MATERNAL mRNA LOCALIZATION IN THE FROG DEVELOPMENT Putting RNAs at the right place in the right time (stage 16) (stage 12) (dorsal view) CLEAVAGE MOVIE ACCURACY AND REPRODUCIBILITY IN ACQUIRING THE CELLULAR FATE WITHIN THE EMBRYO Fig. 3.17 Fate mapping of the early Xenopus embryo. Left pane!: a single cell in the embryo, C3, is labeled by injecting fl uo rescein-d extra n-a mine, which fluoresces green under UV light, Right panel; a cross-section of the embryo, made at the tailbud stage, shows that the labeled cell has given rise to mesoderm cells on one side of the embryo. Scale bar = 0.5 mm. Photograph courtesy off.. Dale. Fate map: lateral view Animal / epidermis \ I / nervous \ / / system \ Ventral —„_J Dorsal ........ somites, ^a^B \blood, kidney"--, heart '. / ^x^^^en d od e rrn^^y^ marginal notochord Vegetal zone 3 MANY CLEAVAGES MOVIE Fate map: dorsal view Animal ^^^^^ Vegetal Fig. 3.IS Fate map of a late Xenopus blastula. The ectoderm gives rise to the epidermis and nervous system. Along the dorso-ventral axis the mesoderm gives rise to notochord, somites, heart, kidneys, and blood. Note that blood can also form in more dorsal regions. In Xenopus, although not in all amphibians, there is also endoderm (not shown here) overlying the mesoderm in the marginal zone. ti .Mu ml Animal pnle GASTRULATION/NEURULATION wtwii ceí Iíí |iiíi Iwlmv ihe centwof ihegray taní IhpfbíHi] lífj oí ihe lulur? blü5lupcw, Q Cfelfe of ihe animal půhe spread (Krt, iJltShiilo íiuríáCO ťpltfi Mow lhář twaiíi ji uri .icrass tíie íbrs.il ftfj. Those ccII s involute nii<< ih< ni\rtnitcí ill*.-ťiribryo, whi>KMhřy if>rrn iht" rrnk>(l(*nri iind nrcsodeirm. ©Tlik muohiiion cntftť*5 ilir? .irdh«)k*riHi :itid (t^im^i ÜK1 lilj^utoel. The doisol lip rwrnj a ťirrlr, wiřh rolí; nu v. in-- m ihi.1 inítítior nil aitaind iho liljutofion?; thr* ydfc pluj; ls vříibk? thnmgh iJn? Ji-^UVii blastapow ImhJckKmui Ectodenn\ Endoderm [torsa! |jpf>f \ egetsl pole TíPíSal Hp I blitsttipore Ydk plug Ventil !ip of blast(jpurr ANIMAL POLE we-nUfll mid lina dorsal lip of blastopore ■ Ji :■■ k:iI midline yolk plug VEGETAL POLE external views blaalocael dorsal lip o1 blastopore no-ural actoderm '!)'■..... r.i ecloderm Oblirtrijirjd bla h1 aeoel yoik cavity af gut andodarm of gut roof (A| 1 mm □ross-secti uns 0 Cwilinuml (IwkpTiwi hí ves rise lo .i niitcH;hurd OfrivdírninmrstxkríTt. Q Üvlwginnin^oflhe nervous system ßreriil AK iJcrivíd in mi vi Fi liI<-i ni M^ildiymp Ectoderm EtiÄn pl,1tc Mesenchyme ot brain Nutochnrd . Dorsal lip of ' Wastopore Mesoderm ventral ectoderm I epidermis; laloral plíi ta I!, endoderm ■..-1 -11 :■ i: i: r i -1 lllULirLlI Ll ĚIIlO notoeryord blfl-alapore lip mhos dorsal Neural platě 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 GASTRULATION/NEURULATION - dorsal surface view Differential mRNA localization to subcelular 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 j. Smilh. CELL-TO-CELL SIGNALING vs.MATERNAL FACTORS IN TISSUE SPECIFICATION Ectoderm and endoderm are specificed by maternal factors in the egg. Versus mesoderm that is induced by vegetal tissue Tissues formed from explants from a Xenopus late blastula Animal animal cap celfs 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 ceils induced mesodermal tissue '---■—:- muscle , . mesenchyme i notochord MATERNAL vs. ZYGOTIC REGULATORS Summary: genes involved in patterning of axes and germ layers Gene activin BMP-4 Brachyury {3-catenin cerberus chordin derriere fibroblast growth factor goosecoid GSK-3 HNF-3& noggin Pintoifavis siamois Maternal/ Type of protein Zygotic Z Z z M Z z z z M Z M/Z Z Z TCF-p family transcription factor transcription factor gene regulatory protein secreted secreted signat molecule TCF-p family secreted signat molecule transcription factor protein kinase transcription factor secreted transcription factor transcription factor Where expressed late blastula early mesoderm egg vegetal egg organ izer vegetal egg blastula organizer egg organizer organizer organizer dorsal blastula Effects mesoderm induction ventralizes mesoderm mesoderm development dorsalizing signal mesoderm inhibition dorsalizes mesoderm mesoderm induction ventral mesoderm induction organizer function suppresses dorsalizing signals org a nizer development dorsalizes mesoderm dorsalizing signal_ VegT Vg-1 M M transcription factor TGF-p" family vegetal egg vegetal egg induces endoderm and mesoderm signals mesoderm induction KVsm-7 Xnot Xnr-7 Xnr-2 Xnr-4 Xwnt- 7 7 Xwnt-8 Z Z Z z z M Z transcription Tactor transcription factor secreted secreted secreted Wnt family Wnt famiiy organizer organizer vegetal egg vegetal egg vegetal egg vegetal egg propective mesoderm notochord specification mesoderm induction mesoderm induction mesoderm induction mesoderm induction ventralizes mesoderm Vgl (TGFp family ligand) Vgl 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 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 nc - notorchord green - muscles arrow - ectopic auditory vesicles VegT RNA ^^^^ 5 F nt ^1 * .........by activation of Xwnt8/ß-catenin pathway Xenopus embryo at the four-cell stage divided into dorsal and ventral halves sperm entry point IP Ventral half lacks Nieuwkoop center dorsal half Nieuwkoop center ventral half The ventral half develops into a ventralized embryo. The dorsal half develops into a dorsalized embryo centralized embryo dorsalized embryo MECHANISMS OF INTRACELLULAR mRNA SORTING II Colocfllizalicm o VLE -Vgl SUK4- kinesin-1 heavy chain IP IP VLE* Vg1* ^9^D 1 2 J 12 3 4\ 5 6 *4 5 6 total oocyte lysate >9GlB mSUK2IC uSUK4 D Injection Localization (%} n IgQ 100 62 0(SUK2 1 102 36 ft3UK4 52 53 Vgl mRNA localization IgG - isotypic control SUK2 -non-neutralizing Ab SUK4 -neutralizing Ab RNA in RNP granules 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 "foor (different to 1st to prevent twisting of spiral) 4'm 2 m®m4 2 Surface of microtubules ~ \s* {passageways} beta-tubulin afpha-tubulin The (Ncr) Kinesin "walking" transportation of cell chemicals KINESIN WALK MOVIE