Establishing a growth axis in fucoid algae

Recent studies indicate that fucoid zygotes establish developmental polarity much earlier than previously thought. A growth axis is first set in place at fertilization, with the site of sperm entry defining the rhizoid pole of the axis. This initial axis is a default axis, which is only used as the final growth axis if the zygote fails to detect spatial cues (such as sunlight) in its intertidal environment. However, the zygote usually senses vectorial information; it then abandons the sperm-induced axis and assembles a new axis de novo in accordance with the perceived vector(s).     

Asymmetrical jelly secretion of zygotes of Pelvetia and Fucus: An early polarization event

Five hours before germination the zygotes of Pelvetia fastigiata adhere to their substrate. A jelly layer covers the entire cell but most of the transparent jelly, artificially outlined by a layer of resin beads, is secreted at the prospective rhizoid pole. If the direction of the growth-orienting light is shifted after the asymmetrical secretion has already started, the direction of the secretion is also shifted. The polarization axis can be predicted by the site of the intensive jelly secretion. The germination of Fucus vesiculosus and F. spiralis is also preceded by an intensive asymmetrical jelly secretion. However, at the rhizoid pole of F. serratus the jelly secretion does not increase until the germinating zygote becomes pear-shaped. Fucoid zygotes do not adhere, neither do they have a jelly cover as long as they develop in sulfate-free sea water.Abbreviations a.f. after fertilization     

Polarity of sperm entry in the ascidian egg

We have investigated the point of sperm entry in denuded eggs of the ascidian Phallusia mammillata. In contrast to what is generally believed, the sperm show a strong tendency to enter the animal hemisphere rather than the vegetal hemisphere. After entry, the sperm nucleus is carried toward the vegetal pole of the egg during the cortical contraction which occurs within a few minutes after fertilization. This polarity of sperm entry is abolished and the entry point is randomized by pretreating the eggs with cytochalasin D. We suggest that cytochalasin may act by randomizing components needed for sperm attachment or fusion, or structures needed for sperm entry.     

Polarity establishment requires dynamic actin in fucoid zygotes

Summary.  Previous work has demonstrated that actin plays important roles in axis establishment and polar growth in fucoid zygotes. Distinct actin arrays are associated with fertilization, polarization, growth, and division, and agents that depolymerize actin filaments (cytochalasins, latrunculin B) perturb these stages of the first cell cycle. Rearrangements of actin arrays could be accomplished by transport of intact filaments and/or by actin dynamics involving depolymerization of the old array and polymerization of a new array. To investigate the requirement for dynamic actin during early development, we utilized the actin-stabilizing agent jasplakinolide. Immunofluorescence of actin arrays showed that treatment with 1–10 μM jasplakinolide stabilized existing arrays and induced polymerization of new filaments. In young zygotes, a cortical actin patch at the rhizoid pole was stabilized, and in some cells supernumerary patches were formed. In older zygotes that had initiated tip growth, massive filament assembly occurred in the rhizoid apex, and to a lesser degree in the perinuclear region. Treatment disrupted polarity establishment, polar secretion, tip growth, spindle alignment, and cytokinesis but did not affect the maintenance of an established axis, mitosis, or cell cycle progression. This study suggests that dynamic actin is required for polarization, growth, and division. Rearrangements in actin structures during the first cell cycle are likely mediated by actin depolymerization within old arrays and polymerization of new arrays. Received July 15, 2002; accepted November 27, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, U.S.A.     

The pattern of cell wall adhesive formation by Fucus zygotes

As a first step in understanding the mechanism of algal adhesion, we describe the adhesive process during early development in Fucus gardneri zygotes. These brown algal embryos adhere to the intertidal substrate shortly after fertilization. Zygotes adhered nonspecifically to hydrophilic and hydrophobic substrates and microspheres. Initial binding of microspheres to the zygote surface coincided with initial zygote adhesion to the substrate. Binding of monodisperse dyed microspheres was used for adhesive localization and quantitation. The timing and extent of adhesive development were variable in populations of synchronously-fertilized zygotes. Small adhesive patches first appeared at 3–6 h, indicating secretion of adhesive components from cytoplasmic vesicles. The zygote hemisphere toward the substrate became sticky by 7–8 h. The entire surface was sticky after rhizoid germination at 12 h. Localization of adhesive at both the outer wall surface and along strands attached to the wall implicates cell wall polymers as a glue component. Loss of microspheres from the rhizoid surface in high salt or chelators indicates that initial adhesive attachment to the wall is noncovalent. Formation of adhesive aggregates in medium showed that the mechanism of adhesive formation includes two separable processes, secretion of adhesive components and extracellular interactions between adhesive components and the wall.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

Membrane recycling occurs during asymmetric tip growth and cell plate formation inFucus distichus zygotes

Summary Zygotes of the brown algaFucus distichus undergo a series of intracellular changes resulting in the establishment of a polar growth axis prior to the first embryonic cell division. In order to examine the dynamics of membrane recycling which occur in the zygote during polar growth of the rhizoid, we probed living Fucus zygotes with the vital stain FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylammo)phenyl)hexatrienyl)pyridinium dibromide. In newly fertilized, spherical zygotes, FM4-64 staining is symmetric and predominantly in the perinuclear region which is rich in endoplasmic reticulum, Golgi, and vacuolar membranes. As rhizoid or tip growth is initiated, this population of stained membranes becomes asymmetrically redistributed, concentrating at the rhizoid tip and extending centrally to the perinuclear region. This asymmetric localization is maintained in the zygote throughout polar growth of the rhizoid and during karyokinesis. Subsequently, FM4-64 staining also begins to accumulate in a central location between the daughter nuclei. As cytokinesis proceeds, this region of stain expands laterally from this central location, perpendicular to the plane of polar rhizoid outgrowth. The staining pattern thus delineates the formation of a cell plate, similar spatially to the accumulation of nascent plate membranes of higher plants. Treatment of Fucus zygotes with brefeldin-A inhibits both asymmetric growth of the rhizoid and formation of a new cell plate. These data suggest that inF. distichus FM4-64 is labeling a Golgi-derived membrane fraction that appears to be recycling between the site of tip growth, perinuclear region, and new cell plate.Abbreviations AF after fertilization - ASW artificial seawater - BFA brefeldin A - ER endoplasmic reticulum - FM4-64 N-(3-triethylam-moniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide     

Role of auxin in induction of polarity in zygotes of Fucus vesiculosus L

We studied the effects of auxin (indolyl-3 acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosus. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the environment. The treatment of developing zygotes with the inhibitor of indolyl-3-acetic acid transport from the cell triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indolyl-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid process. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was fully relieved. It has been proposed that the content of indolyl-3-acetic acid in the environment is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipens

The region of the frog egg that is receptive to fertilization was determined. As an approximation to the site of sperm entry, the start of the male pronuclear penetration path within the egg was made visible externally by bleaching fixed eggs. A bleached egg had a pigment accumulation on its surface corresponding to the start of the penetration path. The accumulation characteristically changed shape with cortical movements prior to first cleavage, and most accumulations (path starts) were within 60° of the animal pole.Localized inseminations and an analysis of the distribution of failures of fertilization at the egg plasma membrane demonstrated that few if any sperm entered the vegetal region of the egg. Localized inseminations, however, demonstrated that sperm entered between 60° from the animal pole and the animal-vegetal margin.Although sperm entry occurred throughout the animal region, most penetration paths started within 60° of the animal pole. To account for this, the sperm nucleus must move towards the animal pole prior to starting the penetration path. This movement appeared to be due to a contraction of the cortex towards the animal pole that occurred 3–4 min after activation of the egg.     

Ca2+ and Calmodulin Dynamics during Photopolarization in Fucus serratus Zygotes

The role of Ca2+ in zygote polarization in fucoid algae (Fucus, Ascophyllum, and Pelvetia species) zygote polarization is controversial. Using a local source of Fucus serratus, we established that zygotes form a polar axis relative to unilateral light (photopolarization) between 8 and 14 h after fertilization (AF), and become committed to this polarity at approximately 15 to 18 h AF. We investigated the role of Ca2+, calmodulin, and actin during photopolarization by simultaneously exposing F. serratus zygotes to polarizing light and various inhibitors. Neither removal of Ca2+ from the culture medium or high concentrations of EGTA and LaCl3 had any effect on photopolarization. Bepridil, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester, nifedipine, and verapamil, all of which block intracellular Ca2 release, reduced photopolarization from 75 to 30%. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-L-naphthalenesulfonamide and trifluoperazine inhibited photopolarization in all zygotes, whereas N-(6-aminohexyl)-L-naphthalenesulfonamide had no effect. Cytochalasin B, cytochalasin D, and latrunculin B, all of which inhibit actin polymerization, had no effect on photopolarization, but arrested polar axis fixation. The role of calmodulin during polarization was investigated further. Calmodulin mRNA from the closely related brown alga Macrocystis pyrifera was cloned and the protein was expressed in bacteria. Photopolarization was enhanced following microinjections of this recombinant calmodulin into developing zygotes. Confocal imaging of fluorescein isothiocyanate-labeled recombinant calmodulin in photopolarized zygotes showed a homogenous signal distribution at 13 h AF, which localized to the presumptive rhizoid site at 15 h AF.     

The relationship between changes in cell wall composition and the establishment of polarity in Fucus embryos

Changes in the appearance and location of fucoidin in the cell walls of Fucus embryos were related to embryo development. Fucoidin was not present in the cell wall until 10–14 hr after fertilization, when the embryos began to incorporate fucoidin preferentially into a localized area of the wall. At this time the site of rhizoid initiation was determined; that is, the embryos had undergone axis commitment. Germination of the single-celled embryo occurred between 12 and 16 hr, after fertilization, with all cell walls from germinated embryos showing fucoidin localization at the rhizoid end of the cell. The percentage of embryos with localized fucoidin at the time of axis fixation equaled the percentage of embryos that subsequently germinated. Culturing the embryos in sea water plus 0.8 M sucrose prevented the outgrowth of the rhizoid, but not the localization of fucoidin in the wall or axis commitment. Cycloheximide, nitroprusside, cytochalasin B, sulfate-free sea water, high levels of Ca²⁺, and a breakdown product of TIBA all prevented rhizoid growth and the specific localization of fucoidin. In addition, axis commitment could not be demonstrated in the presence of these inhibitors. DTNB, PCMBS, TIBA, HgCl₂, Mg²⁺ were ineffective as reversible inhibitors of rhizoid initiation. The authors propose that the fixation of axis commitment is accompanied by localized changes in the cell wall involving the incorporation of fucoidin as a structural component of the wall.     

First cleavage of the mouse embryo responds to change in egg shape at fertilization

Although mouse development is regulative, the cleavage pattern of the embryo is not random. The first cleavage tends to relate to the site of the previous meiosis. Sperm entry might provide a second cue, but evidence for and against this is indirect and has been debated. To resolve whether sperm entry position relates to the first cleavage, we have followed development from fertilization by time-lapse imaging. This directly showed cytokinesis passes close to the site of the previous meiosis and to both the sperm entry site and trajectory of the male pronucleus in a significant majority of eggs. We detected asymmetric distribution of Par6 protein in relation to the site of meiosis, but not sperm entry. Unexpectedly, we found the egg becomes flattened upon fertilization in an actin-mediated process. The sperm entry position tends to lie at one end of the short axis along which cleavage will pass. When we manipulated eggs to change their shape, this repositioned the cleavage plane such that eggs divided along their experimentally imposed short axis. Such manipulated eggs were able to develop to term, emphasizing the regulative nature of their development.     

Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation

Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion.     

Establishment and expression of cellular polarity in fucoid zygotes.

Zygotes of fucoid algae have long been studied as a paradigm for cell polarity. Polarity is established early in the first cell cycle and is then expressed as localized growth and invariant cell division. The fertilized egg is a spherical cell and, by all accounts, bears little or no asymmetry. Polarity is acquired epigenetically a few hours later in the form of a rhizoid/thallus axis. The initial stage of polarization is axis selection, during which zygotes monitor environment gradients to determine the appropriate direction for rhizoid formation. In their natural setting in the intertidal zone, sunlight is probably the most important polarizing vector; rhizoids form away from the light. The mechanism by which zygotes perceive environmental gradients and transduce that information into an intracellular signal is unknown but may involve a phosphatidylinositol cycle. Once positional information has been recorded, the cytoplasm and membrane are reorganized in accordance with the vectorial information. The earliest detectable asymmetries in the polarizing zygote are localized secretion and generation of a transcellular electric current. Vesicle secretion and the inward limb of the current are localized at the presumptive rhizoid. The transcellular current may establish a cytoplasmic Ca2+ gradient constituting a morphogenetic field, but this remains controversial. Localized secretion and establishment of transcellular current are sensitive to treatment with cytochalasins, indicating that cytoplasmic reorganization is dependent on the actin cytoskeleton. The nascent axis at first is labile and susceptible to reorientation by subsequent environmental vectors but soon becomes irreversibly fixed in its orientation. Locking the axis in place requires both cell wall and F-actin and is postulated to involve an indirect transmembrane bridge linking cortical actin to cell wall. This bridge anchors relevant structures at the presumptive rhizoid and thereby stabilizes the axis. Approximately halfway through the first cell cycle, the latent polarity is expressed morphologically in the form of rhizoid growth. Elongation is by tip growth and does not appear to be fundamentally different from tip growth in other organisms. The zygote always divides perpendicular to the growth axis, and this is controlled by the microtubule cytoskeleton. Two microtubule-organizing centers on the nuclear envelope rotate such that they align with the growth axis. They then serve as spindle poles during mitosis. Cytokinesis bisects the axial spindle, resulting in a transverse crosswall. Although the chronology of cellular events associated with polarity is by now rather detailed, causal mechanisms remain obscure.     

Inhibition of the establishment of zygotic polarity by protein tyrosine kinase inhibitors leads to an alteration of embryo pattern in Fucus

Fucoid algae, including the genus Fucus and Pelvetia, are recognized as model systems to study early embryogenesis in plants. In particular the zygotes of these fucoid algae are highly suitable experimental systems for investigating the establishment of polarity and its requirement for later embryogenesis. However, the transduction pathways involved in the initiation of polarization are still poorly understood, and the link between the early polarization processes and embryo long-term patterning has never been experimentally demonstrated. We, therefore, have investigated the putative role of protein phosphorylation in the regulation of early embryogenesis, using a combined pharmacological and biochemical approach. Among the various protein kinase inhibitors tested, a subset of well-known PTK inhibitors, including genistein, prevented germination but had no effect on growth of germinated zygotes and embryos. Inhibition of germination appeared to be a direct consequence of prevention of polarization since genistein and other PTK inhibitors specifically inhibited axis formation in a light-independent manner. Genistein inhibited cellular events associated with polarization such as polarized secretion of cell wall sulfated compounds. Anchorage of F-actin at the rhizoid pole was also inhibited and F-actin redistributed in response to a new light vector. Zygotes inhibited in the polarization process over the period of axis formation recovered from the treatment and displayed differentiated cellular structures after a few days. However, they exhibited a deeply disorganized pattern, suggesting that the early polarization process is essential for normal patterning of the embryo. Western blot analysis of protein phosphorylation showed that the patterns of protein phosphorylation changed during development and were disturbed by treatments with genistein. This drug also inhibited in vitro autophosphorylation. The nature of the genistein-sensitive kinases required for polarization and long-term patterning is discussed in light of these data.     

Calcium and the photopolarization ofPelvetia zygotes

The initially apolar zygotes of the brown algae,Fucus andPelvetia, form their main axes during the hours following fertilization and each cell expresses its axis by germinating at one location. The germinating region is destined to become the rhizoid and the rest of the zygote gives rise to the thallus. In response to unilateral blue light, the zygotes organize their developmental axes so that the rhizoids emerge on the shaded side, away from the light source. In the research reported here, the signaltransduction elements involved in the photopolarization ofPelvetia fastigiata De Toni zygotes have been investigated. It was found that exposure of zygotes to 90or 150-min pulses of unilateral light in the absence of extracellular Ca²⁺ completely eliminated photopolarization; that is, the cells formed their rhizoid-thallus axes randomly with respect to the light direction, while controls similarly exposed to light in normal (10 mM) Ca²⁺ were well polarized. When the cells were incubated in Ca²⁺-free sea water for an hour before being given the light pulse (while still in Ca²⁺-free sea water), they exhibited an unusual negative polarization: they formed their rhizoids on the hemisphere nearer the light source. Organic and inorganic calcium-channel blockers reduced or abolished photopolarization when present during light pulses. Reducing external Ca²⁺ to one-tenth of normal has the paradoxical effect of increasing calcium influx intoPelvetia zygotes. When zygotes were given light pulses in reduced extracellular calcium, the degree of photopolarization was increased substantially. These data are consistent with the idea that the formation of an intracellular gradient of [Ca²⁺] is an essential part of the polarization process. The fungus-derived calmodulin antagonist, ophiobolin A, blocked or greatly delayed germination when present continuously at a concentration of 100–300 nM. However, when present at 300 nM during a brief light pulse, it markedly increased the sensitivity of the cells to light. These results suggest that calmodulin may be the mediator of intracellular [Ca²⁺] gradients in the photopolarization process.     

POLAR GROWTH OF HORMOSIRA BÁNKSII ZYGOTES IN SHAKE CULTURE

Zygotes of the fucalean alga Hormosira banksii initiate rhizoidal outgrowths in stationary culture 15 hr after fertilization and are then recognizably polar. By 24 hr most embryos are two-celled, and a few are four-celled. In a dark-grown population orientation of the developmental axis, as indicated by the direction of the rhizoidal outgrowth, was random around the vertical axis. In a unilaterally illuminated population the rhizoid usually emerged on the shaded side. Zygotes grown in light or darkness in shake culture, where they were continuously reoriented, usually developed as polar embryos, indicating that gradients of environmental factors are not required for initiation of polar growth. Some apolar embryos developed in stationary and shake cultures, but they were most frequent in dark shake cultures.     

Stabilization of the polarity axis in the zygotes of some Fucaceae

Summary The existence of a period of latent but stable polarity, i.e. a period in which the polarity axis has been irreversibly established but no morphological asymmetry can be detected, was studied in germinating populations of zygotes of Pelvetia fastigiata. We found that the time course of the loss of sensitivity to a single polarity-axis-determining light stimulus coincided with the time course of germination (rhizoid outgrowth), up to the germination of about one-third the population, showing that the cells remained responsive to the light stimulus until or almost until the appearance of visible asymmetry. In the later part of the germination period, some of the zygotes may have lost their light sensitivity somewhat before rhizoid outgrowth, suggesting that at least some of the ungerminated cells may at this time possess, for a brief period, a latent but stable polarity axis.The loss of responsiveness with time to an antagonistic, second light stimulus followed the same time course as the loss of sensitivity to a single light stimulus. There was no suggestion of the existence of a latent but stable polarity axis in any members of the population in this experiment.An analysis of data of Jaffe (1968) on polarity-axis determination and germination in P. fastigiata following a single light stimulus yields essentially the same conclusions as our own single-light-stimulus experiments.In contrast, analysis of data of Whitaker and Lowrance (1936) on Fucus furcatus indicated that in these zygotes latent but stable determination of the polarity axes had taken place 3–4 hours before germination. A similar situation emerges from the analysis of another experiment of Jaffe (personal communication) with P. fastigiata zygotes in which the loss of sensitivity to an orienting light stimulus appeared accelerated and germination may have been delayed in comparison with his 1968 data.We conclude, therefore, that populations of Fucaceae zygotes may vary with regard to the existence of a latent but stable polarity axis. However, when existence of such a latent, stable polarity axis can be inferred, its duration usually is brief, and it seems to be in most cases limited to a small fraction of the individuals of the total population at any particular time. In order to infer rigorously the existence of latent but stable polarity axes in populations of germinating zygotes and similar cells, it is essential to obtain the time courses for axis stabilization and for the development of visible asymmetry simultaneously.