Lnduction of Polarity in Fucoid Zygotes

Home , Fucaceae, Fucales, Fucus distichus, Invagination, Pelvetia, Rhizoid

The Plant Cell, Vol. 9, 1O1 1-1 020, July 1997 O 1997 American Society of Plant Physiologists lnduction of Polarity in Fucoid Zygotes

Darryl L. Kropf' Department of Biology, University of Utah, Salt Lake City, Utah 841 12

INTRODUCTION

Early events 'in the vegetative development of higher land Developing embryos of many land plants, including Arabidop- plants are difficult to investigate because the young embryo sis, undergo a similar pattern of cell divisions (Figure 1; see is encased in the ovarian tissue of the previous gametophytic also Laux and Jürgens, 1997, in this issue). and sporophytic generations. Recently, genetic approaches This review highlights recent progress in understanding have succeeded in identifying mutations that alter early devel- the polarization of fucoid zygotes, with an emphasis on opment (Jürgens, 1995; see also Laux and Jürgens, 1997, in early events involved in polarity induction in Pelvetia. this issue), but cellular and physiological analyses of higher Readers are directed to a comprehensive review (Kropf, plant embryogenesis remain tedious. 1992) and to other more specialized reviews (Goodner and Brown algae of the family Fucales (comprising the genera Quatrano, 1993; Kropf, 1594; Berger and Brownlee, 1995; Fucus and Pelvetia) are, however, very tractable for investi- Fowler and Quatrano, 1995; Quatrano and Shaw, 1997) for gating early plant embryogenesis, especially the establish- additional analyses and opinions. ment of developmental polarity. These marine organisms grow attached to rocks in the intertidal zone and are typi- cally 0.25 to 0.5 m in height at maturity. Zygotes, eggs, and sperm are shed from fronds of mature plants in the labora- POLARITY INDUCTION tory and can easily be harvested in gram quantities, free of other tissues. Young zygotes attach firmly to the substratum, and populations of adhering zygotes transit synchro- The induction of polarity in fucoid zygotes is a continuum of nously through early development. overlapping events that occur during the first 10 hr after fer- In the Fucales, mature eggs are radially symmetric, and tilization (AF). For convenience, I consider polarity induction cell polarization does not occur until after fertilization. The to occur in two stages: axis selection, followed by axis am- primary developmental axis is organized early in the first cell plification. Events occurring during these stages are consid- cycle and defines the growth axis of the young embryo. Un- ered below, and models describing polarity induction are like higher plants, in which positional cues are often conveyed presented later. by neighboring cells, fucoid zygotes develop autonomously and therefore rely on environmental signals for spatial information to orient their nascent axes. In the laboratory, an entire population can be induced to establish axes in parallel Axis Selection by the application of externa1 vectors such as unidirectional light (Jaffe, 1968). A few hours after polarization, localized Axis selection is the process by which the position of the de- growth occurs at one pole of the nascent axis, as shown in velopmental axis is specified. To select an axis, a zygote Figure 1. must perceive a signal, transduce that signal, and mark the The first zygotic division is an invariant, asymmetric divi- developmental polarity. sion, with the cell plate oriented transverse to the growth Fucoid eggs have no demonstrable cellular asymmetries, axis. The smaller rhizoid cell contains the growing apex and and axis selection must therefore occur during or after fertil- is the precursor to the holdfast of the alga; the larger thallus ization. The egg pronucleus resides in the center of the egg cell gives rise to the stipe and fronds of the mature plant cell, and microtubules nucleate uniformly from the surface of (Figure 1). After the initial division of the zygote, the rhizoid the pronucleus and radiate toward the cortex (Swope and cell elongates and repeatedly divides transverse to the Kropf, 1993). Short rods of F-actin are randomly oriented growth axis, whereas the thallus cell undergoes proliferative and uniformly distributed throughout the cortex (Kropf et al., divisions, each of which is transverse to the previous division. 1992), and organelles are uniformly distributed throughout the cytoplasm (Brawley et al., 1976b). Glycoproteins are distributed in patches over the entire surface of the egg E-mail [email protected]; fax 801-581 -4668. (Stafford et al., 1992). 1012 The Plant Cell

A derived organelles persist through much of the first cell cy- B cle and have the potential to serve as positional markers. However, preliminary evidence indicates that the nucleus and perinuclear material, as marked by centrosomal position, do not align with the developmental axis until after growth begins (S.R. Bisgrove, unpublished results); therefore, they are unlikely to be involved in ais selection. In- stead, polarity is likely to be perceived and marked in the plasma membrane and cell cortex (see below). Although the mechanisms by which sperm may mark the plasma membrane or cortex are unknown, Ca2+ levels increase just be- C D neath the plasma membrane at the sperm entry site (Roberts et al., 1994), and it is possible that, in turn, this local elevation in Ca2+activity triggers regional structural changes (see Trewavas and Malhó, 1997, in this issue).

Environmental Cues

The putative sperm-induced axis is weak and can be over- ridden by vectorial information in the environment (Jaffe, 1958; Robinson, 1996b). Approximately 3 hr AF, Pelvetia and Fucus zygotes secrete an adhesive symmetrically over the surface of the zygote and attach firmly to the substratum (Vreeland et al., 1993; Vreeland and Epstein, 1996). Once at- Figure 1. Embryogenesis in Pelvetia and Arabidopsis. tached, zygotes begin to sense and respond to an impres- sive number of very different environmental cues. Indeed, (A) to (C) Pelvetia embryogenesis. (A) Unfertilized egg. (B) Two- the developmental axis, and, hence, the position of rhizoid celled embryo displaying localized rhizoid growth and invariant division. (C) Eight-celled embryo with transverse divisions in descen- outgrowth, can be controlled by the application of unidirec- dants of the rhizoid cell. tional light, gradients of ions or ionophores, gravity (G. Muday, (D) Arabidopsis embryo. The young embryo is morphologically simi- personal communication),flow of seawater, temperature gra- lar to the Pelvetia embryo. dients, and many other factors (Jaffe, 1968). Plane polarized Arrows indicate the first division plane of the zygote. light induces the formation of two rhizoids (Jaffe, 1958), indicating that vectorial information does not simply result in ròta- tion of the putative sperm-induced axis. Zygotes are sensitive to each gradient for a restricted period, and the windows of The Role of Sperm Entry: An Open Question sensitivity overlap for many gradients (Bentrup, 1984). The mechanisms by which environmental gradients are The role of sperm entry in inducing polarity in fucoid eggs is perceived and transduced are almost totally unknown in unclear (Roberts et al., 1993). Sperm entry is sufficient for plants (see Trewavas and Malhó, 1997, in this issue). In- polar development; externa1 vectorial information (e.g., light) deed, the diversity of stimuli that can elicit developmental is not required for localized rhizoid outgrowth, oriented cell axis selection in zygotes of the Fucales indicates that multi- division, or subsequent development. These observations ple, converging signal transduction pathways are involved. are consistent with sperm entry inducing an initial axis, and in However, to date, only the photopolarization pathway has a related brown alga, Cystosira, rhizoid growth occurs at the been investigated in detail. Unidirectional ultraviolet or blue sperm entry site in the absence of vectorial cues (Knapp, light induces rhizoid growth from the shaded hemisphere of 1931). However, the spatial relationship between sperm entry the zygote (Hurd, 1920; Robinson, 1996a), and the activa- and rhizoid outgrowth has not been investigated in Pelvetia or tion of cortical photoreceptors (Jaffe, 1958) preferentially Fucus, the organisms in which nearly all of the subsequent stimulates plasma membrane redox chains at the presump- polarity studies have been conducted. tive rhizoid (i.e., shaded) pole (Berger and Brownlee, 1994). If sperm entry induces polarity, then the sperm entry site Photopolarization is inhibited by acidification or alkaliniza- must somehow be marked in the egg cytoplasm. The sperm tion of the cytosol (Kropf et al., 1995) and by disruption of eyespot, mitochondria (Brawley et al., 1976a), and cen- F-actin (Quatrano, 1973), but the roles of redox potential, trosomes (Motomura, 1994) migrate with the sperm pronu- cytosolic ions, and the cytoskeleton in signal transduction cleus and are deposited on the zygotic nucleus at the site of remain a mystery. Likewise, the mechanism by which the pronuclear fusion, as shown in Figure 2A. These sperm- rhizoid pole is marked is unknown. Polarity in Fucoid Zygotes 1013

sperm pronucleus \ F-actin | DHP receptor zygotid an g c eg nuclei A current flow •:vi-.cytosoli gradienn cio t centrosome celB 3 l wall * F2; vitronectin-like protein MtOC '.<- jelly ~ actin mRNA microtubule o secretory vesicle

Figure 2. Summary of Structural and Physiological Changes during the First Zygotic Cell Cycle.

Centrosome. AF r h e (A inherite)On e sar d paternally organellesd an , , microtubules F-actid an , uniformle nar y distribute zygotie th n di c cytoplasm. Zygotes. AF r h ) Fou 7 (B secreto rt e local jelly electricad ,an l current flows throug celle hth . Inward curren jell positioned e ytan ar shadee th n do d side of the zygote. Centrosomes separate and begin to migrate along the nuclear envelope. . F-acti corte(Ce AF dihydropyridinth )d r Seveh n i xan 0 1 no t e receptor plasme th n si a membrane accumulat presumptive th t ea e rhizoid- Se . cretion becomes targeted to this site, depositing the sulfated fucan F2 in the wall and causing local cortical clearing. Gradients of H* and Ca2+ are detectable. (D) Ten to 13 hr AF. The rhizoid begins to elongate, Centrosomes become the major site of microtubule nucleating activity, and microtubules extend preferentially into the rhizoid cortex. nucleue Th . (Es)AF Thirteerotatesr h 6 1 no ,t bringin Centrosomee gth s into axial alignment spindle th ; e forms. (F) Twenty to 24 hr AF. At cytokinesis, the cross-wall forms transverse to the growth axis. F-actin and actin mRNA accumulate at the cross-wall. In the next division, only the rhizoid nucleus rotates, so spindles in the two-celled embryo are aligned transverse to one another. DHP, dihydropyridine; MtOC, microtubule organizing center. 1014 The Plant Cell

Axis Amplification

After axis selection, ther prolongea s ei d period, lasting from ~4 to 10 hr AF, during which there is a gradual reorganization of the cytoplasm, plasma membrane, and cell wall in the young zygote. These rearrangements, which presumabl- yam plifstrengthed yan weae nth k initial axis, overlap temporally and are discussed in their approximate chronological order.

Polar Jelly Deposition

One to 2 hr after Pelvetia zygotes adhere to a substratum and begin to sense environmental cues, they deposit a jelly materia presumptive th t a l e rhizoid nascen e polth f eo t axis, as shown in Figure 3A (Schroter, 1978; Weisenseel, 1979). The jelly, which contains alginate, phenolic compounds, and sulfated fucans (Vreelan Epsteind dan , 1996) deposites i , s da an amorphous, translucent layer outside the cell wall. Fucus zygotes are slightly delayed compared with Pelvetia', uniform adhesiv secretes ei conspicuoud an , d AF ~ r 4h s polar jelly (VreelaniF s A deposite r h 8 t al.do e t , d6 1993) rapidite .Th y with which jelly deposition occurs probably relate funco st - tion rhizoid-localizee th ; d jelly strengthen attachmene sth f o t this region of the spherical zygote to the substratum, usually Figur . Jelle3 y Deposition. intertidae roca th n ki l zone (Vreelan Epsteind dan , 1996). (A) Jelly layer surroundin 4-hr-olga d Pelvetia zygote outlined with Robust and rapid attachment helps prevent the cell from be- fluorescent microspheres. The jelly (arrowhead) extends >5 (j.m ing washed out to sea in the next tidal cycle. fro cele mth l surface (arrowpresumptive th t a ) o n e s rhizoiha t dbu Jelly continue depositee b o st d ove surface rth f Fucueo s measurable thicknes presumptive th t sa e thallus arroe e th Th . wn i Pelvetiad an embryos throughout early development, with upper right corner indicates directio f lighno t treatment initiate1 t da H.ITI5 2 .= r Ba . hrAF greatest accumulations occurring towar rhizoie dth d pole (B) The position of jelly deposition changes during axis realignment. (Schroter, 1978). However, jelly depositio t requireno s ni r dfo In respons ligho et (L1)1 t , jell deposites yi t jellda (J1)y1 . Whee nth early embryogenesis. Growth in seawater lacking sulfate direction of the light is reversed to L2, jelly is deposited at J2. prevents sulfation of fucans and thereby interferes with ionic The images, kindly provided by Whitney Hable, are 1-(jim optical interactions between polysaccharide molecules jelle Th y. sections take lasey nb r scanning confocal microscopy. Fluorescent t depositedlayeno s i r zygotet ,ye s germinat dividd ean e nor- organelles insid celle chloroplastse th sar . mally (Schroter, 1978). The jell likels yi y deposite vesicla dvi e secretion (Vreeland t cleaye e tt r al.whetheno , s i 1993) t i t r ,bu uniforml y distributed vesicles fuse with the plasma membrane locally or between plasma membran celd epresumptivlan e walth t a l e whether a subpopulation of secretory vesicles is preferen- rhizoid are detectable within 1 hr of photopolarization. tially localized at the presumptive rhizoid. Electron microscopy studies of Fucus zygotes provide no evidence for asymmetrie Golgsn i vesiclr o i e distribution thit sa s early stage Ionic Currents of development (Quatrano, 1972; Brawle t al.ye , 1976b)e Th . early secretion of polar jelly in fucoid zygotes is surprising, A transcellular ionic current (Figure 2B), which is generated becaus mosn ei t polarizing cells, secretio lata s eni event, plasme ath t a membrane bees ha ,n thoughtfully discussed occurring only after an axis has been amplified and rein- in numerous articles and reviews (Jaffe et al., 1974; Jaffe forced (Drubi Nelsond nan , 1996). Nuccitellid an , 1977; Robinso McCaigd nan , onl1980s i yd )an Despit lace ef obviouth k o s structural polarit cele t th a l n yi discussed briefly here. In very young zygotes, unstable the time of jelly secretion, there are local physiological patches of inward and outward current (defined as the flow of changes in the cellular cortex that can be detected by plas- positive charge) can be detected, but by 6 hr AF, an inward molysis or measurement of electrical current. When 5-hr-old current stabilize presumptive th t sa e rhizoid pole (Nuccitelli, photopolarized zygotes are placed in hypertonic seawater, 1978). Treatment with cytochalasin D prevents localized cur- plasmolysis occurs preferentially at the presumptive rhizoid rent flux (Brawle Robinsond yan , 1985), indicatin depenga - pole (Ree Whitakerd dan , 1941). These change interactionn si s dence on cortical F-actin. Polarity in Fucoid Zygotes 1O1 5

The current is thought to result in local differences in cyto- though these experiments are reported to investigate axis solic ion concentrations at the presumptive rhizoid and thal- formation, it seems more likely that they address realign- lus poles. Indeed, a fraction of the inward current is carried ment of an axis selected before jelly secretion. by Caz+at 5 to 6 hr AF (Robinson and Jaffe, 1975), yet cytosolic Caz+ gradients have not been detected until 10 hr AF (Berger and Brownlee, 1993; Taylor et al., 1996). By contrast, AXlS FlXATlON a small but measurable cytosolic pH gradient (acidic at the presumptive rhizoid) has been detected at 5 hr AF, and the magnitude of the gradient increases throughout the period Just before germination, polar zygotes become insensitive to of amplification (Kropf et al., 1995). However, the difference external cues, and the site of rhizoid outgrowth becomes in pH between rhizoid and thallus poles is <0.1 pH units and fixed in space. Axis fixation occurs 10 to 12 hr AF and is may not be intense enough to induce regional specialization. marked by the polar secretion of F2 into the wall. For the first time in zygotic development, the secretory apparatus shows marked polarity; Golgi complexes accumulate in the perinu- Late Evenfs clear region on the rhizoid-facing side, and vesicles are transported preferentially toward the site of impending outgrowth Severa1 cellular rearrangements occur later in axis amplifica- (Brawley and Quatrano, 1979; Quatrano and Shaw, 1997). tion, between 7 and 10 hr AF. Dihydropyridine receptors, Cytochalasin treatment causes F2-containing vesicles (F gran- which may be Ca2+ channels that carry part of the inward ules) to accumulate near the Golgi complexes, indicating that current, localize to the plasma membrane at the presump- vesicle transpor? is mediated by F-actin (Brawley and Quatrano, tive rhizoid soon after the stable current is first detectable 1979). However, actin filaments in the region between the (Shaw and Quatrano, 1996a). Shortly thereafter, F-actin Golgi and the cortex have not been observed, perhaps be- (Brawley and Robinson, 1985; Kropf et al., 1989), cytosolic cause F-actin is difficult to preserve. Total mRNA and actin Ca2+ (Berger and Brownlee, 1993), and a clearing between mRNA are excluded from the rhizoid and are abundant at plasma membrane and cell wall (cortical clearing; Peng and the thallus pole at the time of axis fixation (Bouget et al., Jaffe, 1976; Nuccitelli, 1978) become localized to the cell 1996), but the significance of these observations is unclear. cortex at the rhizoid pole, and a sulfated fucan (F2; Novotny Agents that block axis fixation extend the period in which and Forman, 1974) becomes localized to the rhizoid cell wall zygotes are sensitive to external stimuli; zygotes germinate in (Figure 2C). The precise temporal relationships between accordance with the most recently perceived light vector these localizations have not been investigated. when the inhibitor is removed. Secretory inhibition (Shaw and Both cortical clearing and polar plasmolysis may be mani- Quatrano, 1996b), disruption of F-actin (Quatrano, 1973), and festations of localized secretion, which locally disrupts inter- enzymatic removal of the cell wall (Kropf et al., 1988) all actions between the plasma membrane and the cell wall have been shown to prevent axis fixation. lncubation in hy- (Peng and Jaffe, 1976). Polar secretion also may function to pertonic seawater of proper osmolarity also prevents axis deposit F2 into the wall at the rhizoid pole (Novotny and fixation but not rhizoid outgrowth, demonstrating that the Forman, 1974). Because these localizations occur late in two phenomena can be uncoupled (Robinson, 1996b). amplification, they may be important for subsequent events such as axis fixation or rhizoid growth (see below). GROWTH

Axis Realignment At germination, vesicle secretion intensifies, and a rhizoid, The developmental axis is labile and susceptible to realign- which elongates by tip growth, emerges from the chosen ment by applied vectors throughout the period of amplifica- site (Figure 2D). The insertion of secretory vesicles deposits tion (4 to 10 hr AF). Components of the axis become aligned new membrane and wall components, including F2 (Brawley with the most recently perceived vectorial cue, as demon- and Quatrano, 1979) and a vitronectin-like protein (Wagner strated for polar jelly in Figure 38. In addition to jelly (Schroter, et al., 1992), at the elongating tip. Some of the cellular com- 1978), dihydropyridine receptors (Shaw and Quatrano, 1996a), ponents that are localized to the presumptive rhizoid during the ionic current, and cortical clearing (Nuccitelli, 1978) can all axis induction and fixation are important for tip elongation. be repositioned. F2, however, is deposited after the axis can This is the case for F-actin and cytosolic ions, which are no longer be realigned (Fowler and Quatrano, 1995) and is thought to play fundamental roles in the elongation of most, therefore a marker for axis fixation (see below). Repositioning if not all, tip growing cells (Steer and Steer, 1989; Heath, of F-actin, Ca2+,and H+ has not been investigated. 1990; Jackson and Heath, 1993; Hepler, 1997). Traditionally, experiments designed to investigate polarity The Ca2+gradient in Fucus zygotes intensifies at germina- induction are conducted on well-attached, 6- to 8-hr-old zy- tion and is maintained during rhizoid growth (Berger and gotes that are then exposed to an environmental vector. Al- Brownlee, 1993). This gradient likely results from flux through 1O1 6 The Plant Cell

stretch-activated channels that are uniformly distributed but Division plane alignment can be perturbed by pulsed preferentially activated in the rhizoid apex (Taylor et al., treatments with cytochalasins, brefeldin A, or microtubule 1996). In addition, the magnitude of the H+ gradient also depolymerizing agents (e.g., oryzalin or nocodazole; Allen increases at germination (Kropf et al., 1995). These ionic and Kropf, 1992; Shaw and Quatrano, 1996b). These agents gradients appear to function in growth; when they are dissi- cause abnormal nuclear rotations that result in misaligned pated by treatment with H+ (Kropf et al., 1995) or Ca2+ spindles and skewed division planes. Embryos with slightly (Speksnijder et al., 1989) buffers, tip elongation is inhibited. misaligned division planes can develop normally, but more Presumably, the elevated ionic activities localize specific severe misalignment reduces growth rate. Embryos in which physiological processes to the tip. the division plane is displaced 90" and bisects the rhizoid tip Cortical F-actin also has an important role in rhizoid growth. often develop two rhizoids (Shaw and Quatrano, 1996b; F-actin is localized to the elongating tip, and treatment with Quatrano and Shaw, 1997), suggesting that rotational align- cytochalasin results in the immediate cessation of growth ment may have evolved as a mechanism to prevent the for- (Kropf et al., 1989). However, the functions of F-actin during mation of multiple rhizoids on a single embryo. tip growth are not clear. lnhibitor studies suggest that vesicles are transported along F-actin (Brawley and Quatrano, 1979), but this has not been demonstrated directly. Cortical F-actin WORKING MODELS may also provide structural support to reinforce the apex against turgor pressure andor may play a more direct role in generating force for rhizoid expansion (Harold et al., 1996). The working models presented below are intended to provide Recently, cortical F-actin has been shown to be present at a framework for interpreting the diverse findings discussed adhesion sites near the apex of growing Pelvetia rhizoids above and to help clarify the most important questions that (Henry et al., 1996). These wall-membrane adhesions are need to be addressed. visible during plasmolysis of germinated zygotes and are localized to the base of the apical dome, where they form a ringlike structure. They contain F-actin (Henry et al., 1996), Polarity lnduction dihydropyridine receptors (Shaw and Quatrano, 1996a), and Ca2+ (Kropf and Quatrano, 1987) on the cytoplasmic face A developmental axis is selected very early in fucoid zy- and are thought to be manifestations of transmembrane gotes, perhaps even at sperm entry. lnvestigation of the complexes (see below). Although they do not appear to be spatial relationships between the sperm entry site and the needed for tip elongation, these adhesions may play a role sites of jelly secretion and rhizoid outgrowth in the absence in defining and maintaining the growth site (Kropf et al., of externa1 vectors will resolve the role of sperm entry in po- 1993; Henry et al., 1996). larization. If sperm entry is found to induce polarity, the next challenge will be to identify the cortical markers of this site. F-actin is a likely candidate because localized cortical do- DlVlSlON PLANE ALIGNMENT mains of F-actin mark important sites in other polar cells (Drubin and Nelson, 1996; White and Strome, 1996). Appli- cation of high-pressure freezing techniques will aid in pre- The first zygotic division is an invariant, asymmetric division serving cortical F-actin structure, and microinjection of with the wall oriented transverse to the growth axis. This ori- fluorescently tagged actin should provide insight into the dy- entation is determined by spindle alignment, which in turn is namic properties of F-actin during fertilization. determined by the positioning of the centrosomes (Allen and The chain of causality during the early stages of polarity Kropf, 1992). Two centrosomes are inherited paternally (Fig- induction is not well understood. In published models, chan- ure 2A) and separate to opposite sides of the zygotic nu- nel localization, transcellular current, and cytosolic Ca2+ac- cleus before growth (Figure 2B; Motomura, 1994). Soon cumulation are postulated to be causal to polar secretion after germination, the nucleus and centrosomes rotate so (Jaffe et al., 1975; Brawley and Robinson, 1985). Alterna- that the axis defined by the centrosomes aligns with the tively, polar secretion may precede ionic localizations, as growth axis, and the spindle subsequently forms in axial depicted in Figure 4A. In this version of polarity induction, (longitudinal) alignment (Figure 2E; Kropf et al., 1990). Cy- axis selection is followed closely by vesicle secretion at the tokinesis occurs centripetally by invagination and is sup- presumptive rhizoid, which inserts ion channels and/or cy- ported by vesicle fusion (Brawley et al., 1977). The cleavage toskeletal anchoring proteins into the presumptive rhizoid bisects the spindle, resulting in a transverse cross-wall (Fig- membrane (Drubin and Nelson, 1996). These membrane ure 2F). Subsequent divisions in the rhizoid are also trans- asymmetries then give rise to local current influx and Ca2+ verse (Figure 1D) and are thought to involve nuclear rotation localization. ldentification of the spatial and temporal rela- (Allen and Kropf, 1992). Rotational alignment implies that in- tionships among dihydropyridine receptor localization, jelly formation regarding the position of the rhizoid is communi- secretion, and cytosolic gradients by double-labeling stud- cated to the nucleus. ies will help distinguish between these two chronologies. Polarity in Fucoid Zygotes 1O1 7

receptors, to the rhizoid site. This amplification loop increases A A asymmetries in Caz+,H+, F-actin, and polar secretion. This amplification model raises severa1 important issues. First, is a Caz+ gradient causal to the initial stages of axis induction? Previous experiments investigating the require- ment for Ca2+ in early development produced contradictory and equivoca1 results (Kropf and Quatrano, 1987; Hurst and Kropf, 1991; Robinson, 1996b). Manipulating cytosolic Ca2+ in specific cellular domains may help resolve the issue. Cy- tosolic Ca2+ can be locally elevated by microinjection of caged Caz+ followed by local Ca2+ release using laser irradi- ation. Putative interactions between cytosolic ionic activities and the cytoskeleton can be investigated by manipulating ionic activities and examining the effects on cytoskeletal as- sembly and stability. Finally, an investigation of secretory vesicle distribution at the time of jelly secretion should clarify whether vesicles are transported directionally at this early C stage of development.

Axis Fixation and Rotational Alignment

T cytoskeletal anchoring protein o ion Wall-membrane interactions mediated by transmembrane I\ integral meinhrane protein jelly complexes are postulated to be important in axis fixation and rotational alignment of the nucleus. The essence of fixa- A cell wall protein -F-actin tion is thought to be the formation of transmembrane com- 0 secretory vesicle [) ion channel plexes that link F-actin in the rhizoid cortex through the plasma membrane to the cell wall (Figure 4C). F-actin accu- Figure 4. Working Model. mulates in the rhizoid by differential stabilization (see above), and plasma membrane and/or cell wall components (A) and (B) Axis amplification. (A) Polar secretion at the presumptive are localized by the polar secretion of Golgi-derived material rhizoid pole of 4-hr zygotes establishes asymmetries in the plasma membrane by locally inserting membrane proteins, including ion (Fowler and Quatrano, 1995; Shaw and Quatrano, 1996b). channels and cytoskeletal anchoring proteins. (B) The weak asym- Although the components of these proposed complexes metry is amplified by continued local insertion of channels, recruit- have not been identified, proteins that cross-react with anti- ment of channels already in the plasma membrane, F-actin bodies raised against mammalian focal adhesion complex stabilization, and the establishment of locally elevated ionic activities components, such as vinculin, p-1 integrin, and vitronectin at the presumptive rhizoid pole. (Burridge and Chrzanowska-Wodnicka, 1996), have been (C) Axis fixation. Transmembrane complexes (right side of drawing) identified in Fucus (Quatrano et al., 1991; Wagner et al., containing F-actin, an integral membrane protein, and a secreted 1992). However, the vitronectin-like protein and F2 are un- cell wall component fix the developmental axis and are involved in likely to be the cell wall components needed for axis fixation the rotational alignment of the nucleus. For simplicity, putative actin- because fixation proceeds even when localization of these binding proteins of the complex are not shown. molecules is disrupted (Fowler and Quatrano, 1995). It is important to identify the true adhesion components, which may be different from molecules comprising focal adhesions The initial asymmetry is hypothesized to be amplified and in animal cells. Regardless of their composition, the forma- reinforced over the ensuing hours, as depicted in Figure 4B tion of transmembrane complexes is thought to stabilize the (Brawley and Robinson, 1985). Continued vesicle insertion rhizoid cortex and define a target site for vesicle fusion dur- adds new channels and amplifies the local increase in the ing ensuing growth. activity of inward-flowing cations, particularly H+ and Caz+. However, available evidence indicates that there may not F-actin is stabilized locally by elevated H+ and Caz+ and by be a close association of plasma membrane and cell wall at interaction with cytoskeletal-anchoring proteins. This F-actin the time of axis fixation. A clear zone in the cortex at the pre- in turn anchors and stabilizes ion channels and other mem- sumptive rhizoid pole is thought to be caused by displace- brane proteins, which results in further accumulations at the ment of the plasma membrane from the cell wall (Figure 2C; presumptive rhizoid and reinforcement of the vesicle secretion Peng and Jaffe, 1976). Moreover, wall-membrane adhesions site. Furthermore, F-actin may mediate the movement of addi- visualized by plasmolysis are not localized at the presumptive tional plasma membrane components, such as dihydropyridine rhizoid at the time of axis fixation (Henry et al., 1996). 1O1 8 The Plant Cell

Rotational alignment of the nucleus before mitosis is also manuscript. Part of this work was supported by a grant from the Na- thought to involve transmembrane complexes. At the time of tional Science Foundation (No. IBN-9506094)to D.L.K. rotational alignment, wall-membrane adhesions, presumably composed of transmembrane complexes, are localized at the elongating rhizoid tip (Henry et al., 1996), and treat- REFERENCES ments that disrupt adhesions, such as the application of cytochalasin D, cause improper rotation (Allen and Kropf, Allen, V., and Kropf, D.L. (1992). Nuclear rotation and lineage spec- 1992). Treatment with brefeldin A also prevents nuclear rota- ification in Pelvetia embryos. Development 115, 873-883. tion (Shaw and Quatrano, 1996b), perhaps by preventing deposition of the cell wall component of the transmembrane Bentrup, F.W. (1984). Cellular polarity. In Cellular Interactions, H.F. Linskens and J. Heslop-Harrison, eds (Berlin: Springer-Verlag), complexes (Quatrano and Shaw, 1997). pp. 473-490. The current working model for rotational alignment postu- lates that microtubules anchored at the centrosomes grow Berger, F., and Brownlee, C. (1993). Ratio confocal imaging of free cytoplasmic calcium gradients in polarising and polarised fucus into the rhizoid cortex and are captured there (Kropf, 1994). zygotes. Zygote 1,9-15. Although the nature of the microtubule-capturing site is unknown, it is postulated to contain the cortical F-actin associ- Berger, F., and Brownlee, C. (1994). Photopolarization of the Fucus sp. zygote by blue light involves a plasma membrane redox chain. ated with adhesion sites. Motors located at the centrosome Plant Physiol. 105, 519-527. or cortex exert force on the captured microtubules and pull the centrosome apically. Stochastically, one centrosome will Berger, F., and Brownlee, C. (1995). Extracellular matrix and pattern in plant embryos: On the lookout for developmental informa- be closer to the apex and is therefore likely to have more mi- tion. Trends Genet. ll, 344-348. crotubules captured. This centrosome wins the tug-of-war and rotates apically (Figures 2D and 2E). Bouget, F.-Y., Gerttula, S., Shaw, S.L., and Quatrano, R.S. (1996). Localizationof actin mRNA during the establishment of cell polarity Similar mechanisms have been proposed to orient asym- and early cell divisions in Fucus embryos. Plant Cell8, 189-201. metric divisions in Caenorhabdifis elegans embryos (White and Strome, 1996) and in Saccharomyces cerevisiae (Palmer Brawley, S.H., and Quatrano, R.S. (1979). Sulfation of fucoidin in Fucus distichus embryos. IV. Autoradiographic investigations of et al., 1992; Li et al., 1993; Muhua et al., 1994). The emerg- fucoidin sulfation and secretion during differentiation and the ing picture is that a cortical patch of actin and capping pro- effect of cytochalasin treatment. Dev. Biol. 73, 193-205. tein serves as a nucleation site for a dynactin complex and Brawley, S.H., and Robinson, K.R. (1985). Cytochalasin treatment dynein, which pull on the astral microtubules (vallee et al., disrupts the endogenous currents associated with cell polariza- 1995). Whether the same cortical machinery operates in rotation in fucoid zygotes: Studies of the role of F-actin in embryogen- tional alignment in fucoid zygotes remains to be determined. esis. J. Cell Biol. 100, 1173-1184. Brawley, S.H., Wetherbee, R., and Quatrano, R.S. (1976a). Fine- structural studies of the gametes and embryo of Fucus vesiculo- sus L. (Phaeophyta). I. Fertilization and pronuclear fusion. J. Cell FUTURE DIRECTIONS Sci. 20, 233-254. Brawley, S.H., Wetherbee, R., and Quatrano, R.S. (1976b). Fine- Recent technological advances promise further rapid ad- structural studies of the gametes and embryo of Fucus vesiculo- vances in studies of early development in fucoid algae. Re- sus L. (Phaeophyta). II. The cytoplasm of the egg and young porter molecules can be introduced into the cytoplasm of zygote. J. Cell Sci. 20,255-271. living cells by microinjection, and redistributions can be fol- Brawley, S.H., Quatrano, R.S., and Wetherbee, R. (1977). Fine- lowed during polarization (Hepler et al., 1993). lnjection of structural studies of the gametes and embryo of Fucus vesiculo- multiple reporters will permit spatial and temporal relation- sus L. (Phaeophyta). 111. Cytokinesis and the multicellular embryo. ships to be examined. Fluorescent reporters for ionic activity J. Cell Sci. 24,275-294. and for cytoskeleton are currently being injected into zy- Burridge, K., and Chrzanowska-Wodnicka, M. (1996). Focal gotes, and others will soon follow. Application of molecular adhesions: Contractility and signaling. Annu. Rev. Cell Dev. Biol. tools has led to the cloning and spatial characterization of 12,463-519. cytoskeletal sequences (Bouget et al., 1996; Coffman and Coffman, H.R., and Kropf, D.L. (1997).The brown alga Pelvetia fas- Kropf, 1997), and microinjection of antisense RNA and anti- tigiata expresses two alpha tubulin sequences. Plant Physiol. 113, bodies will permit analyses of gene function. 663-664. Drubin, D.G., and Nelson, W.J. (1996). Origins of cell polarity. Cell 84,335-344.

ACKNOWLEDGMENTS Fowler, J.E., and Quatrano, R.S. (1995). Cell polarity, asymmetric division, and cell fate determination in brown alga1 zygotes. In Seminars in Developmental Biology: Simple Systems for the Anal- I thank Tasuku Hishinuma, Whitney Hable, Sherryl Bisgrove, and ysis of lmportant Developmental Problems, D. Kirk, ed (Cam- Mary Slocum for valuable discussions and critical comments on the bridge, UK: Academic Press), pp. 347-358. Polarity in Fucoid Zygotes 1 O19

Goodner, B., and Quatrano, R.S. (1 993). Fucus embryogenesis: Kropf, D.L., Jordan, J.R., Allen, V.W., and Gibbon, B.C. (1992). A model to study the establishment of polarity. Plant Cell 5, Cellular polarity in Pelvetia zygotes: Studies of intracellular pH and 1471-1 481. division alignment. Curr. Top. Plant Biochem. MOI. Biol. Physiol. Harold, R.L., Money, N.P., and Harold, F.M. (1996).Growth and 11, 143-1 52. morphogenesis in Saprolegnia fera: 1s turgor required? Proto- Kropf, D.L., Coffman, H.R., Kloareg, B., Glenn, P., and Allen, V.W. plasma 191, 105-114. (1993).Cell wall and rhizoid polarity in Pelvetia embryos. Dev. Heath, I.B. (1990).The roles of actin in tip growth of fungi. Int. Rev. Biol. 160, 303-314. Cytol. 123, 95-127. Kropf, D.L., Henry, C.A., and Gibbon, B.C. (1995).Measurement Henry, C., Jordan, J.R., and Kropf, D.L. (1996).Localized mem- and manipulation of cytosolic pH in polarizing zygotes. Eur. J. Cell brane-wall adhesions in Pelvetia zygotes. Protoplasma 190,39-52. Biol. 68, 297-305. Hepler, P.K. (1997).Tip growth in pollen tubes: Calcium leads'the Laux, T., and Jürgens, G. (1997).Embryogenesis: A new start in life. Plant Cell9, 000. way. Trends Plant Sci. 2, 79-80. 989-1 Hepler, P.K., Cleary, A.L., Gunning, B.E.S., Wadsworth, P., Li, Y.Y., Yeh, E., Hays, T., and Bloom, K. (1993).Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc. Natl. Wasteneys, G.O., and Zhang, D.H. (1 993). Cytoskeletal dynam- Acad. Sci. USA O100. ics in living plant cells. Cell Biol. Int. 17, 127-142. 90,10096-1 Motomura, T. Electron and immunofluorescence micros- Hurd, A.M. (1920). Effect of unilateral monochromatic light and (1 994). group orientation on the polarity of germinating Fucus spores. copy of the fertilization of Fucus disfichus (Fucales, Phaeo- phyceae). Protoplasma BOt. Ga. 70, 25-50. 178, 97-1 10. Muhua, L., Karpova, T.S., and Cooper, J.A. A yeast actin- Hurst, S.R., and Kropf, D.L. (1991).lonic requirements for estab- (1994). related protein homologous to that in vertebrate dynactin complex lishment of an embryonic axis in Pelvetia zygotes. Planta 185, is important for spindle orientation and nuclear migration. Cell 27-33. 78, 669-679. Jackson, S.L., and Heath, I.B. (1993).Roles of calcium ions in Novotny, A.M., and Forman, M. (1974).The relationship between hyphal tip growth. Microbiol. Rev. 57, 367-382. changes in cell wall composition and the establishment of polarity Jaffe, L.F. (1958).Tropistic responses of zygotes of the Fucaceae to in Fucus embryos. Dev. Biol. 40, 162-1 73. polarized light. Exp. Cell Res. 15, 282-299. Nuccitelli, R. (1978).Ooplasmic segregation and secretion in the Jaffe, L.F. (1968).Localization in the developing Fucus egg and the Pelvetia egg is accompanied by a membrane-generated electrical general role of localizing currents. Adv. Morphol. 7, 295-328. current. Dev. Biol. 62, 13-33. Jaffe, L.F., and Nuccitelli, R. (1977).Electrical controls of develop- Palmer, R.E., Sullivan, D.S., Huffaker, T., and Koshland, D. ment. Annu. Rev. Biophys. Bioeng. 6,445-476. (1992).Role of astral microtubules and actin in spindle orientation Jaffe, L.F., Robinson, K.R., and Nuccitelli, R. (1974).Local cation and migration in the budding yeast, Saccharomyces cerevisiae. J. entry and self-electrophoresis as an intracellular localization Cell Biol. 119, 583-593. mechanism. Ann. N.Y. Acad. Sci. 238,372-389. Peng, H.B., and Jaffe, L.F. (1976).Cell wall formation in Pelvetia Jaffe, L.F., Robinson, K.R., and Nuccitelli, R. (1975).Calcium cur- embryos. A freeze-fracture study. Planta 133, 57-71. rents and gradients as a localizing mechanism. ICN-UCIA Symp. Quatrano, R.S. (1 972). An ultrastructural study of the determined MOI. Cell Biol. 2, 135-147. site of rhizoid formation in Fucus zygotes. Exp. Cell Res. 70,l-12. Jürgens, G. (1995).Axis formation in plant embryogenesis: Cues Quatrano, R.S. (1 973).Separation of processes associated with dif- and clues. Cell 81,467-470. ferentiation of two-celled Fucus embryos. Dev. Biol. 30,209-213. Knapp, E. (1931).Entwicklungsphysiologische Untersuchungen an Quatrano, R.S., and Shaw, S.L. (1997).Role of the cell wall in the Fucaceen-Eieren. I. Zur Kenntnis der Polaritat der Eier von Cysto- determination of cell polarity and the plane of cell division in sira barbata. Planta 14, 731-751. Fucus embryos. Trends Plant Sci. 2,15-21. Kropf, D.L. (1 992). Establishment and expression of cellular polarity Quatrano, R.S., Brian, L., Aldridge, J., and Schultz, T. (1991). in fucoid zygotes. Microbiol. Rev. 56, 316-339. Polar axis fixation in Fucus zygotes: Components of the cytoskel- Kropf, D.L. (1994).Cytoskeletal control of cell polarity in a plant eton and extracellular matrix. Development I (suppl.), 11-16. zygote. Dev. Biol. 165,361-371. Reed, E., and Whitaker, D. (1941).Polarized plasmolysis of Fucus Kropf, D.L., and Quatrano, R.S. (1987).Localization of membrane- eggs with particular reference to ultraviolet light. J. Cell. Comp. associated calcium during development of fucoid algae using Physiol. 18,329-338. chlorotetracycline. Planta 171, 158-1 70. Roberts, S.K., Berger, F., and Brownlee, C. (1993).The role of Kropf, D.L., Kloareg, B., and Quatrano, R.S. (1988).Cell wall is Ca2+in signal transduction following fertilization in Fucus serratus. required for fixation of the embryonic axis in Fucus zygotes. Sci- J. Exp. Biol. 184, 197-212. ence 239,187-190. Roberts, S.K., Gillot, I., and Brownlee, C. (1994).Cytoplasmic cal- Kropf, D.L., Berge, S.K., and Quatrano, R.S. (1989).Actin localiza- cium and Fucus egg activation. Development 120, 155-163. tion during Fucus embryogenesis. Plant Cell 1, 191-200. Robinson, K.R. (1 996a). Fucoid zygotes germinate from their dark- Kropf, D.L., Maddock, A., and Gard, D.L. (1990).Microtubule dis- est regions, not their brightest ones. Plant Physiol. 112, 1401. tribution and function in early Pelvetia development. J. Cell Sci. Robinson, K.R. (1 996b). Calcium and the photopolarization of Pel- 97,545-552. vetia zygotes. Planta 198, 378-384. 1020 The Plant Cell

Robinson, K.R., and Jaffe, L.F. (1 975). Polarizingfucoid eggs drive Taylor, A., Manison, N., Fernandez, C., Wood, J., and Brownlee, a calcium current through themselves. Science 187, 70-72. C. (1996). Spatial organization of calcium signaling involved in cell volume control in the Fucus rhizoid. Plant Cell 8,2015-2031, Robinson, K.R., and McCaig, C.D. (1980). Electrical fields, calcium gradients, and cell growth. Ann. N.Y. Acad. Sci. 339,132-138. Trewavas, A.J., and Malhó, R. (1997). Signal perception and transduction: The origin of the phenotype. Plant Cell 9, 1181-1 195. Schroter, K. (1978). Asymmetrical jelly secretion of zygotes of Pel- vetia and Fucus: An early polarization event. Planta 140, 69-73. Vallee, R., Vaughan, K., and Echeverri, C. (1995). Targeting of cytoplasmic dynein to membranous organelles and kineto- Shaw, S.L., and Quatrano, R.S. (1996a). Polar localization of a chores via dynactin. Cold Spring Harbor Symp. Quant. Biol. 60, dihydropyridine receptor on living Fucus zygotes. J. Cell Sci. 109, 803-81 1. 335-342. Vreeland, V., and Epstein, L. (1996). Analysis of plant-substratum Shaw, S.L., and Quatrano, R.S. (1996b). The role of targeted secre- adhesives. In Plant Cell Wall Analysis: Modern Methods of Plant tion in the establishment of cell polarity and the orientation of the Analysis, Vol. 17, H.F. Linskins and J.F. Jackson, eds (Berlin: division plane in Fucus zygotes. Development 122, 2623-2630. Springer-Verlag), pp. 95-1 16. Speksnijder, J.E., Miller, A.L., Weisenseel, M.H., Chen, T.-H., and Vrseland, V., Grotkopp, E., Espinosa, S., Quiroz, D., Laetsch, Jaffe, L.F. (1989). Calcium buffer injections block fucoid egg W.M., and West, J. (1993). The pattern of cell wall adhesive for- development by facilitating calcium diffusion. Proc. Natl. Acad. mation by fucus zygotes. Hydrobiologia 260/261,485-491. Sci. USA 86,6607-661 1. Wagner, V., Brian, L., and Quatrano, R. (1992). Role of a vitronec- Stafford, C.J., Green, J.R., and Callow, J.A. (1 992). Organisation tin-like molecule in embryo adhesion of the brown alga Fucus. of glycoproteins into plasma membrane domains on Fucus serra- Proc. Natl. Acad. Sci. USA 89, 3644-3648. tus eggs. J. Cell Sci. 101, 437-448. Weisenseel, M.H. (1979). lnduction of polarity. In Encyclopedia of Steer, M.W., and Steer, J.M. (1989). Pollen tube tip growth. New Plant Physiology, Vol. 7, W. Haupt and M.E. Feinleib, eds (Berlin: Phytol. 111,323358, Springer-Verlag), pp. 485-505. Swope, R.E., and Kropf, D.L. (1993). Pronuclear positioning and White, J., and Strome, S. (1996). Cleavage plane specification in C. migration during fertilization in Pelvetia. Dev. Biol. 157,269-276. elegans: How to divide the spoils. Cell 84, 195-198.