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Lh---B Cumuluscells MCB 141 Lecture9 March20,2014 Mammalian(mouse) development The mouseis a eutherianmammal, an amniotewith full placental development. (Non-eutherianmammals are: 1) monotremes (platypus, echidna), which have yolky eggs and no placental development; and 2) marsupials,which have slightly yolky eggs and brief late placentation.) Mammalsare unitedby theirmilk production, hair, and a few othertraits, but not placentation. Simitaitiesof mammati and birdsin their developmeni: .Like birds,mammals have a blastodisc(flat in humans,although in mouseit is bent intoa cup-shape), .a hypoblast(amphibians'have no hypoblast),a primitivestreak, and Hensen'snode, .Likebirds they have 4 extraembryonictissues including a yolk sac even thoughthere is no volk. Differencesof mammalsand birds: .The mammalianegg is very small(100pm) and containsno volk,few stockpiledmaternalmaterials, little or no cell polarityrelated to axes of the embryo,and probablyno germ plasmgranules. .Gene expressionstarts very early,at the 2 cell stage.Axis specificationoccurs by mechanismsinvolving cell interactionsand early cell function,rather than by cytoplasmiclocalizations. .A mammalianinnov ir is the trophobla , an extraembryonictissue which is specialized for placentaldevelopment, i.e., for implantationin the uterinewall, uptakeof nutrients,and waste removal. This ectodermis comoletelyseoarate from embrvonicectoderm and differentiatesmuch earlier. lt replaces and goes beyondthe chorionicectodermpf birds. Germ cells and oogonesls: mammalian germ cells don't have germ plasm granules. Their origin is not understood. They arise in prirnitive gut tissue and migrate through the gut lining and into the gonad. Zou pcllu<ldr . - I (viblUre eovelope) I Cumulus .tI cells &w\mtero' I villi Coosld elB r<o At ovulationthe ooryte is releasedform the fotlicl'e but is slillmvered GRAA.FIAN FOLUCLE lH---b cumuluscells. They fall off soonafter fertilization Eiffi"tB ao earlY I cell em-brYo I into 8 ellg. Combine them 2' 3, or ! i io orieutation. Wetine 4 at a time any celle 0uid240 mosm I Soon tbereafter, Polarize I Att i their contentr go ag to move ++Ala+ cL' trans' k*c HCo; microvilli and certaio membra.ue proteina away from tbe 2<.ll !ta8. site ofcontdct with other cellg. First blastocYst O ther trangmembrane Proteina €vitY (cadherine) collect at the contact (320 boso) Jte. Thev egtabliah aa"aPical' Morub basal polatitY, as have manY epitbelial cells. 3. hatching kinttg ol 'Dla3to<Iet (128 cells) apiel 14.imolantation baslateEl FtcuRE2r ffface surfacc. | . E of ell of ell OaY I \ Developmot of a human embDfo frpm fertiliatior \ ro implantation. Gavitation,or ,.formationof the blastocystcavitlt" oecursat the 32, 64, 128cell stages' Trophoblastcells are tightly joined in an epithelialsheet, and they are polarized(with apical and basalrryer,nbrane regions),-pumping Na* into the intercellularspages. Cl- follows passively. osmoticmovementbf water, as in blastocoelfonnation, inflates the cavity. polari'ty Stepsin establishingthe dorsal-ventral rloM of the mouseembryo (pd4, nanog, Compocted 3?-Cell st ge genes^on) hlii"or. J &<ell stagr (@ss stion) sox2 Step 2 Ebst€tsl .evity+ / fom3tion Trophoblast cell TiEht iuftlions Events in mouse development, pre-implantation. 2 Day 1.5:2 cell stage;gene expression starts. Cleavage continues. r D3y 2.5:early I cellstage,cells toosely ananged in a morula("bunch ot,gr9R9q")-. ' Althoughthere ls a regularrelationship of the spermentry point to the dorsoventralaxis, thb B cells are developmentally equivalentrrvtren isolated; the gctetcan be splitinto 2 quartetsor fodr doubletsin any directionsand all piecesdevelop normally.Atso, you can presstogether 2 or 3 embryos(16 or 24 cells)and these give rtormaFsized"chimeras" having 4 or 6 parents. ln the late'8cellstage, the compactionprocesg begins. Cells polarize (segregate) the surfaceand internalcvtoplasmic-materials such that microvillian-d various surface proteins collect at whateverpart of the cel[srirface is not in contactwith othercellq. Tightjunctions and desmosomes'formbetween ce|ls,makinganepit@betweentheextema|mediumandintercel|ular|iquid. r Mice fnom single culhred cells Plate the cells in a dish of syrtthetic nutrient culhrre medium. I.ct celle Erow as separate elonesthrough 15 mitoiic generatigns to EeCthoirsands ofbells, Pick one clone and replate every I0 datc black mouse parents for-a year or so (nrany hundreds ofcell generations). (fvhite recessiveto black) cellsfrom d? These p.roliferative innercell rnass cells are'called EMBRYONICSTEM riells from a CELLSdT ES CEIIS. reDilovethe 16 inner cell mass -- ili"ei*yii iel' 128 cells), disa ggrpgace the cells' Collec0cells from.the dish . ffi:rd pipet. and draw into a Inject i{iiec{edES cells can developto any embryonic cells into the blastocyst obtaine<l tkieu€or to hypoblast,but hot to trophoblast. from rrhite mouseparents. lfidant bhstocvslInto a whitemouse'fosler . gives white m&hef. whlctrihen birthto a black3nd r t 'pup' (mousebaby). lv--\ *r-----\. -/ ve / MITJ ^+ r/:,t<1 whiCemouse parents baby foster mother Grow up the pup ind crossit with a whi0e mate.' Get some wbolly black mice.and eomewholly white mice. The black babies come frori germ cells descendedfrom the cultured cells. probabbfdid maintain specific (embryonicstem Conclude: 1) These ES cells not ', innercell inasscells cytoplasmlc localizations for the many mitotic generations in culture. cells,catled ES cells) proliferating in a ls generated by the'interadion of cells Morb tikely al lhe organizalion , petridish in nutdentmedium. wilhin the einbryo. 2) This b a g.{ri}dfnetffi ?omake MOSAIC EMBRYOSones contailtkgi$et er mere ldnds of genetically different to strdv cell Transgenesfisin tfte mouse (Gilbertbook, p. 94) ;I*rH@T Crss s.tdmerk @ mouseand Microinjcct I Cni-.ti. wild'tYPc $ansg€ric tl Inncr ccll Clonedgcnc ES cels into mass in wctor host cmbryo | Culturcof Mix cmbryonic with ,@"@, cnrbryonic stm ells -'\ffi*'** stem(ES) cills . clgrcdgcoe (e.9.,me gene enosding HcteozYgoustr Dsgoicmice ore€nfluorescenl and a i@n'erotnoter (g/F) for its 'eroresston.This gerg fl|\ cohesfrom ieltYd$tl --f--^ep i 'uurL-a=:-'--*- tiom€E{€lotls trarcgenic I (!5%l ,/ Y chimericProgeny mlce &^. @= Day3: divisionto 16 cellstage; most cleavage planes are perpendicular,but someare parallelto the 3 em-bryo'ssurface, so mostcet6 1eg.12-13 of them)are stillincluded in the surfaceepithelium but some (eg- 34 of them)are entirelvinternal Divisionscontinue every 12 hr. At 32cell stage, if youremove the outer cell layer, the inner cells can form a new outerlayer; and outer cells can form still form a newinner layer by divisions horizontal to thesurface. By the 64 cellstage,inner and outercells can no longerreplace the otherif removed. Thusan outerlayer of cellsand an innermass are established:the outercells develop as the trophoblast (to placentaltissire which is extraembryonic)and the inner cell mass (lCM)cells develop into the embryoas weil as to other non-placentalextraembryonic tissues. Compaction and cleavagehave_acted as symmetry brcaking mechanisms(ones that do not requirepre-localized spatial inputs but give uniquegeometric outputsi.Trophoblast cells express the cdx gene,whereas ICM cells expressthe oct4, nanog,and sox2 polarity, to genes.The Hippo siqnalinq oathwavresponds to differencesof intercellularjunctions and cell leading cdxgene activation in the outercells. Day3-S: the blastocystcavity formation processbegins and continues in normalenibryo. The "Ner/K niF""u purp', whicfris locatedon the innersurface oi the polarizedtrophoblast ce1ls, pumps sodium ions fromthe'cell interiors into the spacebetween cells. Chlorideand bicarbonateions follow passively through inannetsto balancetfre Lfectrii charge. Tight junctions prevent the ionsfrom escaping, and so the ion concentrationin the intercellularspac-e soon eiceeds that of the externalmedium. Waterenters by osmosis .w"rii the spacebetween celis, creating the btastocystcavity. The surfacelayer of tightlyadjoined trophoblast"no cells exoands, and the innercells delaminate as a clump,attached to one siteof the trophoblast tt;r.- ihi" is noill utastocyst. The site of attachmentof the innercell mass to the trophoblastlayer is Cavityformation acts as a symmettybreaking m.echanism. 'prbuautv random. Thetrophoblast layei now differentiates, making the polar trophoblast over the inner cell mass (lCM) and the muralirophoblast over the iemainder of the blastorystsurface. The mural trophoblast cells become polyploid' multinucleate,very large, and flat. Day4 and 5: The ICMcells continue dividing. ICM cells continue to expressthe oct4, n9!og,and sox2 gJ;"., inO tome additionqtlyexpress the gita6 genewhile interm.ixedwith the others.The gata6-expressing iells then sort out, towardth'e Olistocoet anO aviray from the polartrophoblast, and then someof them mior,aieonto the muraltrophoblastlayer. These cells form the hypoblastlayer, also called the ffimuryonic endodermor.parietal endoderm'. Hypoblast cells that stayin contactwiththeinner cell massare fhe 'visceralendoderm'. Interior cells of the innercell mass become the epiblastor embryonic uJtoCerr, makingthe entireembryo (still expressing the oct4,nanog, and sox2 genes).The few cellsof the muiaflrodnobhsiwhich are mostbisiant from the lCM,and therefore not undedainby innercells (either hypoblastor epiblast),produce "hatching enzyme". ' , Theembryo now his a clearpolarig: polar trophoblast, epiblast, hypoblast, and hatching enzymeproducer cells of themural trophoblast. This relates directly to the dorsal-ventralpolarity
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