From Mouse Egg to Mouse Embryo: Polarities, Axes, and Tissues

ANRV389-CB25-20 ARI 12 September 2009 853

From Mouse Egg to MouseEmbryo Polarities Axesand TissuesMartin H JohnsonDepartment of Physiology Development and Neuroscience and the Center for TrophoblastResearch The Anatomy School Cambridge CB2 3DY United Kingdomemail mhj21camacuk

Annu Rev Cell Dev Biol 2009 25483ndash512

First published online as a Review in Advance onJuly 20 2009

The Annual Review of Cell and DevelopmentalBiology is online at cellbioannualreviewsorg

This articlersquos doi101146annurevcellbio042308113348

Copyright ccopy 2009 by Annual ReviewsAll rights reserved

1081-0706091110-0483$2000

Key Words

trophoblast inner cell mass blastocyst PAR genes ezrin Cdx2

AbstractThis review describes the three classical models (mosaic positional andpolarization) proposed to explain blastocyst formation and summarizesthe evidence concerning them It concludes that the polarization modelincorporates elements of the other two models and best explains mostknown information I discuss key requirements of a molecular basisfor the generation and stabilization of polarity and identify ezrinE-cadherin PAR proteins and Cdx2 as plausible key molecular playersI also discuss the idea of a network process operating to build cell al-locations progressively into committed differences Finally this reviewcritically considers the possibility of developmental information beingencoded within the oocyte and zygote No final decision can be reachedon a mechanism of action underlying any encoded information but acell interaction process model is preferred over one that relies solely ondifferential inheritance

Contents

INTRODUCTION 484THE EXPANDED BLASTOCYST

BACKGROUND 484Time 484Morphological Transitions

Shapes and Axes 485Cell Lineages 486

THE THREE MODELS PROPOSEDTO EXPLAIN HOW ABLASTOCYST IS GENERATED 488The Mosaic Model 488The Positional Model 489The Polarization Model 489

REEVALUATION OF THE THREEMODELS AND THEIRRELATIONSHIPS 491

MOLECULAR BASIS OFPOLARITY GENERATIONAND STABILIZATION 492E-Cadherin β-Catenin Actin

Ezrin and LamininIntegrins 492PAR Proteins 494CDX2 495

SUMMARY 496DOES POSITIONAL

INFORMATION EXISTWITHIN THE EGG ORZYGOTE 497Mechanisms 503

CONCLUSIONS 504

INTRODUCTION

In the mammal fertilization initiates a processof embryogenesis The mature 64- to 128-cellblastocyst (around 4ndash5 days postfertilizationin the mouse) (Figure 1) is the earliest stageat which a group of epiblast cells that couldreasonably be described as embryonic existsSome would argue that even these cells areproto-embryonic and only with the emergenceof definitive epiblast postimplantation are thecells truly embryonic This developmentalstrategy evolved with viviparity to facilitate an

effective sourcing of nutrients for embryonicgrowth via a complex membrane system Thesemembranes establish physical and chemicalcontact with the uterus to provide bothattachment and sustained maternal supportUltimately the membranes form part of theplacenta either a chorio-vitelline placentainvolving the hypoblast derivatives of the blas-tocyst (in monotremes marsupials and earlydevelopment of some eutherians) or a chorio-allantoic placenta involving the trophoblastderivativesmdashthe mature placental form in mosteutherians So although these early develop-mental stages are often called embryogenicthey might equally be called trophoblastogenicor hypoblastogenic This process is summa-rized comparatively with Xenopus in Figure 2It should be noted that the mouse blastocystthe mammalian model is not necessarilytypical in its organization and genesis (seeJohnson 1996 Selwood amp Johnson 2006)

Because the blastocyst is our developmen-tal end point its key features are describedfirst followed by three historical models thathave attempted to explain its genesis Lackof space restricts discussion to a considerationof trophoblast origins although the origins ofhypoblast are equally controversial (Yamanakaet al 2006)

THE EXPANDED BLASTOCYSTBACKGROUND

The first seven cell cycles to an expanded blas-tocyst are cleavage divisions in which there isno growth so cells halve approximately in sizeat each division (Figure 3) (Aiken et al 20042008 Lehtonen 1980) Specific developmentalevents are associated with particular develop-mental cell cycles suggesting operation of someunidentified endogenous clock ( Johnson 2002)The first two cell cycles are approximately18 hours in length and subsequent cycles are12 hours Each round of cell divisions is approx-imately synchronous but with sufficient hetero-geneity that intermediate stages between 2 4

484 Johnson

8 16 32 and 64 cells are increasingly commonas development progresses The time taken toachieve an expanded blastocyst (late 32 to 64cells) is approximately 35 days and approxi-mately 1 day later (128 or more cells) attach-ment to the uterine endometrium occurs In themouse maternal mRNA and protein supportsdevelopment until the mid-two-cell stage andby this point most maternally inherited mRNAis destroyed (Hamatani et al 2006) A few zy-gotic transcripts are synthesized at the late one-cell stage but major transcription follows in twowaves at the mid-two-cell and eight-cell stagesMaternal proteins can persist beyond the blas-tocyst stage (Gilbert amp Solter 1985 Howlett1986 West et al 1986)

Morphological TransitionsShapes and Axes

Two gross morphological transitions occur dur-ing early development (Figure 3) At the eight-cell stage individual cells lose their distinctiveoutlines and maximize intercellular contact(Figure 3)mdasha process called compaction

TadpoleGametogenesis

Includes yolk formation

Fertilization

Includes laying down extraembryonic membranes

Embryonic development

GametogenesisEmbryogenesis Embryonic development

Figure 2Comparison of mouse and Xenopus early development to emphasize the functional differences between themNote that there are also major differences in timescale (a swimming tadpole forms in the time a mouse eggtakes to reach two cells) and size (a mouse egg is approximately 100 μm in diameter compared with the frogegg diameter of 10000 μm)

Mural trophoblast(Cdx2)

Zona pellucida (ZP)

Polar trophoblast(Cdx2)

Epiblast(Oct4 and nanog)

Hypoblast(Gata 46)

Figure 1Schematic sectional view of an expanded blastocyst (64ndash128 cells 4 days) toshow main cell and tissue types ( Johnson amp Selwood 1996) and keytranscription factors that characterize each The zona pellucida (ZP) is anacellular glycoprotein membrane that is produced during oogenesis andsurrounds the oocyte zygote and cleaving embryo It is modified structurallyat fertilization as part of the block to polyspermy and is shed just prior toimplantation

ICM inner cell mass

Then at the early 32-cell stage fluid ac-cumulates between cells and coalesces in asingle expanding blastocoelic cavity (Figure 3)surrounded by mural trophoblast At one endof the cavity lies a cluster of pluriblast cellsknown as the inner cell mass (ICM) which isnot initially exposed to the blastocoelic fluidbecause it is covered by thin trophoblastic

wwwannualreviewsorg bull Egg to Embryo Polarities Axes Tissues 485

Video CLICK TO VIEW

Figure 3(Left) Video showing a time-lapse record of the development of a two-cell mouse embryo to an earlyexpanding blastocyst Note the progressive size reduction (cleavage) in blastomeres as the cells divide fromtwo to eight cells the flattening that occurs at compaction during the eight-cell stage and the appearance andexpansion of the blastocoel at the 32-cell stage In a parallel track (right) the nuclei of the embryo have beencolor-coded to show the disposition of the descendants from each two-cell blastomere in the early cavitatingblastocyst (ECB) Note that division is asynchronous and that there is coherent clonal growth In this embryothe blue-derived descendant cells are largely mural trophoblast with one subclone of 8 cells in the inner cellmass (ICM) and polar trophoblast (top right in the last frame) Video image reproduced with permission fromDevelopment (2008) and created by David-Emlyn Parfitt Marcus Bischoff and Magdalena Zernicka-Goetz

ECB early cavitatingblastocyst

PB polar body

ZP zona pellucida

BS bilateralsymmetry (axis orplane of )

EA embryonic-abembryonic(axis)

processes adluminally and by polar trophoblastexternally (Fleming et al 1984)

Until recently the early cavitating blastocyst(ECB) had been considered spherical How-ever Gardner and colleagues (Gardner 19972001 Gardner amp Davies 2006) have shown thatby the late one-cell stage the zygote becomesan oblate spheroid having in one of its cross-sectional planes a different diameter therebygiving it a plane of bilateral rather than ra-dial symmetry (Figure 4a) With time an in-creasing proportion (60ndash65 or more) of zy-gotes shows bilateral symmetry when viewedwith the second polar body (PB) uppermost butmost of these look circular when viewed side-ways A similar situation is described for the ZPat the two-cell stage although it is not obvi-ous for the embryo itself (Figure 4b) There-after to the ECB stage most embryos (and theirzonae) have a long axis of bilateral symmetry(BS) which at the ECB stage is orthogonal tothe embryonic-abembryonic (EA) axis and theplane of bilateral symmetry and aligned along

the long equatorial axis separating the embry-onic and abembryonic parts of the blastocyst(Figure 4c) We return to the possible devel-opmental significance of these shapes later

Cell Lineages

The fully expanded blastocyst contains tissuesthat are restricted in both their prospectivefate and their developmental potency andseem to be composed of developmentallycommitted cells Indeed trophoblast cellsthroughout blastocyst expansion (32-cell stageECBs) seem unable to contribute cells to ICM-derived lineages (Cruz amp Pedersen 1985 Dyceet al 1987 Pedersen et al 1986 Rossant amp Vijh1980) This trophoblast commitment occursearlier than that of ICM cells Thus ECBs inthe sixth developmental cell cycle contain ICMcells that can readily form trophoblast on theirisolation or aggregation into embryos but havemostly lost this capacity by the late 32-cell stagewhether examined in vitro (Chisholm et al

486 Johnson

1985 Handyside 1978 Hogan amp Tilly 1978Louvet-Vallee et al 2001 Nichols amp Gardner1984 Spindle 1978) or in vivo (Gardner et al1973 Gardner amp Johnson 1973 Papaioan-nou 1982 Rossant amp Croy 1985 Rossantamp Lis 1979 Rossant et al 1983) Similarlyembryonic stem cells (derived from ICMsalthough not from later epiblast tissues seeSchenke-Layland et al 2007) rarely contributetrophoblast derivatives on injection into blas-tocysts (Beddington amp Robertson 1989) Thesuggestion that ICM cells in intact expanded(64 cells or more) blastocysts might regularlycontribute to polar trophoblast (Cruz ampPedersen 1985 Winkel amp Pedersen 1988)remains contested as a possible technicalartifact (Dyce et al 1987) or a result of thelabeling of later dividing 32-cell stage cells(Winkel amp Pedersen 1988) However we donot yet have an agreed exact time during thesixth and seventh developmental cell cycles forICM commitment to a nontrophoblastic fate

Examination of the patterns of expressionof key tissue-distinctive transcription factors(TFs) critical for the activation of downstreamepiblast and trophoblast tissues does not en-tirely relieve this uncertainty Thus expressionof the trophoblast marker Cdx2 is limitedto trophoblast by the end of the 32-cell stage(Dietrich amp Hiiragi 2007 Ralston amp Rossant2008 Strumpf et al 2005) and it can be up-regulated only in ICMs that on isolation formtrophoblast (Suwinska et al 2008) In contrasttwo TFs associated with the pluripotent ICMnamely Oct4 and Nanog (Chambers et al 20032007 Niwa et al 2000 Palmieri et al 1994)are reported to become restricted exclusively toICM cells one to two cell cycles later (Dietrichamp Hiiragi 2007) long after trophoblast com-mitment at the early 32-cell stage Thus at cur-rent sensitivities of detection Oct4nanog ex-pression does not correlate with commitmentbut Cdx2 expression might Evidence thatmutually exclusive expression patterns of Oct4and Cdx2 are essential for commitment (Niwaet al 2005) comes from the analysis of embryosgenetically lacking these TFs Thus bothCdx2- and Oct4-null embryos form early

PBS (ZP) PBS

Zona pellucida

Polar body

Blastocoelic cavity

Figure 4Shapes and axes during early mouse development as proposed by Gardner(Gardner 1997 2001 Gardner amp Davies 2006) The top figure in each panel isrotated 90 to the right to give a lower figure Note that whereas at the one-celland blastocyst stages there are planes of bilateral symmetry in the embryosthemselves at the two-cell stage Gardner claims that only the zona shows thisfeature (Gardner 1997 2001 Gardner amp Davies 2006) A animal pole Vvegetal pole PBS plane of bilateral symmetry ZP zona pellucida ABS axis ofbilateral symmetry of blastocyst EmAb embryonicabembryonic axis

TF transcriptionfactor

blastocysts consisting of both ICM and tro-phoblast tissues which then fail to expandfully or to develop downstream trophoblasticor ICM markers and their tissue derivativesrespectively and they are unable to implantsuccessfully (Nichols et al 1998 Ralston ampRossant 2008) These results also mean that theexpression of zygotically encoded Cdx2 cannotbe required for blastocyst formation (Ralstonamp Rossant 2008) although the same claimcannot be made confidently for Oct4 becausematernally inherited Oct4 is present until thetwo-cell stage in Oct4-null embryos (Nicholset al 1998 Palmieri et al 1994) We returnto the question of how this developmentalrestriction might be achieved when we reviewthe various models advanced to explain howthe blastocyst is generated

Blastocyst 8-cell

Polarization

(Johnson et al 1981) Positional

(Tarkowski andWroblewska 1967)

16-cell 1-cell

Mosaic

(Dalcq 1957)

Figure 5Schematic summary (zona not shown) of the three main hypotheses proposed to explain blastocystformation mosaic polarization and positional In each case the areas shaded green or white indicateputative spatial differences in developmentally significant information Adapted from figure by MadgalenaZernicka-Goetz in Development (2002)

AV animal andvegetal (poles or axis)

THE THREE MODELS PROPOSEDTO EXPLAIN HOW ABLASTOCYST IS GENERATED

Given the apparent simplicity of blastocyststructure its mechanism of formation hasproved contentious (Hiiragi et al 2006) A sim-plified descriptive summary of the three mainmodels proposed historically to explain blasto-cyst formation is shown in Figure 5 and thekey features of each are summarized below

The Mosaic Model

Early ideas about blastocyst formation drewheavily on nonmammalian models in which theselective partitioning of determinants usuallyin association with a standardized cleavage pat-tern was proposed to specify cell fates (Dalcq1957 Mulnard 1992) Given the difficulty ofculturing and experimentally manipulatingmouse embryos in vitro these ideas dependedinitially on observations of fixed embryos Thesame ideas have resurfaced at intervals sincethen using more sophisticated techniques Forexample Antczack amp Van Blerkom (1997) stud-ied leptin and STAT3 distribution in humanand murine oocytes and embryos in relationto the animal-vegetal (AV) axis (Figure 4see Johnson amp McConnell 2004) In oocytesand zygotes their location was described ascortical at the A-pole and by the four-cell stageas characteristically strong in one cell weak

andor variable in two and poor in a fourth thestrongly staining blastomere now remote fromthe A pole a finding explained by a putativecytoplasmic rotation in the late zygote By theblastocyst stage stained cells were observedprimarily in mural trophoblast in continuitywith a small group of eccentrically placedpolar trophoblast cells but not in most polartrophoblast nor in the underlying ICM Is itpossible that one strongly stained four-cell blas-tomere contributed these mural trophoblastcells A similar possibility has been raised forhuman embryos in which reversed-transcribedpolymerase chain reaction (RTPCR) studies onsingle blastomeres isolated from human cleav-ing embryos reported the reciprocal expressionof mRNAs for Oct4 and β-hCG (human chori-onic gonadotrophin) (markers respectively ofICM and trophoblast in the blastocyst) (Hansiset al 2004) and led to speculation that a singlefour-cell animal blastomere might be theprogenitor for trophoblast (Edwards amp Hansis2005)

Although these descriptive accounts mightbe suggestive none of them established formalcontinuity of molecular patterns in the oocytethrough later cell lineages Dynamic experi-ments in which particular blastomeres or partsof blastomeres at the zygotic two-cell or eight-cell stage were marked and their progeny fol-lowed to the blastocyst stage suggested thatzygotes and embryos might contain some sort

488 Johnson

of patterned developmental information Thusinjection of silicone or oil droplets to mark cen-tral or peripheral cytoplasm in two- or four-cellblastomeres resulted in each injection positionbeing associated respectively with a predom-inantly ICM or trophoblast location in theblastocyst suggesting a relationship betweenearly and later positions (Graham amp Deussen1978 Wilson et al 1972) However these dy-namic studies are also correlative and providefate maps not manipulative and they are in-capable of testing for determinative featuresWhen manipulative studies were performedthe mosaic model was not only abandoned butdiscredited

The Positional Model

The sophisticated experimental studies ofearly mammalian development that we take forgranted today became possible by the late 1950sand 1960s through the development of tech-niques of superovulation (Fowler amp Edwards1957) embryo culture (Whitten 1956) andtransfer (McLaren amp Biggers 1958) and laterin vitro fertilization (Whittingham 1968)thereby freeing the mouse embryo from itsuterine environment It allowed pioneering ex-periments in which blastomeres were destroyedor separated (Tarkowski 1959) and in whichgroups of cells from different embryos wereaggregated to form chimaeras (Mintz 19641965 Tarkowski 1961) In 1967 Tarkowski ampWroblewska (1967) reported on the devel-opmental potential of each of the single-cellblastomeres isolated from a single embryo andconcluded that the segregation of develop-mental information required by the mosaicmodel could not be demonstrated In its placethey proposed (on theoretical rather thanexperimental grounds) that blastomeres wereequivalent and totipotent until approximatelythe 30-cell stage at which point some blas-tomeres were enclosed totally by others andthat this microenvironmental positional differ-ence led them to become different and to startthe process of becoming pluriblast (inner) ortrophoblast (outer) tissues This positional (or

inside-outside) model was tested by Hillmanet al (1972) who labeled one or two isolatedfour- or eight-cell blastomeres and aggregatedthem with other unlabeled blastomeres in dif-ferent spatial arrays to show that when placedperipherally they contributed preferentially totrophoblast and centrally to the ICM (see alsoKelly 1977) The demise of the mosaic modelwas further hastened when centrifugationscrambling and removal of zygotic cytoplasmwere shown not to interfere with development(Ciemerych et al 2000 Evsikov et al 1994Tellez et al 1988 Zernicka-Goetz 1998)However formally it remains to be shown thatall individual four- or eight-cell blastomeres arefully developmentally competentmdashthe failureto demonstrate this is usually explained by thedeficiency in cell numbers in the blastocystsresulting from them (Tarkowski et al 2001)

The positional model rapidly gained accep-tance in the mouse and other mammals (egJohnson et al 1995 Willadsen amp Godke 1984)including humans (Van de Velde et al 2008)with the consequence that mammalian devel-opment with its plasticity and regulative prop-erties came to be viewed as highly idiosyncraticand quite different mechanistically from othercommonly studied organisms Of course therewas a considerable interpretative leap involvedbetween the observational data on individualeight-cell blastomeres and the idea of an inter-nal microenvironment two cell cycles later Itwas this mechanistic gap that the polarizationmodel tried to fill

The Polarization Model

If the positional model was correct then itbecame important for the identification of thenature of the putative microenvironmentalstimulus to establish when inside and outsidecells first exist and when differences betweenthem appear The answer to both questionswas the same the early 16-cell morula (Bar-low et al 1972 Graham amp Deussen 1978Handyside 1981 Handyside amp Johnson 1978Louvet et al 1996 Pedersen et al 1986 Suraniamp Handyside 1983) Moreover during the

a Polarization of 8-cell stage b Division to 16-cell stage

c Division to 32-cell stage

EzrinE-cadherin colocalize

Ezrin segregates apically

E-cadherin segregates basolaterally

Apical microvilli ezrinPar6b Cdx2 mRNA aPKC

Basolateral E-cadherinβ-catenin EMK1

Differentiative division = inner + outer cell

Conservative division = 2x outer cells

Outer cells polar ezrin + veflatten on ICs more Cdx2 RNA

Inner cells apolar ezrin ndash veadhesive all over

Figure 6Schematic summary of the polarization model (zona not shown) and the molecular redistributions associated with it (a) During theeight-cell stage (only four cells shown) cells polarize radially in response to asymmetric patterns of cell contacts (b) Elements ofcytocortical polarity persist throughout division to the 16-cell stage divisions are either differentiative ( green) generating two distinctinner (I) and outer (O) populations or conservative (red ) generating two outer cells only (c) The transition to 32 cells is characterizedby three division types one of each is illustrated A further differentiative division of outer cells can occur generating one inner and oneouter cell A conservative division of an inner cell will generate two inner cells Thus the inner cell population is derived in two wavesFinally an outer cell can also divide conservatively to generate two outer cells

preceding eight-cell stage a major transfor-mation in cell phenotype was observed duringwhich each blastomere transformed from aspherical symmetrical cell to a highly polarizedradially oriented cell (Figure 6a) with an apicalmicrovillous face externally and smoother ba-solateral surfaces internally (Handyside 1980Reeve amp Ziomek 1981 Ziomek amp Johnson1980) This radial organization was stablepersisting throughout the ensuing two cleavagedivisions (Figure 6bc) either of which couldbe conservative (generating two outer polarcells) or differentiative (generating an innernonpolar and an outer polar cell) therebyforming two populations that differed in boththeir positions and properties from the momentof their formation (Balakier amp Pedersen 1982Johnson amp Ziomek 1981a Pedersen et al 1986Soltynska 1982 Sutherland et al 1990 Ziomekamp Johnson 1981 1982) These observationsformed the basis for the polarization modelarticulated in 1979 at a meeting in HoustonTexas ( Johnson et al 1981) which proposedthat polarization of eight-cell blastomeres was

the critical event in the initiation of lineagedivergence The model met some resistanceinitially appearing to reinstate a mosaic modelalbeit at a postzygotic stage thus challengingthe notion of plasticity that had led to thepositional model Subsequent experimentsestablished that this challenge was spurious

Thus a range of observations supportedand developed the model (see Johnson ampMcConnell 2004 Yamanaka et al 2006) Itwas shown that the orientation of the axisof polarization in each eight-cell blastomerewas determined by the pattern of asymmetricintercellular contacts it experienced (Adleramp Ziomek 1986 Johnson amp Ziomek 1981b)The cytocortex was identified as the ma-jor route to and locus of positional polarmemory secondarily imposing polarity onthe cytoskeleton and cytoplasm ( Johnson ampMaro 1985 1986) Indeed the critical polarfeature was identified as the structurally stableapical pole of microvilli This polar regionfunctions like an outer cell determinant in thatany cell inheriting all or part of it becomes

490 Johnson

polar ( Johnson et al 1986b Wiley amp Obasaju1988) Consequently the generation of innercell populations requires at least some of thepolarized eight-cell blastomeres to dividedifferentiatively to generate one inside andone outside cell Whether or not a cell dividesdifferentiatively is affected primarily by thesize of its determinant cortical pole rather thanits contact patterns or shape immediately priorto division (Pickering et al 1988) Related tothis observation cells in an eight-cell embryothat were more advanced through the cell cycletended to assume a shape and organization thatfavored a smaller pole and led to more differen-tiative divisions (Garbutt et al 1987) therebycontributing more cells to the ICM (Barlowet al 1972 Kelly et al 1978 Piotrowska et al2001 Surani amp Barton 1984 but see Alarconamp Marikawa 2005 Fujimori et al 2003)The numbers of inside 16-cell blastomeresgenerated varies among embryos most studiesagreeing on a range of three to seven (meanapproximately five) (Balakier amp Pedersen 1982Bischoff et al 2008 Fleming 1986 Handyside1981 Johnson amp Ziomek 1981a Pedersen et al1986 Suwinska et al 2008) although others(Barlow et al 1972 Dietrich amp Hiiragi 2007Graham amp Lehtonen 1979) report only one ortwo inner cells As might be expected eight-cellblastomeres rarely if ever contribute two insidecells (Bischoff et al 2008 Pedersen et al 1986)

The different adhesive properties of insideand outside cells reinforce and maintain theirrelative positions and that of their descen-dants with rare exceptions (Bischoff et al 2008Kimber et al 1982 1982 Pedersen et al 1986Soltynska 1982 Ziomek amp Johnson 1981) In-deed when inside and outside cells are delib-erately mixed up most sort to their originallocation (Surani amp Handyside 1983 Suwinskaet al 2008) However this capacity to sort doesnot mean these cells are committed Thus in-side cells at the 16-cell stage can if retainedexperimentally in an outside position polarizeand become outside cells (Suwinska et al 2008Ziomek amp Johnson 1982 Ziomek et al 1982)a property that persists to the early 32-cellstage (see above) Similarly although outside

16-cell-stage cells do not depolarize and musttherefore contribute to the trophoblast lineage(which may be the default pathway) they canundergo a second round of differentiative divi-sions the extent to which they do so depend-ing on their shape as modified by cell interac-tion patterns ( Johnson amp Ziomek 1983) Theselater differentiative divisions actually occur insitu which means that the ICM is achievedin two distinct cell allocations (Figure 6c)mdashmost (on average 75) deriving from thefourth cleavage descendants but some deriv-ing from the fifth cleavage (Bischoff et al 2008Fleming 1986 Johnson amp Ziomek 1983Pedersen et al 1986) These two inner cell pop-ulations differ (Chisholm amp Houliston 1987)and might therefore contribute differentially toepi- and hypo-blast (Yamanaka et al 2006)

REEVALUATION OF THE THREEMODELS AND THEIRRELATIONSHIPS

There has been a tendency to emphasize oneof the above models and discount the othersby setting them up in mutual opposition Thisstrategy is helpful in stimulating experimentaltests of each but unhelpful if it becomes simplydogmatic The polarization model is a refine-ment of the positional model Thus althoughinside and outside cells differ phenotypicallyand functionally from the moment of their al-location at the 16-cell stage they do respondto their different positions by further divergentdifferentiation as defined by many markers andcharacteristics and ultimately by a restrictionof their developmental plasticity For examplecells in both populations express Cdx2 Nanogand Oct 4 at the 16- and early 32-cell stages andfirst achieve exclusivity of tissue expression inthe blastocystmdashbut only as long as they remainin distinct relative positions (Dietrich amp Hiiragi2007 Palmieri et al 1994 Ralston amp Rossant2008 Suwinska et al 2008) Thus relative po-sition remains important for the progressive di-vergence to commitment of the differently allo-cated cells as Tarkowski amp Wroblewska (1967)proposed

The polarization model also reinstated arole for cytoplasmic determinants in the mam-mal albeit not a determinant that is locatedin the egg or zygote but one that is gen-erated de novo at the eight-cell stage in theform of the apical pole The relatively lateappearance of this determinant coupled withthe abilities of polar cells to generate nonpolarones by differentiative divisions at the fourthand fifth cleavage divisions and the ability ofnonpolar 16- or 32-cell blastomeres to polar-ize later if exposed to asymmetric contact pat-terns also accommodate the plasticity of mousedevelopment demonstrated by blastomere de-struction rearrangement and aggregationexperiments

Nonetheless despite understanding the roleof polarity in early development we still do notfully understand the molecular basis of its gen-eration and stabilization how the orientationof cleavage planes is controlled or exactly howthe two newly formed cell subpopulations allo-cated to different positions become committedto their restricted developmental fates I con-sider clues to address this deficit below

MOLECULAR BASIS OFPOLARITY GENERATIONAND STABILIZATION

Many studies have described the segregationor enrichment of particular macromoleculesto inward-facing (cell-contacted) domains andoutward-facing (noncell-contacted) domains(for recent examples see Herr et al 2008Ohsugi et al 2008) However for such asymme-tries to be developmentally significant for po-larity generation four key features are relevantFew macromolecules currently satisfy any or allof these criteria

1 Their asymmetric distribution should be-come independent of continuing intercel-lular contacts once the stable cortical poleis established

2 They should be asymmetrically dis-tributed at division to the inner and outer16 cells

3 Their disturbance experimentally shoulddisturb polarity generation andorstability

4 Any initial changes of distribution or ac-tivity in them must be regulated post-translationally because remarkably theprocess of polarization does not requireproximate transcription or translationbut it is regulated through posttrans-lational control mechanisms includingphosphorylation (Bloom 1991 Bloomamp McConnell 1990 Levy et al 1986Winkel et al 1990)

E-Cadherin β-Catenin Actin Ezrinand LamininIntegrins

The homotypic Ca2+-dependent E-cadherinmolecule has long been implicated in cuingblastomere polarity Its immunological neutral-ization and the manipulations of external orintracellular calcium levels impairs polariza-tion and its pattern of distribution changes atpolarization to become stably restricted to ba-solateral membranes in which location is alsoposttranslationally modified (Hyafil et al 1980Johnson et al 1986 Pey et al 1998 Sefton et al1992 1996 Shirayoshi et al 1983 Vestweberet al 1987) E-cadherin links via β-cateninwhich also shows distributional and posttrans-lational changes at polarization (Goval et al2000 Ohsugi et al 1999 Pauken amp Capco1999 Sefton et al 1996) to the actin cytoskele-ton and actin-containing microvilli are lostbasolaterally and stabilized apically ( Johnsonamp Maro 1984 1985 1986 Reeve amp Ziomek1981) In addition coassociations of E-cadherinwith fodrin calmodulin and the serine proteaseepithin have been noted and the manipulationof epithin and calmodulin activities affectsE-cadherin distribution and compaction(Khang et al 2005 Pey et al 1998) Finallyat polarization the actin microfilament-stabilizing protein ezrin becomes stablylocalized to the pole concurrent with itsposttranslational modification and it is one ofthe few macromolecules that remain localized

492 Johnson

throughout subsequent cell division (Louvetet al 1996 Louvet-Vallee et al 2001) It thusseems to be a key marker for polar stability andperhaps a key agent of it

Zygotic expression of β-catenin is detectedat the late 2-cell (mRNA) and 4- to 8-cell(protein) stages and zygotic expression ofE-cadherin is observed at the late 4- (mRNA)and 16-cell (protein) stages (de Vries et al2004) but the presence of long-lived mater-nally inherited proteins complicates the inter-pretation of genetic knock-out studies whichindicated no adverse effects until long afterpolarization (Larue et al 1994 Riethmacheret al 1995 Torres et al 1997) Knock-out(E-cadherin) or N-terminal truncation (β-catenin) of maternally inherited proteins hasshown that in the absence of either or bothintercellular adhesion is delayed until sufficientzygotic synthesis of both proteins is achieved bythe 16-cell stage (de Vries et al 2004) Unfortu-nately the impact of these genetic maternal ma-nipulations on polarization was not describedbut the photographs suggest that basolateral lo-calization of neither molecule had occurred bythe eight-cell stage which might be predictedif polarization had failed It would also be in-teresting to know what happens at the 16-cellstage when the cells do compact These studiessupport a key role for E-cadherin in the cuingof cell contact patterns How it does so is un-clear although it may involve interactions withezrin

Ezrin is codistributed with E-cadherinaround the whole cell surface during cleavageprior to polarization when the two segregateto apical (ezrin) and basolateral (E-cadherinand β-catenin) domains (Figure 6a) At thistime total ezrin levels decline suggesting thatit may be destabilized basolaterally leavinglargely the phosphorylated isoform associatedwith the microvillous pole (Dard et al 2004Louvet et al 1996 Louvet-Vallee et al 2001)Associated with these distributional changes arechanges in the lipid composition of the baso-lateral and apical membranes (Pratt 1985) andin the detergent extraction properties of the

membranes (Clayton et al 1993) Point mu-tation of threonine-567 a key phosphoryla-tion site for ezrinrsquos actin cross-linking activ-ity interferes with the loss of microfilamentsbasolaterally and their restriction apically andezrin is no longer excluded from basolat-eral sites E-cadherin-mediated cell adhesion isalso blocked and its restriction basolaterally isseverely disturbed (Dard et al 2004)

Finally intercellular signaling is often me-diated developmentally via extracellular matrixThe earliest detected matrix molecule in mousedevelopment is laminin with two B chain iso-types that are synthesized by the 8-cell stagealthough synthesis of all three chains occurs atthe 16-cell stage only (Cooper amp MacQueen1983 Dziadek amp Timpl 1985 Leivo et al 1980Shim et al 1996) Laminin can influence thedistribution of cadherins (Klaffky et al 2006)and recent functional studies by Chung et al(2008) have provided suggestive evidence for arole for laminin in polarization Thus cultureof blastomeres in a medium rich in laminindisrupted polarizationmdashas evidenced by disor-dered tight junctions and the lack of polarizedmicrovilli These observations suggest that inthe normal embryo the deposition of lamininbetween blastomeres might stimulate throughits asymmetric distribution the redistributionof E-cadherin and initiate the polarization ofthe blastomeres ( Johnson 2008) By surround-ing an isolated blastomere with laminin mightthis asymmetric positional signaling be lost andthus polarization disturbed Tantalizing thoughthese observations are we need to know moreabout the time course and nature (and the pre-vention or reversal) of polarity disruption andwhich isotypes of each chain are involved In-terestingly Roberts et al (2009) have reportedthat partial deletion of beta 4-integrin disturbsdevelopment to the morula interblastomericadhesion and the normally observed colocal-ization of integrins and laminin between in-terblastomere surfaces

Taken together these studies suggest a pos-itive feedback model for driving polarization inwhich

PKC protein kinaseC

1 Posttranslational changes to ezrin andorcadherin and catenin affect their planarinteractions to favor their mutual exclu-sion to distinct microdomains of the cellmembrane

2 These domains eventually become de-fined as apical and basolateral throughthe selective stabilization of cadherinand catenin complexes basolaterally viaan increased capacity for transcellu-lar homotypic cross-linking therebymediating compaction (Clayton et al1993) Whether laminin-integrin signal-ing might also be involved remains to beseen

3 Finally the progressive exclusion of phos-phorylated ezrin to outward-facing mem-brane drives the apical stabilization ofactin microvilli and thus generates thestable pole whereas elsewhere microvilliare lost further favoring intercellular flat-tening via transcellular interaction of cad-herins in a virtuous feedback loop

If this model is correct then a key issuebecomes What triggers the posttranslationalchanges and why does it happen at the eight-cell stage It is unlikely that any of the keymolecular players discussed above are limit-ing until this stage because polarization canbe initiated in the absence of protein synthe-sis (Levy et al 1986) Indeed premature com-paction and polarization can be induced in four-cell blastomeres by inhibiting protein synthesisor by activating either protein kinase C (PKC)or the rho-family GTPase (CDC42)mdashthe lat-ter a known regulator of cadherin-mediated ac-tions (Clayton et al 1999 Cui et al 2007 Levyet al 1986 Natale amp Watson 2002 Ohsugiet al 1993 Winkel et al 1990) These resultssuggest that all the proteins required for com-paction and polarization are made prior to theeight-cell stage (maternally andor zygotically)and await activation posttranslationally Whatmight lead to their activation and why it occursat the eight-cell stage remain to be determinedThe identity of a possible player has come fromthe study of PAR proteins

PAR Proteins

The six PAR genes were discovered duringgenetic screens for regulators of cytoplasmicpartitioning in early Caenorhabditis elegansdevelopment (reviewed in Goldstein amp Macara2007) but homologs have now been foundin diverse animals including the mouse PARproteins have been implicated in the regulationof cell polarization and via positioning effectson the spindle the control of asymmetric celldivision PAR genes encode elements of anintracellular signaling system involving serine-threonine kinases and associated proteinswhich tend to be cortically enriched and local-ized asymmetrically often dynamically so in aself-organizing hierarchy that then affects otherdownstream cell components Exactly how PARproteins become segregated to different corticaldomains is not resolved but evidence fromC elegans implicates the centrosome in directsignaling to a cortical microfilament scaffoldto induce asymmetric contractions that shiftthe PAR protein complexes into asymmetricdistributions Likewise it is unclear how PARproteins once asymmetrically distributed thenmediate downstream actions although severalpathways may operate The local activities ofdifferent kinases either PAR kinases themselvesor via PAR interaction with atypical PKCs(aPKC) may provide one important pathwaybut interactions via the rho-family GTPaseCDC42 and with microtubules also seemimportant

Clarification of these mechanisms is im-portant for early mouse development becausemurine PAR homologs and aPKCs are ex-pressed asymmetrically in oocytes and embryoswhere their manipulation affects polarization(Duncan et al 2005 Jedrusik et al 2008 Grayet al 2004 Plusa et al 2005a Thomas et al2004 Vinot et al 2004 2005) The mouse ho-mologs of PAR3 and 6 each have three splicevariants whereas EMK1 (PAR1 homolog) hastwo isoforms and is a serine-threonine kinase(also a member of the microtubules affinityregulating kinases family) All three PAR pro-teins are detectable in eight cells EMK1 and

494 Johnson

PAR3 are maternally inherited (Vinot et al2005) Blastomere polarization is associatedwith a change in the distribution of EMK1and PAR6b [but Vinot et al (2005) could notdetect PAR3 distribution immunocytochemi-cally until the blastocyst stage] Thus beforepolarization EMK1 and PAR6b were entirelynuclear during interphase and localized to thespindle in M-phase However during polar-ization EMK1 became localized basolaterallywhereas PAR6b associated apically These dis-tinctive localization patterns once establishedwere stable to cell contact pattern disruptionand persisted throughout division to 16 cellsThereafter EMK1 relocated to the nucleusbut aPKC became associated with PAR6b api-cally although some PAR6b was also foundbasolaterally in outer cells and all around thecortex of inner cells

These findings suggest a dynamic andchanging role for the PAR proteins in the po-larization process (Vinot et al 2005) A less de-tailed report led to a similar broad conclusionalthough with variations in detail a differenceunresolved experimentally (Plusa et al 2005a)PAR3 and aPKC were studied and both be-came apically localized during polarization Ofparticular interest was the finding that neutral-ization of Par3 by dsRNA injection into someblastomeres increased their relative contribu-tion to the ICM as did the dominant negativeform of aPKC This shift in relative contri-bution was attributed to two mechanismsfirst an increase in differentiative divisions inthe injected cells thereby contributing moreprogeny to the inside presumably the resultof smaller poles being formed (Pickering et al1988) or systematic effects on spindle orienta-tion or organization (Louvet-Vallee et al 2005Vinot et al 2005) and second an increase in theinternalization of injected cells andor all theirprogeny such that all were insidemdashpresumablyowing to a failure of the eight-cell blastomereto polarize stably Even though further clarifi-cation of distribution patterns of PAR proteinsis required it seems that PAR family proteinsshow changes in distribution associated withpolarization at least one (PAR6b) of which

shows polar stability during subsequent mitoticdivision The fact that neutralization of PARproteins can affect cell allocation presumablythrough effects on polarization and subsequentcleavage patterns argues for a crucial role ofthis family Whether or how the PAR proteinsdirectly or indirectly affect the ezrin andcadherin family or vice versa remains to be es-tablished but the kinase activities of many PARfamily members and their formative roles else-where suggest a possible line of investigation

The Cdx family of transcription factors con-sists of three mouse homologs of the Drosophilacaudal homeobox genes which are involved inspecifying cell position along the fly antero-posterior axis with similar functions in the latermouse embryo (Chawengsaksophak et al 19972004) Cdx2 is also required for commitmentof outer cells to trophoblast but not for theearlier allocation of cells to the outside posi-tion (Ralston amp Rossant 2008 Strumpf et al2005) Although not required until the blas-tocyst stage and evidently lacking any mater-nally inherited Cdx2 mRNA or protein Cdx2is first expressed zygotically in trace mRNA lev-els as early as the four-cell stage ( Jedrusik et al2008) and as nuclear protein by the eight-cellstage where the proportion of Cdx2-positiveeight-cell blastomeres in an embryo increasesas the cell cycle progresses (Dietrich amp Hiiragi2007 Jedrusik et al 2008) Experimental ma-nipulations of Cdx2 levels in two- and four-cellblastomeres have indicated that although it isnot essential for polarization it can nonethe-less influence it ( Jedrusik et al 2008) Thusthe experimental modulation of Cdx2 levelsup or down in one four-cell blastomere ledto proportionately increased or decreased con-tributions respectively by its progeny to tro-phoblast These altered contributions were inturn identified as being due to increased or de-creased incidences of conservative divisions atboth 8- to 16-cell and 16- to 32-cell transitions

How Cdx2 affects division orientation is un-clear but a clue comes from the observation that

aPKC expression is upregulated in cells withincreased Cdx2 expression leading to largermore intensely staining poles As Pickeringet al (1988) showed a larger pole makes a con-servative division more likely The identifica-tion of a Cdx2-consensus-binding site in theaPKC promoter supports an effect via aPKCThus Cdx2 has the capacity to influence cell al-location to different positions although it is notessential Moreover Cdx2 mRNA was found topolarize to the subcortical region during the 8-cell stage and like ezrin and PAR6b remainedpolarized during the subsequent division to 16cells leading to a greater distribution of Cdx2mRNA to outer than inner cells ( Jedrusik et al2008) This unequal distribution may underliethe difference in Cdx2 protein levels follow-ing differentiative divisions noted by Dietrichamp Hiiragi (2007) Thus there seems to be apositive feedback loop involving Cdx2 and cellpolarization which should facilitate the forma-tion of a stable epithelium In this context it isof interest that eight-cell blastomeres vary nat-urally in the expression levels of Cdx2 protein(Dietrich amp Hiiragi 2007 Jedrusik et al 2008Strumpf et al 2005) and there is some evi-dence consistent with those blastomeres withnaturally higher levels of Cdx2 contributingmore progeny to trophoblast (Bischoff et al2008)

Finally a recent paper from Nishioka et al(2009) sheds more light on the molecular mech-anisms by which cell populations that are al-located to inner and outer positions becomecommitted to ICM and trophoblast Thus acomplex involving TEAD4 and Yap seems to berequired to stabilize Cdx2 expression in outercells In inner cells in contrast signaling viathe HippoLats pathway phosphorylates Yapand prevents its nuclear localization leading tothe failure of TEAD4 complex formation anddownregulation of Cdx2 HippoLats signalingdepends on the inner cells remaining totally en-closed but the molecular nature by which suchenclosure renders Hippo signaling effective isunclear Plausibly the differential distributionof E-cadherin consequent upon division of po-larized cells is critical

SUMMARY

A full molecular explanation for polarity gen-eration and stabilization is lacking (Figure 6)Plausible key molecular players have been iden-tified in ezrin PAR family proteins and Cdx2CDC42 and E-cadherin β-catenin and Hippoare strongly implicated and laminin and inte-grins less convincingly so How might theseplayers interact Polarization involves a mas-sive posttranslationally regulated reorganiza-tion of the cell and all the evidence pointsto the cell cortex as being the dominant locusof this process Thus at the outset the axis ofpolarization is set by cortical contact patternsand terminally the locus of the polar memoryis cortical Cytoplasmic reorganization occurssecondary to cortical reorganization and al-tered gene expression patterns are far down-stream of it Early cleavage to the eight-cellstage progressively puts in place all the molec-ular elements required to effect polarizationA triggering device the nature of which re-mains obscure but that is likely to involve ac-tivation of kinase activities then initiates theprocess PAR family proteins seem to dependon cortical changes for their segregation andchanges in the patterns of interaction amongezrin cadherin and actin might provide sucha cortical change indeed when cell interac-tion patterns are disturbed PAR protein lo-calizations are adversely impacted (Vinot et al2005)

Equally PAR kinases might contribute tothe driving force for the cortical changes them-selves producing another example of a cu-mulative positive feedback system driving thecell toward polarity One aspect of this polar-ity is the unequal distribution of informationalmolecules such as Cdx2 mRNA which can actto further reinforce polarity perhaps throughan influence on PAR proteins Thus ratherthan trying simply to prove a serial hierarchyof regulatory factors it is perhaps more usefulto think of the molecular mechanics of earlymouse development as a reinforcing networkprocess This approach to thinking about earlydevelopment makes dissection of that network

496 Johnson

challenging It also sets up a framework forthinking about the topically thorny issue ofwhether there exists within the egg or zygoteinformation that affects subsequent cell alloca-tions and embryo organization because suchinformation might contribute to such a networkbut still admit regulatory capacity The once-slain beast of mosaicism has recently raised itshead yet again and we now confront it with anetwork process in mind

DOES POSITIONALINFORMATION EXIST WITHINTHE EGG OR ZYGOTE

The traditional mosaic model of Dalcq (1957)invoked a role for the selective partitioningof zygotic cytoplasmic tissue determinantsmdashusually in association with a standardized pat-tern of cleavage Three recent claims that reac-tivate a form of mosaic organization are morecomplex and relate more to morphological axesthan to tissue lineages per se although the twoare necessarily linked The first claim is thatthe plane of first cleavage is influenced by theAV axis and the sperm entry point The sec-ond claim which is often conflated with thefirst is that the plane of first cleavage alignswith the equatorial axis of bilateral symme-try (BS axis) of the blastocystmdashorthogonal tothe EA axis The third claim is that the pat-tern and sequence of the two second cleav-age divisions influence the relationship betweenthe plane of first cleavage and the BS axis ofthe blastocyst and the developmental potentialand properties of individual four-cell blas-tomeres These are significant claims becausethe blastocyst BS axis has itself been claimedto correlate with the antero-posterior axis ofthe developing embryo-fetus (Gardner 2000Gardner et al 1992 Smith 1980 1985 Weberet al 1999) Thus the larger claim here isthat the organization within the oocyte or zy-gote can be related to axial development inthe embryo or fetus Each of these claims iscontested

Within the developmental biology com-munity a passionate reductionism that sees

embryos as either mosaic or regulative seemsto recur episodically This passion surfacedbriefly when the polarization model was pro-posed and then as now it was misplacedEven the most lineage-driven of developmen-tal models C elegans has some regulatory ca-pacity and most types of embryo use a mixThe issue therefore is whether the mammalis so different that no vestige of organiza-tional information remains within the egg orzygote to influence development There is noevidence currently available to suggest thatif such information exists it is determina-tive and determinism is not part of these re-cent claims despite curious attempts by crit-ics to disprove determinism (eg Motosugiet al 2005) However critics also say that ifsuch information as exists is nondeterminativeit is irrelevant to our understanding of earlymouse development I reject this view in lightof the network process proposed above In-deed understanding how zygotic informationmight operate mechanistically to nudge devel-opment in certain directions is fundamentallywhat research on mouse development is aboutThus a better question to ask is is the oper-ation of positional information in the zygoteexplicable through mechanisms compatiblewith the polarization model or does it requirethat model to be amended or replaced Perhapsthe example that follows will help to explain

Earlier the effect of artificially elevatingCdx2 levels on the increased allocation of cellsto an outer position and thus ultimately to thetrophoblast lineage was described and a mech-anism for achieving it was identified within thepolarization model It was also suggested thenthat the natural variation among eight-cell blas-tomeres in the expression levels of Cdx2 mightnormally influence allocation of progeny to tro-phoblast This natural variation in Cdx2 lev-els has been described as being ldquostochasticrdquo(Dietrich amp Hiiragi 2007) It is difficult to knowwhat stochastic means in this context otherthan a way of saying that we do not yet knowhow or why something is happening In factevidence was presented recently that the varia-tion in Cdx2 expression levels at the eight-cell

a Zygote b 2-cell stage

Figure 7Does the plane of first cleavage align with the animal-vegetal (A-V) axis andorthe site of sperm entry (SEP) (Zona not shown)

ME meridional andequatorial (divisionplanes)

SEP sperm entryposition

stage is not stochastic but lineage related Thusthe pattern of cleavage by which each eight-cell blastomere is formed naturally affects thelevels of its Cdx2 mRNA and protein ( Jedrusiket al 2008) Those eight-cell blastomeres thatwere derived from a second cleavage divisionorder in which a meridional division precededan equatorial one (ME) showed significantlyhigher levels of Cdx2 mRNA and protein inthe descendants of the E-dividing two-cell blas-tomere E-derived blastomeres in EM-derivedeight-cell embryos did not show this effectThis result suggests that patterns of prior cleav-age had affected the expression of a develop-mentally critical gene and raises two questionsIs this observation real as some deny If it isreal how can we explain it First we addressthe evidence for the three contested claims totest their reality

Claim 1 Does the plane of first cleavage alignwith the AV axis andor the site of sperm en-try (Figure 4 Figure 7 and SupplementalTable 1 follow the Supplemental Materiallink from the Annual Reviews home page athttpwwwannualreviewsorg)

Gardner (1997) claimed (albeit on indirect ev-idence) that in most zygotes the plane of firstcleavage is aligned meridionally along the AVaxis of the zygote as marked by the polar bodySubsequently Zernicka-Goetz and coworkersclaimed that the site of sperm entry also influ-ences the orientation of the first cleavage di-vision within this AV meridional plane (Grayet al 2004 Piotrowska amp Zernicka-Goetz 2001

Plusa et al 2002b) a claim challenged by Daviesamp Gardner (2002) Previous papers cited insupport (Howlett amp Bolton 1985) or against(Eviskov et al 1994) these claims are largelyqualitative and unhelpful A priori the reli-ability of both the PB and the SEP as sta-ble cortical markers is open to doubt givenclear evidence of bulk membrane flows to-ward the cleavage furrow (Davies amp Gardner2002 Pratt amp George 1989) capable of drag-ging cortical sites with them to give theappearance of being in a meridional planeIndeed Hiiragi amp Solter (2004) measuredsignificant PB movement at first cleavage di-rectly in 48 out of 108 (44) zygotes ashave others [Piotrowska amp Zernicka-Goetz2001 (3337 zygotes) Piotrowska-Nitsche ampZernicka-Goetz 2005 (1116 zygotes) Plusaet al 2005b (1664 zygotes)] Piotrowska ampZernicka-Goetz (2001) also described rota-tional movements prior to or during first cleav-age that could displace both the PB and SEPand further cast doubt on their reliability as po-sitional markers Given these doubts about thephenomenon itself a mechanistic explanationwould be helpful

The position and orientation of the spin-dle determines division plane orientation andposition (eg Vinot et al 2004) so are theseinfluenced by the AV axis andor SEP Hiiragiamp Solter (2004) suggested that the orientationof the first cleavage plane is set just prior tospindle formation by the plane of appositionbetween the approaching pronuclei This sug-gestion implies that the sites of female and malePN formation each related to PB extrusion andSEP respectively will tend normally to set thecleavage plane unless of course there is sub-sequent rotation of the apposed PNs or morecritically of the spindle that forms after theirapposition Plusa et al (2002a) described micro-tubules extending from the first mitotic spin-dle poles toward the cortical midbody remain-dered from the meiotic divisions at the A-poleThese microtubules they suggested could thenalign the metaphase plate along the AV axisThis observation provides a mechanism for ex-plaining how an AV cleavage plane is observed

498 Johnson

frequently regardless of concerns about thevalue of surface marker stability

What about the SEP and plane of cleav-age If we accept that the dynamics of mid-body microtubule and mitotic spindle interac-tion tend to favor an AV cleavage plane thenany SEP in the vegetal or animal thirds ofthe zygote will automatically lie close to thatcleavage planemdashespecially given the propen-sity of membrane to flow toward the furrowData on the axial position of SEPs in zona-intact eggs are limited and contradictory ThusPiotrowska amp Zernicka-Goetz (2001) describedthe distribution as 16 animal 47 vege-tal and 37 equatorial (n = 73) whereasMotosugi et al (2006) reported distributions of29 17 and 54 (n = 405) On these fig-ures without any particular influence of SEP onthe cleavage plane 46ndash63 of the SEPs tendto lie close to that plane the upper of thesevalues being similar to those reported (Sup-plemental Table 1 follow the SupplementalMaterial link from the Annual Reviews homepage at httpwwwannualreviewsorg) Forequatorial SEPs to provide an additional influ-ence would require that they cause the mitoticspindle to rotate around its AV axis so that itsmetaphase plate is aligned along a line diametri-cally projected from the SEP Gray et al (2004)suggested that a slight actin-dependent corticalcontraction centered on the SEP occurs withsperm penetration and changes oocyte shapefrom spherical to spheroidal the lesser diameterbeing centered on the SEP This narrowing itwas suggested might provide a mechanism forinfluencing spindle orientation but mechanis-tic details are lacking However artificial flat-tening imposed on the zygote externally didlead to an increased incidence of cleavage planesacross the lesser diameter of the zygote regard-less of PB or SEP position (Gray et al 2004Plusa et al 2005b) Thus cell shape can over-ride any influence either of these might have onthe cleavage plane

I conclude that this first claim may be correctbut is not proved A preferred AV cleavage planeis plausible mechanistically if not proven deci-sively Evidence that the SEP also influences the

cleavage plane actively rather than incidentallyremains disputed

A final comment A firm conclusion on thisfirst claim is only important for the claim thatis considered next in that it is often conflatedwith it to imply that the AV axis andor SEPnot the plane of first cleavage is the critical axialfeature relative to the BS axis of the blastocystIf the cleavage plane was critical then the PBand SEPs may be red herrings So what of thissecond claim

Claim 2 Does the plane of first cleavage alignequatorially along the BS axis of the blasto-cyst and orthogonal to the EA axis (Figures 4and 8 and Supplemental Table 2 followthe Supplemental Material link from theAnnual Reviews home page at httpwwwannualreviewsorg)

Two experimental approaches have been used totest this claim One approach uses focal markersof the cell surface or its overlying ZP to map inthree dimensions the zygotic and two-cell bilat-erality onto the blastocyst By far the strongestof these approaches used three distinctivelyplaced axial markers injected into the ZP at dif-ferent coordinates with respect to the under-lying late two-cell embryo cleavage plane andPB and has provided evidence for coalignmentof the plane of first cleavage and the BS axis inrelatively large numbers of embryos with rel-atively few excluded embryos and to high lev-els of significance (Gardner 2001) This studyshowed that the EA axis was orthogonal to theplane of first cleavage in 151 of 182 (82) blas-tocysts analyzed

The second approach starts from our knowl-edge that cell proliferation is coherently clonalto the ECB stage after which more cell mix-ing occurs routinely (Garner amp McLaren 1974)Thus if the prediction is true that first cleav-age does demarcate a plane that will align withthe BS axis of the blastocyst then distinctivemarking of each of the two-cell blastomeresfollowed by examination of the distribution oftheir progeny at the ECB stage should showthem to be distributed broadly on opposite sidesof the BS axis that is at opposite ends of the

First cleavageplane

Early cavitatingblastocyst ECB

Polar body

Blastocoelic cavity

Figure 8Proposed alignment between the plane of firstcleavage and the axis of bilateral symmetry (ABS) inthe ECBmdashorthogonal to the embryonicabembryonic (EmAb) axis The red lines indicatethat the alignment may be tilted 20ndash30 in eitherdirection with respect to ABS (zona not shown)

EA axis Piotrowska et al (2001) found this to bethe case for between 60 and 80 of embryosstudied but suggested that there was a devia-tion of 20ndash30 from exact coalignment of theclonal boundary and BS axis (Figure 8) An ex-planation for this tilt was offered by a time-lapsestudy in which the clonal descendants of eachof the four eight-cell sister blastomeres derivedfrom each two-cell blastomere were identifiedtracked and mapped to the ECB stage (Bischoffet al 2008 and Figure 3) In 61 of embryosstudied one out of four of the eight-cell em-bryos from each two-cell blastomere crossedthe BS axis at its opposite ends The tilt patternis strikingly similar to the stat3leptin stainingpattern described by Antczak amp Van Blerkom(1997 see Mosaic Model section above)

In the previous section we saw that chang-ing zygotic shape by compression changed theorientation of the cleavage plane Plusa et al(2005b) applied this approach to 20 zygotes toforce a first cleavage plane orthogonal to the AVaxis then marked each two-cell blastomere withvital dye DiI or DiD cultured them to the ECBstage (although total cell numbers averaged 22which is low) and undertook a clonal analysis

by confocal sectioning They reported that in17 of 20 there was clear evidence of the clonalboundary respecting the BS axis These resultssuggest that it is the first cleavage plane not AVaxis that influences cell allocation along the EAaxis

Between them these studies seem to providestrong evidence to favor the claim Howeverthe claim has been both supported (Ciemerychet al 2000 Fujimori et al 2003 Gardner1997 Gardner amp Davies 2006 Piotrowska ampZernicka-Goetz 2001 Plusa et al 2005b) anddisputed (Alarcon amp Marikawa 2003 2005Chroscicka et al 2004 Motosugi et al 2005)Technical issues might explain some of the dis-crepancies (see Gardner 2006) but again thelegitimacy of this disputation is better addressedby asking whether plausible mechanisms areon offer to convert descriptive phenomena intofunctional understanding

Gardner does not offer us a clear mecha-nistic interpretation but some of his critics doThus Motosugi et al (2005) focused on therole of the spheroidally shaped ZP imposingshape on the embryo (see also Kurotaki et al2007) They described an experiment in whichthe impact of compressing the two-cell embryoin each of two orientations throughout cultureto the blastocyst is assessed by analysis of the EAaxis in relation to the first cleavage plane Whenthe two-cell embryo was compressed laterallyin a direction that exaggerated that suggestedto be imposed naturally by the ZP (Figure 9a)the EA axis indeed formed orthogonal to thefirst cleavage plane in 17 out of 18 (94) aneven higher frequency than the 82 claimedby Gardner (2001) However when the com-pression was at 90 to the first cleavage plane(Figure 9b) the EA axis was aligned parallelto the first cleavage plane in 12 of 13 embryos(92) Thus changing the shape imposedon the embryo had changed the alignmentof axial patterns in the ECB Motosugi et al(2005) building on a suggestion by Alarconamp Marikawa (2003) concluded that normallytherefore the form of the ECB was imposedmechanically at the blastocyst stage by the

500 Johnson

naturally spheroidal shape of the ZP ratherthan through any intrinsic information withinthe embryo itself

Gardner (2007) tested this explanation di-rectly by either softening the ZP at the two-cell stage or removing it altogether prior toblastocoel formation and found that despite be-ing freed from the suggested zona constraintsthere remained significant alignment of the firstcleavage and ECB BS axis Gardnerrsquos resultssuggest that the mechanical constraint expla-nation is not adequate

So this second claim remains contestedand the issues remain unresolved Howeverperhaps the observations of Motosugi et al(2005) following compression do contain a clueabout mechanisms Might the different out-comes of each type of compression reflect animmediate impact namely the different con-tact patterns imposed between the two-cellblastomeres Thus the first compressionmethod will tend to reduce intercellular con-tacts between two-cell blastomeres and increasethe ratio of greater to lesser contact diameters inthe cleavage plane whereas the second methodwill increase intercellular contact and reducethe ratio of greater to lesser diameters in thecleavage plane (Figure 9) These changes tothe contacts and shapes of the individual two-cell blastomeres probably impact the patternsof subsequent second cleavages as well as blas-tomere packing postcytokinesis Thus the firstcompression pattern seems more likely to re-sult in two meridional second cleavage divi-sions (with respect to the plane of the first)whereas the second compression pattern seemsmore likely to result in two equatorial secondcleavage divisions However leaving this sec-ond claim unresolved this possibility does leadus directly to the third controversial claim

Claim 3 Does the order and pattern of thesecond cleavage divisions influence subsequentdevelopment

This claim divides into two parts (a) Thereis a regular pattern to the second cleavagedivisions and (b) the patterns observed have

Polar body

Blastocoelic cavity

Figure 9Compression of two cells (zona not shown) in different orientations results indifferent outcomes (based on data from Motosugi et al 2005) (a) When thetwo-cell embryo was compressed laterally in a direction that exaggerated thatsuggested by Motosugi et al to occur naturally the embryonicabembryonic(EmAb) axis indeed formed orthogonal to the first cleavage plane in 94 ofembryos (b) When the compression was at 90 to the first cleavage plane theEmAb axis was aligned parallel to the first cleavage plane in 92 of embryos

developmental consequences Gardner (2002)analyzing only those four-cell blastomeres thatformed tetrahedrons with a PB located betweenthree of the cells (estimated as 70ndash85 offour-cell blastomeres) suggested that the mostcommon pattern of second cleavage is onemeridional plus one equatorial division (82n = 65) of which it was inferred from twoexperiments that the sequence was ME in 60(n = 81) and 48 (n = 48) ndash (average 56)These second division plane orientations wereinferred after the cleavage events rather thanobserved directly and were defined with re-spect to a presumptive AVndashfirst cleavage planeaxis as assessed retrospectively by PB position

N = 460 EM39

N = 88N = 60

Figure 10Relationships between second cleavage patterns and later development as proposed by Zernicka-Goetz andcolleagues Four-cell blastomeres were classified according to the cleavage pattern by which they formedwhere both the orientation of the plane of cleavage (E equatorial M meridional with respect to the PB) andthe sequence (ME or EM) are recorded For equatorial divisions the two E blastomeres can be furtherclassified as primarily from the animal end (A) or the vegetal end (V) The percentages are those recorded byPiotrowska-Nitsche amp Zernicka-Goetz (2005) (N = 460 and N = 88) and Bischoff et al (2008) (N = 60) asshowing cleavage patterns and the ECB patterns illustrated respectively Note that the relationships claimedare not absolute and vary between the two experiments Note also that the E blastomeres from ME (but notEM) embryos contribute disproportionately to trophoblast and that MM- and EE-derived blastocysts showthe axial alignment of the first cleavage plane with the axis of bilateral symmetry less frequently thanME- and EM-derived blastocysts

and after prelabeling the presumptive V pole onone two-cell blastomere Piotrowska-Nitscheamp Zernicka-Goetz (2005) extended this studyusing a similar approach with dye-markedcells to assess the nature and sequence ofdivisions in the 90 of two-cell blastomeres inwhich the PB came to lie in the plane of firstcleavage They also found that 81 (n = 460)of four-cell blastomeres were tetrahedronswith a PB between three cells and were ableto classify these into four categories by thesequence and orientation of second cleavage

planes (see Figure 10 for details) confirmingand extending Gardnerrsquos findings

Both of the above studies examined four-cell embryos once formed Two studies haveattempted to examine the process of four-cellformation Bischoff et al (2008) used time-lapseanalysis with serial optical sections of nonma-nipulated embryos and were able to track everyindividual blastomere through 3D coordinatesThey concluded that the frequencies of divi-sion patterns were 36 ME 33 EM 20MM and 7 EE (n = 66 Figure 10) which

502 Johnson

given the smaller numbers is not very differ-ent from the proportions found by Piotrowska-Nitsche amp Zernicka-Goetz (2005) HoweverLouvet-Vallee et al (2005) challenged theseinterpretations based on their own time-lapseanalysis on whole mount dividing two-cell em-bryos observed in a single optical plane inwhich tubulin-GFP and Hoechst dye was usedto visualize the spindle and chromosomes re-spectively on a bright field background Theorientations of both the second cleavage spin-dle and the plane of cytokinesis were assessedwith reference to the plane of maximum contactbetween sister two-cell blastomeres (ie firstcleavage plane) Metaphase and anaphase spin-dles were described as being anchored firmlyto the cytocortex by polar microtubules inthe orientation in which they initially formeduntil cytokinesis at which point the spindleaxis could change in relation to the adjacentcell

However this change in orientation was de-scribed as being due to the relative movementof daughter cells not to the internal rotationof the spindle itself Significantly metaphasespindles were described as being oriented ran-domly in both blastomeres but the terminalrotation of cytokinetic cells gave the appear-ance under bright-field images of the moresystematic MEEM patterns reported above us-ing that end point Indeed Piotrowska-Nitscheet al (2005) using surface-marked blastomereshave indicated that 50 of newly formed equa-torially derived four-cell pairs in ME embryosrotate through up to 180 during or soon aftertheir formation The data from Louvet-Valleeet al (2005) which need independent confirma-tion offer a different sort of explanation for theobservations described earlier Thus presum-ably these cytokinetic rotations occur to facili-tate cell packing within the constraints of theintra-ZP space If this were the explanationthen two-cell embryos freed of the ZP stabi-lized by gelation of the intra-ZP space or iso-lated as single two-cell embryos should differin their apparent cleavage patterns from thoseheld naturally within the ZP Gardner (2002)

addressed this issue by gelation when he found60 EM plus ME and by use of (marked) iso-lated blastomeres when he found 45 ME plusEM divisions both values lower than those re-ported earlier Earlier studies by Graham ampDeussen (1978) and Suzuki et al (1995) hadshown that removal of the ZP decreased thenumbers of intercellular contacts at the four-cell stage which is consistent with the abovefindings Taken together these results suggestthat the explanation offered by Louvet-Valleeet al (2005) might be plausible

A decision on which explanation is cor-rect is important given the developmentalsignificance that Zernicka-Goetz and her col-leagues have accorded to the patterns of sec-ond cleavage Thus they have presented alarge body of evidence suggesting that ECBsderived from different four-cell cleavage pat-terns (ME-EM-MM-EE) show proximate dif-ferences depending on whether E- or M-derived and for E-derived whether from nearthe A or the V pole These differences includedevelopmental capabilities (but not potential)of four-cell blastomeres (Piotrowska-Nitscheet al 2005) and differences in epigenetic mod-ifications to chromatin (Torres-Padilla et al2007) Presumptively consequential differencesare also described for example the differencesin Cdx2 expression levels one cell cycle later( Jedrusik et al 2008) and different incidencesof various patterns of clonal organization andaxes at the ECB stage (Bischoff et al 2008Piotrowska-Nitsche amp Zernicka-Goetz 2005)Many of these later differences in cell behav-iors are explicable mechanistically at least inpart through the polarization mode

Mechanisms

At the center of these elegant studies liesa large explanatory holemdashmechanistically atleast Given the weight of evidence now restingon the perceived consequences of the regular-ity of cleavage patterns it becomes importantto be sure exactly what is happening at sec-ond cleavage and how There seem to be two

types of theoretical explanations but neitherof them is robustly mechanistic The explana-tion favored by Gardner and Zernicka-Goetz intheir various publications is that there is somesort of partitioning of oocytic and zygotic de-velopmental information during the first twocleavage divisions that generates cells differingin composition specifically differences in levelsof A and V type information It would seemintuitively likely given the claimed influenceof division planes and sequence that any suchinformation is cytocortically encoded IndeedPratt (Pratt 1989 Pratt amp George 1989) hassuggested that the pattern of new membraneinsertion during early cleavage divisions mightencode spatio-temporal information that is de-velopmentally important for guiding cells latertoward inside-outside differences These ideasbear closer examination in the context of theserecent claims because they provide possiblemechanistic explanations

However the evidence for both first andsecond cleavage divisions being regular in re-lation to the AV axis is open to question andcan certainly be overridden simply by manipu-lating the orientation of the cleavage plane ex-perimentally So an alternative explanation forthe developmental consequences of differentapparent cleavage patterns is that they arise notbecause the cells inherit different informationalcontent but because once formed their con-tact patterns differ depending on division orderand planes These differences then lead to mi-nor differences in for example gene expressionpatterns subsequent interaction patterns with

other cells and so on Indeed there is alreadyevidence that the nature and number of cellcontact patterns at the four-cell stage as wellas the sequence in which four cells form caninfluence subsequent development significantly(Garbutt et al 1987 Graham amp Deussen 1978Graham amp Lehtonen 1979 Kelly et al 1978Piotrowska et al 2001a Surani amp Barton 1984Suzuki et al 1995)

Perhaps most likely there is a role forboth types of mechanism operating interac-tively through cytocortically encoded informa-tion and cell contact-mediated interaction in aform of networking feedback process of the sortdescribed earlier The challenge experimentallyfor all of us is to provide testable mechanisticmodels

CONCLUSIONS

Despite over 50 years of research the mecha-nisms underlying the early development of themouse remain to be explained The polariza-tion model has proved sufficiently resilient andadaptive as a viable explanation for the eventsleading to cell allocation to different positionsand their subsequent commitment to differentlineages but the molecular basis of this modelis yet to be elucidated fully The relative roles ofinherited and positionally generated differencesat the two- and four-cell stages is the subject ofongoing investigation Whether the polariza-tion model will also be able to accommodatethe outcome of these investigations remains tobe seen

SUMMARY POINTS

1 Three main models (mosaic positional and polarization) have been proposed to explainthe generation of cell diversity in the blastocyst

2 Of these the polarization model currently provides the most complete mechanistic ex-planation and in doing so incorporates some features of the other two models

3 The mechanistic explanation is broadly satisfactory at a cellular level but not at a molec-ular level

4 Strongly implicated molecular players include ezrin and E-cadherin PAR proteins andCdx2

504 Johnson

5 The suggestion that axial developmental information may be present in the oocyte andzygote remains controversial and awaits identification of underlying mechanisms

FUTURE ISSUES

1 What times the onset of polarization

2 What is the exact molecular basis of polarity generation and stabilization at the eight-cellstage

3 How are the orientations (differentiative or conservative) of cleavage planes at 8- to16-cell and at 16- to 32-cell stages controlled

4 What is the exact molecular basis of commitment of inside and outside cells

5 Do first and second cleavage divisions segregate inherited oocytic and zygotic informationand generate different contact patterns to influence later cell allocations and if so how

6 Is the mouse truly a model for all other mammalsmdasheutherian marsupial and monotreme

DISCLOSURE STATEMENT

The author is not aware of any biases that might be perceived as affecting the objectivity of thisreview

ACKNOWLEDGMENTS

I wish to thank David-Emlyn Parfitt for making available the video

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Edwards RG Hansis C 2005 Initial differentiation of blastomeres in 4-cell human embryos and its significancefor early embryogenesis and implantation Reprod BioMed 11206ndash18

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Plusa B Hadjantonakis A-K Gray D Piotrowska-Nitsche K Jedrusik A et al 2005b The first cleavage ofthe mouse zygote predicts the blastocyst axis Nature 434392ndash95

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Tarkowski AK 1961 Mouse chimaeras developed from fused eggs Nature 190857ndash60Tarkowski AK Ozdzenski W Czolowska R 2001 Mouse singletons and twins developed from isolated diploid

blastomeres supported with tetraploid blastomeres Int J Dev Biol 45591ndash96Tarkowski AK Wroblewska J 1967 Development of blastomeres of mouse eggs isolated at the 4- and 8-cell

stage Development 18155ndash80Tellez V Ahumada A Muro J Sepulveda S Izquierdo L 1988 Centrifugation of 2-cell mouse ova cytoplasm

stratification and recovery Dev Genes Evol 197360ndash65

Thomas FC Sheth B Eckert JJ Bazzoni G Dejana E et al 2004 Contribution of JAM-1 to epithelialdifferentiation and tightjunction biogenesis in the mouse preimplantation embryo J Cell Sci 1175599ndash608

Torres M Stoykova A Huber O Chowdhury K Bonaldo P et al 1997 An alpha-E-catenin gene trap mutationdefines its function in preimplantation development Proc Natl Acad Sci USA 94901ndash6

Torres-Padilla M-E Parfitt D-E Kouzarides T Zernicka-Goetz M 2007 Histone arginine methylationregulates pluripotency in the early mouse embryo Nature 445214ndash18

Van de Velde H Cauffman G Tournaye H Devroey P Liebaers I 2008 The four blastomeres of a 4-cell stagehuman embryo are able to develop individually into blastocysts with inner cell mass and trophectodermHum Reprod 231742ndash47

Vestweber D Gossler A Boller K Kemler R 1987 Expression and distribution of cell adhesion moleculeuvomorulin in mouse preimplantation embryos Dev Biol 124451ndash56

Vinot S Le T Maro B Louvet-Vallee S 2004 Two PAR6 proteins become asymmetrically localized duringestablishment of polarity in mouse oocytes Curr Biol 1452ndash55

Vinot S Le T Ohno S Pawson T Maro B et al 2005 Asymmetric distribution of PAR proteins in the mouseembryo begins at the 8-cell stage during compaction Dev Biol 282307ndash19

Weber RJ Pedersen RA Wianny F Evans MJ Zernicka-Goetz M 1999 Polarity of the mouse embryo isanticipated before implantation Development 1265591ndash98

West JD Leask R Green JF 1986 Quantification of the transition from oocyte-encoded to embryo-encodedglucose phosphate isomerase in mouse embryos Development 97225ndash27

Whitten WK 1956 Culture of tubal mouse ova Nature 17796Whittingham DG 1968 Fertilization of mouse eggs in vitro Nature 220592ndash93Wiley LM Obasaju MF 1988 Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage

blastomeres fused with 8-cell- and 16-cell-stage blastomeres Dev Biol 130276ndash84Willadsen SM Godke RA 1984 A simple procedure for the production of identical sheep twins Vet Rec

114240ndash43Wilson IB Bolton E Cuttler RH 1972 Preimplantation differentiation in the mouse egg as revealed by

microinjection of vital markers Development 27467ndash79Winkel GK Ferguson JE Takeichi M Nucitelli M 1990 Activation of protein kinase C triggers premature

compaction in the four-cell stage mouse embryo Dev Biol 1381ndash15Winkel GK Pedersen RA 1988 Fate of the inner cell mass in mouse embryos as studied by microinjection

of lineage tracers Dev Biol 127143ndash56Yamanaka Y Ralston A Stephenson RO Rossant J 2006 Cell and molecular regulation of the mouse blasto-

cyst Dev Dynamics 2352301ndash14Zernicka-Goetz M 1998 Fertile offspring derived from mammalian eggs lacking either animal or vegetal

poles Development 1254803ndash8Ziomek C Johnson MH 1980 Cell surface interaction induces polarization of mouse 8-cell blastomeres at

compaction Cell 21935ndash42Ziomek CA Johnson MH 1981 Properties of polar and apolar cells from the 16-cell mouse morula Dev

Genes Evol 190287ndash96Ziomek CA Johnson MH 1982 The roles of phenotype and position in guiding the fate of 16-cell mouse

blastomeres Dev Biol 91440ndash47Ziomek CA Johnson MH Handyside AH 1982 The developmental potential of mouse 16-cell blastomeres

J Exp Zool 221345ndash55

512 Johnson

AR389-FM ARI 14 September 2009 1458

Annual Reviewof Cell andDevelopmentalBiology

Volume 25 2009

ContentsChromosome Odds and Ends

Joseph G Gall 1

Small RNAs and Their Roles in Plant DevelopmentXuemei Chen 21

From Progenitors to Differentiated Cells in the Vertebrate RetinaMichalis Agathocleous and William A Harris 45

Mechanisms of Lipid Transport Involved in Organelle Biogenesisin Plant CellsChristoph Benning 71

Innovations in Teaching Undergraduate Biologyand Why We Need ThemWilliam B Wood 93

Membrane Traffic within the Golgi ApparatusBenjamin S Glick and Akihiko Nakano 113

Molecular Circuitry of Endocytosis at Nerve TerminalsJeremy Dittman and Timothy A Ryan 133

Many Paths to Synaptic SpecificityJoshua R Sanes and Masahito Yamagata 161

Mechanisms of Growth and Homeostasis in the Drosophila WingRicardo M Neto-Silva Brent S Wells and Laura A Johnston 197

Vertebrate Endoderm Development and Organ FormationAaron M Zorn and James M Wells 221

Signaling in Adult NeurogenesisHoonkyo Suh Wei Deng and Fred H Gage 253

Vernalization Winter and the Timing of Flowering in PlantsDong-Hwan Kim Mark R Doyle Sibum Sung and Richard M Amasino 277

Quantitative Time-Lapse Fluorescence Microscopy in Single CellsDale Muzzey and Alexander van Oudenaarden 301

Mechanisms Shaping the Membranes of Cellular OrganellesYoko Shibata Junjie Hu Michael M Kozlov and Tom A Rapoport 329

The Biogenesis and Function of PIWI Proteins and piRNAs Progressand ProspectTravis Thomson and Haifan Lin 355

Mechanisms of Stem Cell Self-RenewalShenghui He Daisuke Nakada and Sean J Morrison 377

Collective Cell MigrationPernille Roslashrth 407

Hox Genes and Segmentation of the Hindbrain and Axial SkeletonTara Alexander Christof Nolte and Robb Krumlauf 431

Gonad Morphogenesis in Vertebrates Divergent Means to aConvergent EndTony DeFalco and Blanche Capel 457

From Mouse Egg to Mouse Embryo Polarities Axes and TissuesMartin H Johnson 483

Conflicting Views on the Membrane Fusion Machinery and the FusionPoreJakob B Soslashrensen 513

Coordination of Lipid Metabolism in Membrane BiogenesisAxel Nohturfft and Shao Chong Zhang 539

Navigating ECM Barriers at the Invasive Front The CancerCellndashStroma InterfaceR Grant Rowe and Stephen J Weiss 567

The Molecular Basis of Organ Formation Insights from theC elegans ForegutSusan E Mango 597

Genetic Control of Bone FormationGerard Karsenty Henry M Kronenberg and Carmine Settembre 629

Listeria monocytogenes Membrane Trafficking and LifestyleThe Exception or the RuleJavier Pizarro-Cerda and Pascale Cossart 649

Asymmetric Cell Divisions and Asymmetric Cell FatesShahragim Tajbakhsh Pierre Rocheteau and Isabelle Le Roux 671

Indexes

Cumulative Index of Contributing Authors Volumes 21ndash25 701

Cumulative Index of Chapter Titles Volumes 21ndash25 704

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articlesmay be found at httpcellbioannualreviewsorgerratashtml

viii Contents

Annual Reviews Online

Search Annual Reviews

Annual Review of Cell and Developmental Biology13 Online

Most Downloaded Cell and Developmental Biology Reviews13

Most Cited Cell and Developmental Biology Reviews

Annual Review of Cell and Developmental Biology13 Errata

View Current Editorial Committee

All Articles in the Annual Review of Cell and Developmental Biology Vol 25

Chromosome Odds and Ends

Small RNAs and Their Roles in Plant Development

From Progenitors to Differentiated Cells in the Vertebrate Retina

Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells

Innovations in Teaching Undergraduate Biology and Why We Need Them

Membrane Traffic within the Golgi Apparatus

Molecular Circuitry of Endocytosis at Nerve Terminals

Many Paths to Synaptic Specificity

Mechanisms of Growth and Homeostasis in the Drosophila Wing

Vertebrate Endoderm Development and Organ Formation

Signaling in Adult Neurogenesis

Vernalization Winter and the Timing of Flowering in Plants

Quantitative Time-Lapse Fluorescence Microscopy in Single Cells

Mechanisms Shaping the Membranes of Cellular Organelles

The Biogenesis and Function of PIWI Proteins and piRNAs Progressand Prospect

Mechanisms of Stem Cell Self-Renewal

Collective Cell Migration

Hox Genes and Segmentation of the Hindbrain and Axial Skeleton

Gonad Morphogenesis in Vertebrates Divergent Means to a Convergent End

From Mouse Egg to Mouse Embryo Polarities Axes and Tissues

Conflicting Views on the Membrane Fusion Machinery and the Fusion Pore

Coordination of Lipid Metabolism in Membrane Biogenesis

Navigating ECM Barriers at the Invasive Front The Cancer CellndashStroma Interface

The Molecular Basis of Organ Formation Insights from the C elegans Foregut

Genetic Control of Bone Formation

Listeria monocytogenes Membrane Trafficking and LifestyleThe Exception or the Rule

Asymmetric Cell Divisions and Asymmetric Cell Fates

Contents

INTRODUCTION 484THE EXPANDED BLASTOCYST

BACKGROUND 484Time 484Morphological Transitions

Shapes and Axes 485Cell Lineages 486

THE THREE MODELS PROPOSEDTO EXPLAIN HOW ABLASTOCYST IS GENERATED 488The Mosaic Model 488The Positional Model 489The Polarization Model 489

REEVALUATION OF THE THREEMODELS AND THEIRRELATIONSHIPS 491

MOLECULAR BASIS OFPOLARITY GENERATIONAND STABILIZATION 492E-Cadherin β-Catenin Actin

Ezrin and LamininIntegrins 492PAR Proteins 494CDX2 495

SUMMARY 496DOES POSITIONAL

INFORMATION EXISTWITHIN THE EGG ORZYGOTE 497Mechanisms 503

CONCLUSIONS 504

INTRODUCTION

In the mammal fertilization initiates a processof embryogenesis The mature 64- to 128-cellblastocyst (around 4ndash5 days postfertilizationin the mouse) (Figure 1) is the earliest stageat which a group of epiblast cells that couldreasonably be described as embryonic existsSome would argue that even these cells areproto-embryonic and only with the emergenceof definitive epiblast postimplantation are thecells truly embryonic This developmentalstrategy evolved with viviparity to facilitate an

effective sourcing of nutrients for embryonicgrowth via a complex membrane system Thesemembranes establish physical and chemicalcontact with the uterus to provide bothattachment and sustained maternal supportUltimately the membranes form part of theplacenta either a chorio-vitelline placentainvolving the hypoblast derivatives of the blas-tocyst (in monotremes marsupials and earlydevelopment of some eutherians) or a chorio-allantoic placenta involving the trophoblastderivativesmdashthe mature placental form in mosteutherians So although these early develop-mental stages are often called embryogenicthey might equally be called trophoblastogenicor hypoblastogenic This process is summa-rized comparatively with Xenopus in Figure 2It should be noted that the mouse blastocystthe mammalian model is not necessarilytypical in its organization and genesis (seeJohnson 1996 Selwood amp Johnson 2006)

Because the blastocyst is our developmen-tal end point its key features are describedfirst followed by three historical models thathave attempted to explain its genesis Lackof space restricts discussion to a considerationof trophoblast origins although the origins ofhypoblast are equally controversial (Yamanakaet al 2006)

THE EXPANDED BLASTOCYSTBACKGROUND

The first seven cell cycles to an expanded blas-tocyst are cleavage divisions in which there isno growth so cells halve approximately in sizeat each division (Figure 3) (Aiken et al 20042008 Lehtonen 1980) Specific developmentalevents are associated with particular develop-mental cell cycles suggesting operation of someunidentified endogenous clock ( Johnson 2002)The first two cell cycles are approximately18 hours in length and subsequent cycles are12 hours Each round of cell divisions is approx-imately synchronous but with sufficient hetero-geneity that intermediate stages between 2 4

484 Johnson

Fertilization

Zona pellucida (ZP)

Hypoblast(Gata 46)

ICM inner cell mass

Video CLICK TO VIEW

PB polar body

ZP zona pellucida

Cell Lineages

486 Johnson

PBS (ZP) PBS

Zona pellucida

Polar body

Blastocoelic cavity

Blastocyst 8-cell

Polarization

16-cell 1-cell

Mosaic

(Dalcq 1957)

The Mosaic Model

488 Johnson

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Fertilization

Zona pellucida (ZP)

Hypoblast(Gata 46)

ICM inner cell mass

Video CLICK TO VIEW

PB polar body

ZP zona pellucida

Cell Lineages

486 Johnson

PBS (ZP) PBS

Zona pellucida

Polar body

Blastocoelic cavity

Blastocyst 8-cell

Polarization

16-cell 1-cell

Mosaic

(Dalcq 1957)

The Mosaic Model

488 Johnson

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Video CLICK TO VIEW

PB polar body

ZP zona pellucida

Cell Lineages

486 Johnson

PBS (ZP) PBS

Zona pellucida

Polar body

Blastocoelic cavity

Blastocyst 8-cell

Polarization

16-cell 1-cell

Mosaic

(Dalcq 1957)

The Mosaic Model

488 Johnson

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

PBS (ZP) PBS

Zona pellucida

Polar body

Blastocoelic cavity

Blastocyst 8-cell

Polarization

16-cell 1-cell

Mosaic

(Dalcq 1957)

The Mosaic Model

488 Johnson

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Blastocyst 8-cell

Polarization

16-cell 1-cell

Mosaic

(Dalcq 1957)

The Mosaic Model

488 Johnson

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

490 Johnson

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

492 Johnson

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

PKC protein kinaseC

PAR Proteins

494 Johnson

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

SUMMARY

496 Johnson

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

498 Johnson

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

First cleavageplane

Polar body

Blastocoelic cavity

500 Johnson

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Polar body

Blastocoelic cavity

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

N = 460 EM39

N = 88N = 60

502 Johnson

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Mechanisms

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

CONCLUSIONS

SUMMARY POINTS

504 Johnson

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

FUTURE ISSUES

ACKNOWLEDGMENTS

LITERATURE CITED

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

506 Johnson

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

508 Johnson

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

510 Johnson

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

512 Johnson

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Volume 25 2009

Joseph G Gall 1

Indexes

Errata

viii Contents

Indexes

Errata

viii Contents

Documents

From Mouse Egg to Mouse Embryo: Polarities, Axes, and Tissues