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     

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.     

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.     

DETERMINATION OF RHIZOID ORIENTATION BY LIGHT AND DARKNESS IN GERMINATING SPORES OF ONOCLEA SENSIBILIS

When spores of the fern, Onoclea sensibilis L., are allowed to germinate in darkness, the rhizoid and the protonema are positioned at close to a right angle. If the spores are exposed initially to light and allowed to germinate, the rhizoid and protonema are positioned nearly axially, at opposite ends of the spore. The greater the duration and intensity of the initial illumination, the greater the tendency towards axial arrangement. All colors of light are active to some degree, and the effects are intensity-dependent. The response occurs in a uniform light field and is not dependent on a directional stimulus; the phenomenon reflects the relative arrangement of one part of the gametophyte to another part but not the orientation of growth with respect to an external stimulus. Direct tests show that neither the relative rhizoid orientation nor initial polarity of germination are affected by unilateral white light or polarized red light; the subsequent growth of the protonema, however, is oriented perpendicular to the plane of light polarization. The effects of light in determining the positional relationship between rhizoid and protonema are interpreted in terms of a hypothesis proposing light-induced changes in the structure and mechanical properties of the spore wall.     

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.     

Sperm entry induces polarity in fucoid zygotes

Fucoid zygotes establish a rhizoid-thallus growth axis in response to environmental signals; however, these extrinsic cues are not necessary for polarization, suggesting that zygotes may have inherent polarity. The hypothesis that sperm entry provides a default pathway for polarization of zygotes cultured in the absence of environmental signals was tested, and was supported by several lines of evidence. First, an F-actin patch, a cortical marker of the rhizoid pole, formed at the sperm entry site within minutes of fertilization. Second, the sperm entry site predicted the site of polar adhesive secretion (the first morphological manifestation of the rhizoid pole) and the position of rhizoid outgrowth. Third, when fertilization was restricted to one hemisphere of the egg, rhizoid outgrowth always occurred from that hemisphere. Fourth, delivery of sperm to one location within a population of eggs resulted in polarization of both adhesive secretion and rhizoid outgrowth toward the sperm source. Finally, induction of polyspermy using low sodium seawater increased the frequency of formation of two rhizoids. Sperm entry therefore provides an immediate default axis that can later be overridden by environmental cues.     

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.     

Localization of membrane-associated calcium during development of fucoid algae using chlorotetracycline

During the first day of development, fertilized eggs of fucoid algae generate an embryonic axis and commence rhizoid growth at one pole. Using Fucus distichus (L.) Powell, F. vesiculosus L. and Pelvetia fastigiata (J.Ag.) DeTony we have investigated the role of calcium in axis formation and fixation as well as in tip growth. The intracellular distribution of membrane-associated calcium was visualized with the fluorescent calcium probe chlorotetracycline (CTC). Punctate fluorescence associated with organelle-like structures was found in conjunction with diffuse staining at all developmental stages. This membrane-associated calcium remained uniformly distributed throughout the cortical cytoplasm while the axis was established, but increased in the rhizoid protuberance at germination. In subsequent development, fluorescence was restricted to the cortical cytoplasm at the elongating tip and at sites of crosswall biosynthesis.The requirement for Ca²⁺ uptake during development was investigated through inhibition studies; influx was impaired with transport antagonists or by removal of extracellular calcium. Both treatments curtailed germination and tip elongation but had little effect on axis polarization. Reductions in external calcium that interfered with elongation also markedly reduced the apical CTC fluorescencence, indicating that calcium uptake and localization are prerequisites for tip growth. This apical Ca²⁺ is probably involved in the secretory process that sustains tip elongation. By contrast, calcium was not implicated in the generation of an embryonic axis.Abbreviations ASW artificial seawater - CTC chlorotetracycline - DU developmental unit - EGTA erhylene glycol bis(amino-ethyl ether) N,N,N¹,N¹–tetraacetic acid - NPN N-phenyl-1-napthylamine     

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).     

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.     

Cortical actin filaments form rapidly during photopolarization and are required for the development of calcium gradients in Pelvetia compressa zygotes

Previous research has shown that cortical gradients of cytosolic Ca(2+) are formed during the photopolarization of Pelvetia compressa zygotes, with elevated Ca(2+) on the shaded hemisphere that will become the site of rhizoid germination. We report here that the marine sponge toxin, latrunculin B, which blocks photopolarization at nanomolar concentrations, inhibited the formation of the light-driven Ca(2+) gradients. Using low concentrations of microinjected fluorescent phalloidin as a tracer for actin filaments, we found that exposure to light induced a striking increase in actin filaments in the cells as indicated by an increase in fluorescence. The increase was quantified in the cortex, where it was most apparent, and the fluorescence there was found to increase by about a factor of 3. This increase in cortical phalloidin fluorescence was inhibited by latrunculin B at the same concentration required to inhibit Ca(2+) gradient formation and photopolarization. The distribution of the increasing phalloidin fluorescence was uniform with respect to the developing rhizoid-thallus axis during the formation of the axis, and no intense patches of fluorescence were observed. After germination, fluorescence suggestive of an apical ring of actin filaments was seen near the rhizoid tip. Finally, inhibitor studies indicated that myosin may be involved in the photopolarization process.     

Photopolarization of the Fucus sp. Zygote by Blue Light Involves a Plasma Membrane Redox Chain

Zygotes of fucoid algae are photopolarized by unidirectional blue light (BL). Polar axes are formed, fixed, and expressed by germination of a rhizoid. Hexacyanoferrate(III) ions (HCF) specifically inhibit transduction of the BL signal. HCF reduction by Fucus sp. zygotes occurs on the outer surface of the plasma membrane at higher rates in BL than in dark. These observations suggest that BL signal transduction involves a redox chain in the plasma membrane. Low doses of HCF (<50 pmol cell-1) inhibit photopolarization but not germination, hence uncoupling both processes. Exposure during the photosensitive period to higher doses of HCF together with BL significantly inhibits germination. Further results suggest that BL transduction is dependent on photosynthetic products that could also interact with redox processes.     

A COMPARATIVE STUDY OF CELL DIVISION PATTERNS DURING GERMINATION OF SPORES OF ANEMIA,LYGODIUM AND MOHRIA (SCHIZAEACEAE)

Cell division patterns during germination of spores of Anemia (A. hirsuta, A. munchii, A. phyllitidis), Lygodium (L. circinatum, L. flexuosum, L. japonicum, L. salicifolium) and Mohria caffrorum have been examined by light microscopy of glycol methacrylate embedded materials. Spores of all species in a genus exhibited a constant pattern of division under different conditions of germination. In spores of species of Anemia, following an asymmetrical division, the proximal cell differentiated into the protonemal cell while the distal cell divided to produce the rhizoid. A similar division sequence was found in spores of Mohria caffrorum, but the fate of cells formed was reversed. In Lygodium spores, a proximal cell formed by an initial division of the spore cut off a protonemal cell, a rhizoid and a wedge-shaped cell by walls parallel to the polar axis. Our results contradict earlier observations on cell division sequence during germination of spores of these genera based on whole mount preparations.     

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.     

Cell wall deposition during morphogenesis in fucoid algae

Cell wall deposition was investigated during morphogenesis in zygotes of Pelvetia compressa (J. Agardh) De Toni. Young zygotes are spherical and wall is deposited uniformly, but at germination (about 10 h after fertilization) wall deposition becomes localized to the apex of the tip-growing rhizoid. Wall deposition was investigated before and after the initiation of tip growth by disrupting cytoskeleton, secretion or cellulose deposition; effects on wall strength and structure were examined. All three were involved in generating wall strength in both spherical and tip-growing zygotes, but their relative importance were different at the two developmental stages. Much of the wall strength in young zygotes was dependent on F-actin, whereas cellulose and a sulfated component, probably a fucan (F2), were most important in tip growing zygotes. Some treatments had contrasting effects at the two developmental stages; for example, disruption of F-actin or inhibition of secretion weakened walls in spherical zygotes but strengthened those in tip-growing zygotes. Transmission electron microscopic analysis showed that most treatments that altered wall strength induced modifications of internal wall structure. Received: 12 June 2000 / Accepted: 26 July 2000     

Cytochalasin treatment disrupts the endogenous currents associated with cell polarization in fucoid zygotes: studies of the role of F-actin in embryogenesis

We determined the distribution of F-actin in fucoid (Pelvetia, Fucus) embryos with nitrobenzoxadiazole-phallacidin, and studied the effect of cytochalasin upon the endogenous currents associated with cell polarization by using the vibrating probe. F-actin is not localized at the presumptive rhizoid immediately after experimental induction of the polar axis with a light gradient; however, a preferential distribution of F-actin develops at the presumptive rhizoid by the time the position of the polar axis is fixed. F-actin continues to be localized at the tip of the rhizoid after germination, except during cytokinesis, when the furrow is the only brightly staining region of the embryo. Incubation with cytochalasin can result in either an enhanced or a diminished pool of F-actin in the embryonic cortex (see Results). Cytochalasin D (100 micrograms/ml) significantly reduces the inward current at the rhizoid pole (n = 11) after a 2.5-h incubation. This drop is concentration dependent and occurs within approximately 30 min at 100 micrograms/ml and approximately 60 min at 10 micrograms/ml. Cytochalasin treatment eliminates the pulsatile component of the current. Preliminary results suggest that 100 micrograms/ml cytochalasin D prevents development of inward current at the presumptive rhizoid but does not completely delocalize this locus if added after photopolarization. We conclude that microfilaments are required for the establishment and maintenance of the pattern of endogenous currents observed during early embryogenesis. This suggests a new model for axis formation and fixation.     

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     

Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii

A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 m sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 m sorbitol layer. Regeneration occurred over a period of 10–24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater. Received: 26 September 1997 / Revision received: 17 January 1998 / Accepted: 7 February 1998     

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.