4/19/15&1&Regional&Specifica3on/Pa5ern&forma3on&How&to&make&a&fruit&fly:&anteriorAposterior&pa5ern&Learning&objec3ves:&1. Understand&the&basis&for&body&pa5erning&in&Drosophila:&&coordinated&ac3vity&of&three&pa5erning&systems.&&One&for&anteriorAposterior&(composed&of&three&different&subsystems),&one&for&dorsalAventral,&and&one&for&specifying&the&iden33es&of&segments.&2. Understand&the&different&roles&of&three&proteins&in&the&egg&that&act&as&cytoplasmic&determinants&to&set&the&AAP&pa5ern.&3. Know&the&three&sets&of&embryonicallyAexpressed&genes&that&act&sequen3ally&to&divide&the&embryo&into&stripes&of&gene&expression&that&lead&to&the&specifica3on&of&segment&number,&spacing&and&polarity.&4. Understand&the&example&of&how&transcrip3onal&regula3on&establishes&the&stripes&of&gene&expression.&pie$1&mutant!ABaABpEMSEMSnormal& parA1& mexA5/6&par&mutants&mex'5/6+Posterior&PARàMEXA5/6à&PIEA1&P&granules&How&are&pieA1&and&P&graunules&localized?&pie'1+MEXA5&pie-1 mutant required for specifying How are pie-1 and P graunules localized? when par-1 and mex-5/6 were missing the gene failed to localize posterior Par -> Mex 5/6 -> Pie 1 P granules4/19/15&2&Ques3ons&we&will&address&today:&&1) How&do&cells&come&to&be&different&in&appropriate&and&reproducible&places?&(Regional!Specifica.on)&2) How&do&similar&cell&types&organize&into&different&shapes&and&pa5erns?&(Pa0ern!forma.on)&How&do&you&make&a&fruit&fly?&1. Introduc3on&to&the&fruit&fly&model.&2. Gene3c&approach&to&understanding&development:&satura3on&screening&3. Oocyte&(maternal)&contribu3on&to&fruit&fly&embryonic&pa5ern&forma3on&&4. Embryonic&contribu3on&to&fruit&fly&embryonic&pa5ern&forma3on&5. Linking&pa5ern&forma3on&to&transcrip3onal&regula3on&–&how&does&the&fly&ac3vate&the&evenAskipped&gene&in&a&pa5ern&of&stripes?&iclicker question: Bug-1 -> Ant-3 -> Grub-5 B is dependent on A Outcomes: Ant-3 and Grub-5 proteins are mislocalized in bug-1 mutants, and Grub-5 is mislocalized in ant-3 mutants E) more than one is correct4/19/15&3&Easy&to&culture&&Grows&in&bo5les&with&yeast&for&food&&Rapid&development&&24&hours&to&hatch&&Simple&gene3cs&&4&chromosomes&15,000&genes&&Sophis3cated&gene3c&and&molecular&biology&tools&&More&than&a&century&of&gene3cs&combined&with&tools&for&isola3ng&DNA&corresponding&to&genes&iden3fied&by&muta3on.&Completely&sequenced&genome.&&embryo starts out with 15 germ cells dumps into oocyte4/19/15&4&Drosophila&starts&life&as&a&syncy.um*.&Syncy.al!blastoderm**!Cellular!blastoderm!Cytoplasmic&determinants&provided&in&the&egg&&Transcrip3onal&cascades&Intercellular&signaling&*Synci3um&=&Mul3ple&nuclei&in&a&common&cytoplasm.&**Blastoderm&=&Insect&blastula&Gut&and&posterior&terminus&Gut&and&anterior&&terminus&Crude&fate&map&of&A/P&cell&fates&nuclei divide but cells don't divide 3rd figure: what nucleus makes goes into the common cytosol 3rd figure: syncytial blastoderm transition between syncytial and cellular blastoderm: cell boundaries comes down and the cells connect to become a cellular blastodermnuclei can communicatenuclei have to communicate with intercellular signalinglarva: allow researchers to ask how development is going wronggrey areas: giving rise to terminal structures (very important structures)4/19/15&5&1. Introduc3on&to&the&fruit&fly&model.&2. Gene3c&approach&to&understanding&development:&satura3on&screening&3. Oocyte&(maternal)&contribu3on&to&fruit&fly&embryonic&pa5ern&forma3on&&4. Embryonic&contribu3on&to&fruit&fly&embryonic&pa5ern&forma3on&5. Linking&pa5ern&forma3on&to&transcrip3onal&regula3on&–&how&does&the&fly&ac3vate&the&evenAskipped&gene&in&a&pa5ern&of&stripes?&Chris3ne&NussleinAVolhard&and&Eric&Wieschaus&won&the&1995&Nobel&Prize&in&Physiology&and&Medicine&for&their&work&on&embryonic&pa5erning&in&Drosophila.&Forward&gene3c&“satura3on”&screens&&Muta3ons&that&altered&the&pa5ern&of&the&1st&larval&stage&cu3cle.&&• Maternally&expressed&genes&(loaded&into&oocyte)&• Embryonically&expressed&genes&(transcribed&from&the&embryonic&genome)&changes in cutical pattern, easy to see and interpret and what went wrong4/19/15&6&1. Introduc3on&to&the&fruit&fly&model.&2. Gene3c&approach&to&understanding&development:&satura3on&screening&3. Oocyte&(maternal)&contribu3on&to&fruit&fly&embryonic&pa5ern&forma3on&&4. Embryonic&contribu3on&to&fruit&fly&embryonic&pa5ern&forma3on&5. Linking&pa5ern&forma3on&to&transcrip3onal&regula3on&–&how&does&the&fly&ac3vate&the&evenAskipped&gene&in&a&pa5ern&of&stripes?&Gene3c&screens&revealed&egg&components&that&specified&the&anterior/posterior&polarity&of&the&embryo.&&Egg=polarity!genes!maternal: m-RNA or n-RNA that are placed into the egg and control embryonic pattern formationlooking for changes in the cuticalar pattern that affect the polarity of the embryo^ removed all 3 systems < 1 mutant can act independently4/19/15&7&Events&during&oogenesis&establish&asymmetric&distribu3ons&of&key&cytoplasmic&determinants&in&the&early&embryo&Torso&AA&receptor&tyrosine&kinase&Bicoid&–&regulator&of&transcrip3on&Dorsal&Nanos& Bicoid&Graded&morphogen&Torso&Nanos&–&regulator&of&transla3on&Field&of&equipoten3al&cells&Graded!morphogen!Cell&fate&is&dependent&upon&level&of&morphogen&Thresholds&A&B&C&D&B&C& D& E&A&Posterior System: main component called NanosPosterior System: localized mRNA is located in the posteriorGraded Morphogen:How it works: different levels across an equal potential fields of cells lets scientists make different copies of Bicoid4/19/15&8&Evidence&that&Bicoid&acts&as&a&graded&morphogen&comes&from&finding&that&changing&the&gradient&results&in&changes&in&the&pa5ern.&Increasing&copies&of&bicoid&gene&Posterior&shio&of&dorsal&fold&1&copy&2&copies&3&copies&4&copies&Events&before&ovula3on&establish&asymmetric&distribu3ons&of&key&cytoplasmic&determinants&in&the&early&embryo&Torso&AA&receptor&tyrosine&kinase&Nanos&–&regulator&of&transla3on&Bicoid&–&regulator&of&transcrip3on&Dorsal&Nanos& Bicoid&Graded&morphogen&Torso&higher number of Bicoid -> granulate changesBicoid acts as a Morphogen degranulate isn't localized to posteriorNanos: creates a protein gradient but doesn't function as a gradient morphogen4/19/15&9&How!can!a!receptor!(torso)!act!as!a!cytoplasmic!determinant?!Satura3on&screens&also&iden3fied&egg&components&required&for&specifying&fates&along&the&dorsal/ventral&axis.&&(You&do&not&need&to&know&how&this&works&only&that&this&D/V&pa5erning&system&exists.)&yellow thing in center: