mice” as a source of ubiquitous green cells. FEBS Lett, 1997; 407:313–319.
33.
RamezaniA, HawleyTS, and HawleyRG. Performance- and safety-enhanced lentiviral vectors containing the human interferon-β scaffold attachment region and the chicken β-globin insulator. Blood, 2003; 101:4717–4724.
34.
SekkaliB, TranHT, CrabbeE, et al.Chicken β-globin insulator overcomes variegation of transgenes in Xenopus embryos. FASEB J, 2008; 22:2534–2540.
35.
AllenBG, and WeeksDL. Transgenic Xenopus laevis embryos can be generated using φC31 integrase. Nat Methods, 2005; 2:975–979.
36.
CummingG, FidlerF, and VauxDL. Error bars in experimental biology. J Cell Biol, 2007; 177:7–11.
37.
DunnNR, and HoganBLM. How does the mouse get its trunk?. Nat Genet, 2001; 27:351–352.
38.
Martin-BelmonteF, and Perez-MorenoM. Epithelial cell polarity, stem cells and cancer. Nat Rev Cancer, 2012; 12:23–38.
StemmlerMP. Cadherins in development and cancer. Mol Biosyst, 2008; 4:835–850.
41.
YuW, DattaA, LeroyP, et al.β1-Integrin orients epithelial polarity via Rac1 and laminin. Mol Biol Cell, 2005; 16:433–445.
42.
O'BrienLE, JouTS, PollackAL, et al.Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat Cell Biol, 2001; 3:831–838.
43.
MinerJH, and YurchencoPD. Laminin functions in tissue morphogenesis. Annu Rev Cell Dev Biol, 2004; 20:255–284.
44.
DuJ, TakeuchiH, Leonhard-MeliefC, et al.O-Fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation. Dev Biol, 2010; 346:25–38.
45.
KemlerR, HierholzerA, KanzlerB, et al.Stabilization of β-catenin in the mouse zygote leads to premature epithelial–mesenchymal transition in the epiblast. Development, 2004; 131:5817–5824.
46.
KunathT, Saba-El-LeilMK, AlmousailleakhM, et al.FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development, 2007; 134:2895–2902.
47.
MarkevichNI, HoekJB, and KholodenkoBN. Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. J Cell Biol, 2004; 164:353–359.
48.
BetschingerJ, NicholsJ, DietmannS, et al.Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell, 2013; 153:335–347.
49.
RobsonP, SteinP, ZhouB, et al.Inner cell mass-specific expression of a cell adhesion molecule (PECAM-1/CD31) in the mouse blastocyst. Dev Biol, 2001; 234:317–329.
50.
FurusawaT, OhkoshiK, HondaC, et al.Embryonic stem cells expressing both platelet endothelial cell adhesion molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in a chimeric embryo. Biol Reprod, 2004; 70:1452–1457.
51.
BedzhovI, GrahamSJ, LeungCY, et al.Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo. Philos Trans R Soc Lond B Biol Sci, 2014; 369:20130538.
52.
PeltonTA, SharmaS, SchulzTC, et al.Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development. J Cell Sci, 2002; 115:329–339.
53.
KrensSF, Corredor-AdamezM, HeS, et al.ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis. BMC Genomics, 2008; 9:196.
54.
AkhtarN, and StreuliCH. An integrin–ILK–microtubule network orients cell polarity and lumen formation in glandular epithelium. Nat Cell Biol, 2013; 15:17–27.
55.
VespaA, D'SouzaSJ, and DagninoL. A novel role for integrin-linked kinase in epithelial sheet morphogenesis. Mol Biol Cell, 2005; 16:4084–4095.
56.
OhtsukaS, Nishikawa-TorikaiS, and NiwaH. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development. PLoS One, 2012; 7:e45220.
57.
BryantDM, and MostovKE. From cells to organs: building polarized tissue. Nat Rev Mol Cell Biol, 2008; 9:887–901.
58.
de SouzaN. Many routes to pluripotency. Nat Methods, 2009; 6:864–865.
59.
HuangDW, ShermanBT, and LempickiRA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc, 2009; 4:44–57.
60.
PapaioannouVE, and BehringerRR. Early embryonic lethality in genetically engineered mice: diagnosis and phenotypic analysis. Vet Pathol, 2012; 49:64–70.
61.
DoreyK, and AmayaE. FGF signalling: diverse roles during early vertebrate embryogenesis. Development, 2010; 137:3731–3742.
62.
DubrulleJ, and PourquieO. fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature, 2004; 427:419–422.
63.
KieckerC, and NiehrsC. A morphogen gradient of Wnt/β-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development, 2001; 128:4189–4201.
64.
GuptaS, SchoerRA, EganJE, et al.Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci U S A, 2004; 101:1927–1932.
65.
GossenM, FreundliebS, BenderG, et al.Transcriptional activation by tetracyclines in mammalian cells. Science, 1995; 268:1766–1769.
66.
FireA, XuS, MontgomeryMK, et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998; 391:806–811.
67.
PrattAJ, and MacRaeIJ. The RNA-induced silencing complex: a versatile gene-silencing machine. J Biol Chem, 2009; 284:17897–17901.
68.
YinH, KanastyRL, EltoukhyAA, et al.Non-viral vectors for gene-based therapy. Nat Rev Genet, 2014; 15:541–555.
69.
KogureT, KawanoH, AbeY, et al.Fluorescence imaging using a fluorescent protein with a large Stokes shift. Methods, 2008; 45:223–226.
70.
YonemuraS. Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment. PLoS One, 2014; 9:e112922.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
