MATERNAL mRNA LOCALIZATION IN THE FROG DEVELOPMENT Putting RNAs at the right place in the right time (stage 16) (stage 12) (dorsal view) GASTRULATION/NEURULATION kjjfelnil.il Kill I XT I us wtwi cieä Ii jusl below ihecaitwoflliegrjy (bfrti lliefkjfsJil llfj uf the Jutuve btalopore, 3 Cell* of the JinirmI pole spread out, (jushiiiji liiirfart' rt*lls Miiw lliflF toward 3 kI acrosi the ctorsal lip, Those cell i involule in lo ihc iftlwiof ül ih* embryo, whwdwy /«tu ilw emiexlerm .iitil nwitic-rm, © I ins Inviiliiimn mutes ilia.irchGntwon .irniidMn.ivMllH'lil.iMin LM-'t Nlinlnisil lip kiimi 2 ^irrlu, with col Is moving in lln; inltii™ .ill jKumr.1 iho bbskifjow-; the yflllt pluß is vbibb ifuougli du? RlastDcocI Animal pole Ebnddc r""--Düf«l lip (it Vegetal potle ANIMAL POLE .ArdKntcrom Mfsoh-mt Arthenlcron Ec toderni \ Rndodvnn \ \ -L. .^otochord [)or$al lip of Vvrsal lip of bli»topoff Volk pbp Ventrjilipof blast apon1 dorsal lip oT blastopore idi:.| Kill midline: y:i t plug VEGE1 AL POLE external views dorsal lip o1 blastopore nti-ural rjcloderrn nonrMUrai ectoderm cavity of gut (A] I mm cress- secti on s ondode-r of gut roef rnesode-mi I Continued (Woj)ffle^ jaws rise to. ro|f)fh()[(l derived from mesoderm. Q The beginning!ol' the nervous system (green) are derive torn Cloderm. Mtstfldiyaie Ectoderm a Endoderm N™] phfc McsMclwme ofl)™ Neural pUte Notochord , Dorsal lip oi ir bldislüporo Mesoderm ventral lateral plats v «nrjr>derrn IS. ectoderm lapi dermis) ectoderm (neural plain) --1:1. :■■ -I I ■: ■ -: I dorsal ACCURACY AND REPRODUCIBILITY IN ACQUIRING THE CELLULAR FATE WITHIN THE EMBRYO GASTRULATION MOVIE 1 - internal cell movements GASTRULATION MOVIE 2 - dorsal surface view - blastoporus closure Dorso-ventral axis is set-up by site of sperm entry Fertilization of egg sperm Animal pigmented /Cortex A Cytoplasm \ V\ cortex heavy yolk Vegetal 30° cortical rotation Direction of cortical rotation Late biastula Spemann Ü W \#\ f blastocoel \ä organizer blastopore Tailbud stage embryo GASTRULATION/NEURULATION - dorsal surface view Differential mRNA localization to subcellular compartments -allows for spatial regulation of gene expression -essential for polarity set-up in oogenesis -patterning during embryogenesis -in Xenopus: localized maternal mRNAs generate developmental polarity along the animal/vegetal axis. Fig. 2.4 The unfertilized egg of Xenopus. The surface of the animal half (top) is pigmented and the paler, vegetal half of the egg is heavy with yolk. Scale bar - 1 mm. Photograph courtesy of}. Smith. CELL-TO-CELL SIGNALING vs.MATERNAL FACTORS IN TISSUE SPECIFICATION Ectoderm and endoderm are specificed by maternal factors in the egg Mesoderm is induced by vegetal tissue Tissues formed from explants from a Xenopus late blastuta Animal animal cap cells Ventral Dorsal ectoderm vegetal cells Vegetal mesenchyme notochord mesoderm epidermis blood muscle neural tube endoderm undifferentiated vegetal tissue Vegetal tissue induces mesoderm in animal cap animal cap cells vegetal cells induced mesodermal tissue ,---1_ muscle . . mesenchyme notochord MATERNAL vs. ZYGOTIC REGULATORS Summary: genes involved in patterning of axes and germ layers Gene Maternal/ Type of protein Where Effects Zygotic expressed activin Z TGF-(3 famity ? mesoderm induction BMP-4 Z transcription factor late blastula ventralizes mesoderm Brachyury Z transcription factor early mesoderm mesoderm developmer /3-catenin M gene regulatory protein egg dorsalizing signal cerberus Z secreted vegetal egg mesoderm inhibition chord in Z secreted signal molecule organizer dorsalizes mesoderm derriere z TGF-p family vegetal egg mesoderm induction fibroblast z secreted signal molecule blastula ventral mesoderm growth induction factor goosecoid z transcription factor organizer organizer function GSK-3 M protein kinase egg suppresses dorsaMzing signals HNF~3{3 z transcription factor organizer org a nizer development noggin M/Z secreted organizer dorsalizes mesoderm Pintatlavis Z transcription factor organizer ? siamois z transcription factor dorsal blastula dors3liz!na sianal VegT M transcription factor vegetal egg induces endoderm and mesoderm signals Vg-1 M TGF-p family vegetal egg mesoderm induction Xlim- 1 Z transcription factor organizer Xnot Z transcription factor organizer notochord specification Xnr-7 Z secreted vegetal egg mesoderm induction Xnr-2 z secreted vegetal egg mesoderm induction Xnr-A z secreted vegetal egg mesoderm induction Xwnt-11 M Wnt family vegetal egg mesoderm induction Xwnt-8 z Wnt family propective mesoderm ventralizes mesoderm VegT (T-box family transcription factor) A - stage I oocytes B - stage IV oocytes C - ovulated egg D - stage 9.5 embryo E - stage 9.5 embryo (vegetal pole view) F - stage 10.25 embryo (vegetal pole view) G - stage 10.5 embryo (vegetal view) H - stage 12.5 embryo (posterior view) I- mid neural fold embryo (stage 16) VegT RNA injection into vegetal/ventral blastomeres can induce secondary exis via induction of dorsal fate....... I - primary axis II - secondary axis nt - neural tube notorchord green - muscles arrow - ectopic auditory vesicles .........by activation of Xwnt8/ß-catenin pathway Xenopus embryo at the four-cell stage divided into dorsal and ventral halves sperm entry point Übt IN Ventral half lacks Nieuwkoop center dorsal hall Nieuwkoop center ventral half The ventral 'half develops into a ventralized embryo. The dorsal halt develops into a dorsalized embryo ventralized embryo dorsalized embryo Vgl (TGF(3 family ligand) Vg1 depletion by morpholinos delayes gastrulation and mesoderm induction with loss of head structures, absence of notochord and fusion of somites (arrow) A C .....via loss of the induction of the mesodermal markers F mesoderm markers res taps alone caps on caps on wt bases Vg1- bases mesoderm t Vg1 MECHANISMS OF INTRACELLULAR mRNA SORTING Colonization VLE - Vg1 SUK4- kinesin-1 heavy chain IP IP SIO lgGaSUK4 SlQ IgG «SUM 12 3 12 3 4«^ 6 ^4 5 6 total oocyte lysate uSUK2|C m D t Injection Localization (%) n igG 100 62 aSUK2 102 36 aSUK4 52 53 Vg1 mRNA localization ixSUK4 IgG - isotypic control SUK2 -non-neutralizing Ab SUK4 -neutralizing Ab XKHC - kinesin 1 Red - injected Vg1 RNA XKHC-T591 - rigor mutant (can not move - binds tight to microtubules) XEg5-T105N - rigor mutant of kinesin unrelated to XKHC Control XKHC XKHCT92I XEgS XEg5-TlQ5N Construct Expressed KINESIN TRANSPORT 1 full cycle ■ uses 2 ATP - moves 16 nanometers Kinesin Spiral motor protein ("walks" along microtubules} Rotation of 1 st leg" and "foot" "Cargo" being carried to destination Sliding action of 2nd, leg" and "foot11 (different to 1st to prevent twisting of spiral) 1BOB3 * 4 1M■3 4 16 nmtrs, t Surface of microtubules \f (passageways) beta-tubulin alpha-tubulin The (Ncr) Kinesin "walking" transportation of cell chemicals Xcat2-RNP (early pathway gene) (transport over long distances - both late and early pathway genes hitch a ride)