doi:10.1006/scdb.2001.0262, available online at http://www.idealibrary.com onseminars in CELL & DEVELOPMENTAL BIOLOGY , Vol. 12, 2001: pp. 345–351

Choosing sides: establishment of polarity in zygotes of fucoidalgae

Colin Brownlee a,∗, Francois-Yves Bouget b and Florence Corellou a,b

The acquisition and expression of polarity during earlyembryogenesis underlies developmental pattern. In manymulticellular organisms an initial asymmetric division of thezygote is critical to the determination of different cell fatesof the early embryonic cells. Zygotes of the marine fucoidalgae are initially apolar and become polarized in responseto external cues. This results in an initial asymmetricdivision of the zygote. Subsequent divisions occur in a highlyordered spatial and temporal pattern. A combination of cellbiological and biochemical studies is providing new details,and some controversies concerning the mechanisms by whichzygotic polarity is acquired and amplified. Here, we discusssome of the more recent studies that are allowing improvedunderstanding of polarization in this system.

Key words: polarity / Fucus / Pelvetia / actin / cell cycle

c© 2001 Academic Press

Introduction

Polarity can have functional or developmentalsignificance for many cell types. Functional polarityallows specialized cell types to carry out specializedtasks. Good examples in plants include pollen tubesand root hairs. Pollen tubes are highly polarized inorder to grow though the female style to reach theovary. Root hairs are specially elongated to increasethe surface area of the root in order to exploit soilnutrient and water sources. Developmental polarity isan absolute requirement for multicellular organisms.

From the aMarine Biological Association, The Laboratory, CitadelHill, Plymouth, PL1 2PB, UK and bStation Biologique, UMR 1931,CNRS, F-29680, Roscoff, France. *Corresponding author.E-mail: cbr@mba.ac.uk

c©2001 Academic Press1084–9521/01/050345+ 07/$35.00/0

All dividing cells need to become polarized into twohalves before partitioning into two daughter cells. Inmany cases, cell division results in two unequal daugh-ter cells and the division is thus referred to as asym-metric. Critical to our understanding of developmentis the realization that asymmetric division invariablyresults in different fates of the daughter cells.1–4

Zygotic polarity may be pre-determined in theunfertilized ovum or, more rarely, it can be acquiredde novo following fertilization. Pre-determined polar-ity is often the rule in many animal and plant zygotes.Thus, for example, eggs of ascidians are organizedinto distinct domains prior to fertilization that definepermissive zones within which gastrulation can occurduring subsequent development.5 In many higherplants such as Arabidopsis, the growing pollen tubeenters the ovary at a precise location and the siteof fusion of the egg and sperm is dictated by thesurrounding structures.6,7 Subsequent polarizeddevelopment of the zygote and embryo is influencedby the surrounding tissues. Higher plant embryosand zygotes are not amenable to direct manipulationand biochemical and physiological studies thatrequire large numbers of synchronously developingzygotes are difficult, though this disadvantageis, of course, countered by the availability of anincreasing number of mutants defective in polarityand early development.

Fucoid algae as a paradigm for polarity studies

Examples of plant or algal cells that can be polarizedde novo include protoplasts of mosses such asPhyscomitrella and Ceratodon 8 and zygotes of the fucoidalgae. The moss system has the advantage that it isamenable to genetic manipulation9 and offers greatpotential for the study of single cell polarization,though so far our knowledge of the mechanisticcellular processes underlying acquisition of polarity

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Perception of environmentalasymmetries

Anchorage proteins

actin

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(a)

(b)

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Figure 1. Putative localization mechanisms for actin at therhizoid pole. Two possibilities are presented. In (a), local-ization of actin precedes the localization of proteins thatare required for stable anchorage of actin. The activity oftyrosine kinase-like proteins (PTK) brings about the local-ization of anchorage proteins and stabilization of the polaraxis. In (b) both actin and anchorage proteins co-localizeindependently but their interaction leading to stableactin localization depends on tyrosine kinase-like activity.Localized secretion of sulphated compounds at the rhizoidpole follows stabilization of F-actin (see Reference 43).

is more extensive in the fucoid algae such as Fucusand Pelvetia. This largely reflects the ease with whichgametes and zygotes can be obtained, their suitabilityfor cellular imaging studies and microinjection,coupled with the ability to carry out biochemicalanalyses of large numbers of synchronously develop-ing zygotes. In this review we will outline some of theessential features of polarization in zygotes of fucoidalgae, paying particular attention to some recentdevelopments and controversies. More detailedreviews of polarization and early development in thefucoid algae can be found elsewhere.4,10–12

Detecting asymmetric cues: photopolarization

Fertilization of fucoid eggs occurs externally inthe sea between motile sperm and eggs thatpossess no intrinsic polarity. The resulting zygote

can be polarized by a range of external vectors.Polarization progresses through a distinct sequenceof events4,10–12 that begin with a selection phaseduring which external polarizing vectors are detectedand translated into intracellular asymmetries.This phase normally occurs between 4 and 10 hfollowing fertilization, depending on the species.This is followed by an amplification phase wherethe initial labile asymmetries (axis formation)become increasingly irreversible (axis fixation)leading to polarized structural changes, culminatingin germination of a rhizoid several hours later.The asymmetric first division of the zygote occurstransverse to the polar axis producing two cellswith different fates–a rhizoid cell which developsinto the holdfast and a thallus cell which forms thephotosynthetic and reproductive fronds.

The most commonly used polarizing vector in thelaboratory, and the one that is likely to be mostsignificant in the natural environment, is unilaterallight. The mechanism by which unilateral light istranslated into cellular asymmetries is still unclear. Ithas long been accepted that photopolarization occursin response to photoreceptor activation at the levelof the plasma membrane13 This has been shownto be associated with enhanced redox transport atthe cell surface on the side of the zygote facingaway from the light source.14 Plasma membraneredox transport was shown to be enhanced by bluelight in Fucus zygotes and externally applied electronacceptors such as hexacyanoferrate (HCF) renderedzygotes unable to sense the direction of the incidentlight. This effect was specific to the photosensitivephase of polarization. The same level of inhibitorapplied at later stages of polarization did not disrupta previously fixed polar axis and did not preventrhizoid germination or growth. This suggests thata light-sensitive redox transport process is an earlycomponent of the signal transduction chain leadingto photopolarization.

An alternative, though not necessarily exclusivemodel, for fucoid photopolarization has beenpresented by Robinson and co-workers15,16 basedon the demonstration of the existence of retinalin Pelvetia zygotes. In animal cells, retinal is knownto be exclusively associated with opsins with whichit forms rhodopsin-like light-sensitive complexes.This led these workers15,16 to propose, by analogy,that spatial variation in the level of cyclic GMP inresponse to a light gradient within the zygote couldbe one of the first asymmetries established duringphotopolarization. This was supported by evidence

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that blue light exclusively elevated cyclic GMPlevels by approximately two-fold in Pelvetia zygotes.Pharmacological inhibition of guanylyl cyclasealso resulted in inhibition of photopolarization,as did treatment with a permeant cyclic GMPanalogue. Cyclic GMP is known to promote actindepolymerization in several systems.17,18

Robinson and co-workers proposed that elevatedcyclicGMP on the side of the zygote facing the lightsource would result in preferential depolymerizationof actin in that region with actin becoming increas-ingly polymerized towards the rhizoid pole facingaway from the light direction. Validation of thesemodels for photopolarization will require accuratemeasurements of the light gradient around thezygote. Simple models for photopolarization basedon a single differentially activated photoreceptorand spatial second messenger gradients do not easilyexplain how photopolarization can still occur at veryhigh light levels where conceivably all photoreceptormolecules are saturated leading to uniform secondmessenger levels throughout the zygote. A morecomplex photoreceptor system is likely to operatethat allows discrimination of light gradients at alllevels of irradiance.

Role of actin in photopolarization

The role of actin in polarization is well-establishedin yeast and animals.19 In budding yeast, regulationof actin polymerization at specific sites either duringcell division or budding in response to matingpheromone is essential for polarized cell shapechanges.20 It is likely that the activity of Rho familyGTPases such as Cdc42, Rho and Rac regulate thelocation and timing of organization and dynamicsof the actin cytoskeleton. The guanidine nucleotideexchange factor (GEF) cdc24 is normally sequesteredin the nucleus by complexation with a nuclearlocalization signal Far1. Activity of the cell cyclecontroller CDC28/cln in G1 phase triggers Far1degradation and subsequently the release of cdc24that can translocate to the plasma membrane whereit associates with and activates a rho-GTPase (cdc42)involved in actin polymerization at specific sitesmarked by BUD gene products. The factors thatlocalize BUD gene products are not clear. Cdc24can also be released from the nucleus in responseto a MAP kinase signalling pathway activateddownstream of pheromone-receptor interactions atthe plasma membrane. This involves the association

of Far1/cdc24 with a nuclear export factor Msn5 andlocalization to the bud site via interaction with theGβγ subunits of a heterotrimeric G-protein arisingfrom pheromone-induced G-protein activation.While such detailed molecular interactions have yetto be characterized in the fucoid algal system, thelarger size of Fucus and Pelvetia zygotes makes themwell suited to studies of the spatial aspects of actinlocalization and the interaction between cytoskeletonand other intracellular signals such as Ca2+ (seelater). Localization of actin in the Fucus zygote marksone of the first steps in the patterning of the complexmulticellular embryo. However, while evidence existsfor the early localization of actin during polarization,there are conflicting reports about when this actuallyoccurs and about the precise pattern of localization.

In the absence of a polarizing light vector, Pelvetiazygotes will germinate a rhizoid from the site of spermentry.21 This was shown by monitoring the site ofsperm pronucleus entry into the egg in relation tothe site of rhizoid outgrowth and polar secretion ofadhesive which occurs at the rhizoid pole prior togermination. Hable and Kropf21 were also able tomanipulate the site of rhizoid outgrowth by selectiveapplication of sperm to one side of eggs. Significantly,these workers also showed that a patch of corticalactin, visualized by staining living cells that hadbeen briefly permeabilized to allow entry of thefluorescent actin probe rhodamine phalloidin, couldbe observed at the site of sperm entry within minutesof fertilization. This work was extended further in adetailed study of actin localization in response to apolarizing light vector.22 This showed that a corticalactin patch could be detected at the future rhizoidpole in response to a polarizing light vector within 3 hof fertilization, corresponding to the early stages ofphotopolarization and well before the period duringwhich the axis becomes fixed. This also showed thatan environmental vector could override the sperm-induced actin localization. After 4 h in unilaterallight, the position of the cortical actin patch could bereversed approximately 40–50 min after reversing thelight direction. This relocation of the actin patch wasshown to arise by de novo synthesis of a new patch atthe new rhizoid pole.

A contrasting result was obtained by Pu andco-workers23 who imaged fluorescent phalloidinthat had been microinjected into live cells priorto photopolarization of Pelvetia zygotes. This studyshowed that cellular actin levels increased within1 h of the onset of a polarizing light vector. Nocortical actin patch was observed in this study and

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only a very slight increase in actin levels was observedat the future rhizoid pole after 2 h of unilaterallight. Localized actin at the rhizoid pole was onlyobserved after germination with the appearanceof a sub-apical actin ring near the rhizoid apex. Itwas suggested23 that methodological differencescould explain the appearance of cortical actinpatches in the study by Alessa and Kropf.22 Despitethese differences, this work, together with inhibitorexperiments showing an absolute requirement foractin in photopolarization,23 firmly indicates theabsolute necessity for actin in photopolarization.

The role of Ca2+ in fucoid zygote polarization

While there has been some disagreement overthe absolute requirements for Ca2+ in thephotopolarization of fucoid zygotes,4,24 Ca2+

has been shown to be elevated at the apex of severalpolarized growing plant cells, including pollen tubes,root hairs and rhizoids of Fucus and Pelvetia. TheseCa2+ gradients are known to be intimately associatedwith apical growth in pollen tubes and root hairs.25–27

Spatial buffering of Ca2+ gradients in polarizingPelvetia zygotes by injection of BAPTA-type Ca2+

buffers prevents polarization.28 Moreover, Ca2+ hasbeen shown to be elevated at the future rhizoid polein Fucus zygotes prior to germination.29 More recentconfocal imaging studies23,30 provided evidence forthe timing of the establishment of elevated Ca2+ atthe future rhizoid pole. These workers were able todetect a significant Ca2+ gradient after only 1 h ofexposure to polarizing light, which was significantlyearlier than any asymmetries in actin distribution.However, the actin depolymerizing agent latrunculinB prevented the appearance of the Ca2+ gradient,indicating an absolute requirement for actin.23

These apparently contradictory observations ledto the speculation that an actin framework wasnecessary for the putative asymmetric distributionof Ca2+ channels in the rhizoid apex but that theputative localized insertion of channels into theplasma membrane at the rhizoid apex was effected byanother polarized molecule such as myosin.23 Whilelocalized binding of fluorescent dihydropyridines hasbeen observed at the rhizoid pole of polarizing fucoidzygotes,31 it is not clear whether this really representsthe asymmetric distribution of Ca2+ channelssince the specificity of dihydropyridines for Ca2+

channels in plants or algae has not been conclusivelydemonstrated. Moreover, patch clamp studies of

ion channel distribution in polarized Fucus zygoteshave not revealed any preferential accumulation ofCa2+-permeable channels in the rhizoid apex.32 Ca2+

levels in the Fucus rhizoid are dynamic, respondingto osmotic fluctuations and involving both Ca2+

influx and release of Ca2+ from intracellular storesin apical and sub-apical regions.33,34 Release of Ca2+

from intracellular stores in the rhizoid apex may wellplay a significant role in amplifying any localizedplasma membrane channel-mediated elevation ofCa2+ levels. A simple Ca2+ gradient model based onthe distribution of Ca2+ channels may only partlyexplain the development of Ca2+ signals that mayamplify initial asymmetries.

Several studies point to a role for calmodulinas a mediator of the cytosolic Ca2+ gradientat the rhizoid pole. However, the relative rolesof calmodulin in axis formation, fixation andgrowth are still unclear. Calmodulin inhibitorssuch as ophiobilin A were shown to inhibitphotopolarization albeit in a complex concentration-dependent manner24 suggesting that calmodulinwas involved in photopolarization, germinationand rhizoid growth. Further evidence for a rolefor calmodulin in mediating the Ca2+ gradienthave come from experiments using microinjectedcalmodulin. This was shown to either amplifythe photopolarization response35 or to overcomethe inhibition of photopolarization followinginjection of heterologous calmodulin antibodies.30

However, while these experiments indicated a rolein photopolarization, localization of microinjectedfluorescent calmodulin to the rhizoid pole wasapparent only during the later stages of rhizoidgermination,35 though earlier detection may havebeen limited by the sensitivity of the assay. Thus,while calmodulin appears to play a fundamentalrole in polarization the precise stage at which thisoperates remains unclear.

The role of secretion in polar axisdetermination

Several studies have demonstrated an essential rolefor localized secretion and membrane recycling infucoid zygote polarization.36–39 Localized secretionof jelly occurs at the presumptive rhizoid pole duringthe photopolarization period.10 After photopolar-ization and prior to rhizoid germination, secretionof a sulphated fucan (F2) occurs preferentially atthe rhizoid pole.10,39 This continues during rhizoid

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22-24 h 0 h

F

CDK

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+?

Y Y

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Figure 2. The first cell cycle in Fucus comprises well-defined G1, S, G2 and M phases. CDK activities are required forG1/S and G2/M. The transcription of Histone H3 in S phase and synthesis of CDKs from maternal mRNAs occurs afterfertilization. Transcription, before 10 h AF, of the genes encoding activating proteins is required for mitotic activity ofCDKs. CDKs are maintained inactive in G2 by inhibitory phosphorylation on tyrosine residues before being activated inmitosis by a cdc25-like phosphatase. A DNA replication checkpoint prevents mitosis, including chromatin condensationand spindle formation, through inactivation of mitotic CDKs by inhibitory phosphorylation. A spindle assembly checkpointprevents progression through mitosis, including chromatin decondensation, by inhibiting inactivation of CDKs by anunknown mechanism. Photopolarization corresponds to early S phase while axis fixation and germination occurs in G2(see References 49 and 50).

growth. The requirement for localized secretionof cell wall components in zygote polarization hasled to models for polar axis determination basedon the formation of axis stabilizing complexescomprising trans-plasma membrane links betweena sulphated cell wall polysaccharide in the cell walland the cytoskeleton.40,41 While adhesions betweenthe plasma membrane and cell wall clearly play acritical role in polar axis specification and rhizoidgermination, the unequivocal identification of thecomponents of these complexes remains a greatchallenge for future studies.

Tyrosine kinase activity appears to be required forpolarity establishment as well as for polar secretion ofF2 into the rhizoid pole which in turn is dependenton the anchorage of actin. Direct disruption of theactin cytoskeleton leads to inhibition of polarizedF2 secretion.42 A similar inhibition of F2 secretioncan be observed following inhibition of tyrosinekinase-like activity.43 Several proteins have beenshown to change in their levels of phosphorylationon tyrosine residues during the first 24 h of Fucus

development could be altered by inhibition oftyrosine phosphorylation. Interestingly, inhibition oftyrosine kinase-like activity with a range of inhibitorsdid not affect actin localization but did prevent thestable anchorage of actin to the rhizoid pole, sothat actin could become re-localized to the oppositepole by changing the light direction up to 24 hlater. When applied continuously to developingembryos from the photopolarization stage onwards,tyrosine kinase inhibitors produced non-polarmuliticellular embryos. In contrast, inhibition oftyrosine phosphorylation did not affect the growthof pre-germinated rhizoids. This identifies separateactin localization and anchorage processes that aredifferentially sensitive to tyrosine phosphorylation(Figure 1).

Polarization and the cell cycle

During normal development, the first asymmetriccell division occurs only after the polar axis has

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been established and the rhizoid has germinated(Figure 2). This suggests that a tight coupling occursbetween the control of cell cycle and polarization.However, it is also clear that cell division can occurwhen polarization has been prevented. Treatmentwith sucrose prevents rhizoid germination but doesnot prevent polar axis fixation or asymmetric mRNAlocalization or cell division.44 Inhibitors of Golgivesicle transport,38 tyrosine kinase activity43 andphospholipase C45 can all produce spherical embryoswith no rhizoids but which have divided severaltimes, though often with disrupted pattern (seeReference 38). Taken together, these studies suggeststhat cell cycle progression does not depend absolutelyon polarization but is regulated by endogenous orother developmental cues.

A combination of biochemical, inhibitor andmicroinjection studies, is beginning to shed morelight on the regulation of the Fucus cell cycle. Inanimal and plant somatic cells and in yeast, a rigidseries of checkpoints ensures that the cell cyclecannot progress until events such as DNA replicationand spindle pole assembly have been completed.46,47

This ensures that each cell receives its correctcomplement of DNA. The cell cycle passes througha sequence of G1, S, G2 and M phases. However, inmany animal embryos, these checkpoints and theG1 and G2 phases are often reduced or absent.48

This is associated with rapid embryo cell division.The Fucus zygote, however shows distinct G1 andG2 phases and clear checkpoints that operate inS and M phases.49,50 These checkpoints involveregulation of cyclin dependent kinases (CDKs). Thespindle assembly checkpoint in Fucus involves themaintenance of high levels of CDK activity and itappears that inactivation of CDKs is required forchromatin condensation and exit from mitosis.50

While it is clear that cell division can occur in theabsence of zygote polarization, the reverse may notbe true. Recent work in our laboratories has shownthat specific inhibition of CDK at the G1/S transitioncan block polarization. This does not occur if thecell cycle is blocked in S phase through inhibition ofDNA polymerase or when CDKs are inhibited afterthe G1/S transition. This suggests that CDK activityis required for polarity establishment. Whether CDKspositively control polarization or are part of a polaritycheckpoint that operates to prevent polarizationwhen the G1/S transition is blocked is not known(Corellou et al., unpublished). These emergingfindings are indicating that a tight regulation betweenpolarization and cell division operates during early

development to ensure correct patterning of theembryo. However, while polarization appears todepend on early cell cycle progression, the cell cycleis able to proceed independently when polarization isblocked, suggesting that a developmentally regulatedpathway controls these events independently.

Conclusions

The Fucus zygote and embryo continue to providenew insights into the processes regulating polariza-tion from a single celled zygote to a multicellularplant. There are still uncertainties surrounding theprecise timing of events associated with polarizationand many of their interactions are still unclear.Particular attention needs to be paid to actinlocalization and comparisons with other systems.Major advances have been made and are likely tocontinue in our understanding of the interactionsbetween the cytoskeleton and second messengerssuch as Ca2+. A combination of cell biological andbiochemical approaches has also provided newinsights into the regulation of the cell cycle duringzygotic polarization, indicating the presence ofcomplex interactions between these processes.

Acknowledgements

Supported by the NERC and BBSRC (CB) and CNRS(FYB) and Association de le Recherche sur le Cancer(FC).

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