2 gene protein cell organ animal Extra-cellularenvironment • • • • •Drosophila,C.elegans,zebrafish, medaka fish,mouse •Mouse Drosopila,C.elegans (saturationmutagenesis) GeneticdissectionofA-Ppatterningofthebody 1. 2. ( 3. 4. Christiane Nüsslein-Volhard and EricWieschaus usedgeneticstoidentify proteinsthatsetupthe embryonicbodyplan 9 Nobelprizephysiologyandmedicine 1995 24h Fig.2.1 2010 PrinciplesofDevelopment4e V D 3 1. EMS 2. 3 m 3. 1 2 m m PrinciplesofDevelopment4e m m m Wolpert/TickleCopyright©2011byOxfordUniversityPress Screenfordevelopmentalmutants(Drosophila) Lethalhits=100%(essentialgenes- ca.5.000) (efficiencyofmutagenesis=numberofhitspergene) zygoticmutants embryonal-lethalmutants withmorphologicaldefects - segmentationdefects(AP) - tissuetypesdefective(DV) female-sterilemutants witheffectsonembryogenesis (=maternal-effectmutants) - antero-posteriorpattern - dorso-ventralpattern 100% 25% 3% 0.5% 0.5% 8% 2% 0.4% 0.3% (male-sterilemutants) ca.2%ofallgenesinvolvedinembryopatternformation (ca.100of>15.000protein-encodinggenes,only5.000essentialgenes) 1 wildtype leu1 Xleu2 Wieschaus and Nüsslein-Volhard collected mutants that affect the fly body plan until they did not see anymore new phenotypes leu1 leu1 X leu+ X X leu2 X leu+ leu2 Allele leu- leu+ 2 Alleles:differentversionofagene A. B. Lossoffunction(lf)alleles • hypomorphs – someactivityremains • amorphs - phenotypicnulls,tight,strong • null- nogeneproduct Gainof function(gf)alleles Generallyassociatedwithmisregulation ofageneproduct’sactivity,andare generallydominant. • Hypermorph - extraactivity • Neomorph - newactivity • antimorph - dominantnegative segment3 Wildtype Threeexamples Slight-lf hypomorphs Moderate-lf Nullmutant amorphs Antennapedia Antenna primordia antenna Leg primordia leg wildtype antenna antenna leg leg dorsal mutant cactus mutant dorsalized ventralized Antplf A7 A8 A6 A5 A2 A3 A4 T3 A1 T1 Antpgf T2 Antennapedia isexpressedin thesecondthoracicsegment wherethesecondlegwillform. Wild type Epistasis Epistasis Loss-of-functionalleles spz Toll pelle tube dl phenotype dorsalized dorsalized dorsalized dorsalized dorsalized cact ventralized Doublemutants spz cact Toll cact pelle cact tube cact dl cact ventralized ventralized ventralized ventralized dorsalized spz,Toll, pelle,tube, cact dl Dorsal ventralized Tollgf spz ventralized dorsalized dorsalized dorsalized pelle Tollgf tube Tollgf dl Tollgf spz Toll dlisepistatic tocact =dlhasamore predominantrole thancact. pelle tube cact dl Toll amnio serosa pelle tube dorsal ectoderm neuro-ectoderm cact dl mesoderm Dorsal Dl nuclear protein bicoid Tollgf Toll spz Wild type Whatdoesthisadditionalinformation tellyouabouttheorderoffunction? toll mutant cactus mutant PrinciplesofDevelopment4e Wolpert/TickleCopyright©2011byOxfordUniversityPress PrinciplesofDevelopment4e Wolpert/TickleCopyright©2011byOxfordUniversityPress Kyotolargescalemutagenesisscreenusingmedaka (1998-2004) F2 families screened: 1137 (1588 genomes) • F2 mated pairs: 6088 • Clutches screened: 24887 • Total eggs screened: 261617 • people involved: • embryonic lethals: m 脊椎動物での初めての神経系形成に関す る 細胞系譜解析 （メダカ 13-44時間） Hirose, Varga, Kondoh, Furutani-Seiki., Development 2004 組織の配置 m m m m Furutani-Seikiatal.,medaka specialissue, Mech.Dev. 121:647-58.2004 組織の 三次元化 Micropipett aspiration ES/iPS cell Furutani-Seiki et al. Nature YAP (Yes associatedprotein) Yes:Yamaguchisarcomavirusoncogene • 核に移行して、転写 因子と結合し、標的遺伝 子の転写活性化により細 胞増殖を促進する。 • YAPの核への移行は、 上流のがん抑制Hippoシ グナルによってYAPがリ ン酸化され、抑制される in press 2310 • mutations/ F2 family: 2.03 • mutations with specific phenotypes: 312 (118 loci) 19 • • • • 38 covers ca 80% of the genome m i i 3D 3D 神経板 の形成 位置情報 の付与 神経板 の形成 組織張力の 調整 ES3D 3D 位置情報 の付与 組織張力 の調整 ES3D Sasai Y.,Nature493:318(2013) Sasai Y.,Nature493:318(2013) i 1917) YAP 3D 神経板 の形成 位置情報 の付与 3D 組織張力 の調整 YAP ES3D Sasai Y.,Nature493:318(2013) Dongetal.,Cell,2007 1wk 4wks 8wks 11wks 1. 2. 3. YAP/TAZ 50% YAP/TAZ P.196,197 Neoblasts:stemcells EDFig.19.5 MBC:Fig.22-36 Adultstemcells( • : (stemcellniche) (cancerstemcells) Fig.23-31,MBC / Yoshiki Sasai, Next-Generation Regenerative Medicine: Organogenesis from Stem Cells in 3D Culture MBC:Fig.22-1,6 miniorgan CRISPR(ClusteredRegulatoryInterspacedpalindromicRepeats) genome (Church,Science339:823,2013) • 1 4 • • / • 3Dspheroid Takebe etal,.CellStem Cell16,2015 CRISPR ClusteredRegularlyInterspacedShortPalindromicRepeat(CRISPR) 1988-2005 F1000PrimeRep. 2014eCollection 2014.
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