Fucoid Zygotes Research Articles

Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii (Fucales, Phaeophyceae) Zygotes.

We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination.

Effects of sediment on early life history stages of habitat-dominating fucoid algae

The effects of sediment on settlement and early post-settlement survival of habitat-dominating fucoid algae have not been extensively investigated. Two separate experiments tested whether different size-fractions, abundances and types of sediment differentially affect the attachment of Hormosira banksii and Cystophora torulosa zygotes under laboratory conditions, and whether alteration of the sediment environment differentially affects early post-settlement survival of fucoid zygotes placed into the natural environment. In the laboratory, an average of c. 59% of Hormosira zygotes were able to attach to primary substratum in the presence of sparse sand and silt, compared to 28% (silt) and 39% (sand) of Cystophora zygotes. In the presence of dense sand or silt, <8% of either species attached to primary substratum, with the great majority of zygotes of both species attaching to sediment grains. In the field, Hormosira zygotes across three treatments survived for the entire two week experimental period, whereas no Cystophora zygotes survived beyond 8 days. For both species there was a gradient of survival across treatments, from the poorest survival on bare rock, slightly better survival in coralline turf that contained sediments, and greater survival in coralline turf with the sediment removed, although there were significant interactive effects of treatment and time. After two weeks, an average of 22% of Hormosira zygotes survived on bare rock, around 38% on coralline turf with sediment, and 40% on coralline turf minus sediment, but with high variation in the treatments. On day 5 of the field experiments, when zygotes of both species remained across treatments, there were significant differences between species and between treatments which mirrored results across the whole experiment. Overall, the experiment showed that the sediment environment influenced both the attachment of fucoid zygotes and early post-settlement survival. The experiments highlighted key differences between species in their method of attachment and subsequent interactions within the abiotic environment, with the larger stickier zygotes of Cystophora more readily attaching to sediment grains, and the less sticky Hormosira zygotes often rolling through the sediments and attaching to the primary substratum. These early life history characteristics of key species and their interaction with those of microsites in which they settle clearly have importance to the replenishment and maintenance of populations.

Photopolarization of Fucus zygotes is determined by time sensitive vectorial addition of environmental cues during axis amplification.

Fucoid zygotes have been extensively used to study cell polarization and asymmetrical cell division. Fertilized eggs are responsive to different environmental cues (e.g., light, gravity) for a long period before the polarity is fixed and the cells germinate accordingly. First, it is commonly believed that the direction and sense of the polarization vector are established simultaneously as indicated by the formation of an F-actin patch. Secondly, upon reorientation of the zygote, a new polar gradient is formed and it is assumed that the position of the future rhizoid pole is only influenced by the latter. Here we tested these two hypotheses investigating photopolarization in Fucus zygotes by reorienting zygotes 90° relative to a unilateral light source at different time points during the first cell cycle. We conclude that fixation of direction and sense of the polarization vector is indeed established simultaneously. However, the experiments yielded a distribution of polarization axes that cannot be explained if only the last environmental cue is supposed to determine the polarization axis. We conclude that our observations, together with published findings, can only be explained by assuming imprinting of the different polarization vectors and their integration as a vectorial sum at the moment of axis fixation. This way cells will average different serially perceived cues resulting in a polarization vector representative of the dynamic intertidal environment, instead of betting exclusively on the perceived vector at the moment of axis fixation.

Rac1 signaling in the establishment of the fucoid algal body plan.

Fucoid zygotes use environmental vectors, including sunlight, to initiate a growth axis a few hours after fertilization. The first division is then transversely oriented by the growth axis, producing daughter cells of distinct fates. The tip growing rhizoid cell gives rise to the holdfast, anchoring the alga to the intertidal substratum, while the opposite thallus cell mainly generates the photosynthetic and reproductive stipe and fronds. Elaboration of this simple growth axis thus establishes the basic body plan of the adult; and elucidating the mechanisms responsible for formation of the growth axis is paramount to understanding fucoid morphogenesis. Recent studies have culminated in a model whereby sunlight, and perhaps other environmental cues, activate the signaling protein Rac1 at the rhizoid pole. Here it sets in motion nucleation of a patch of actin filaments that in turn, targets ions, proteins, and cellular processes to the future growth site. At germination, Rac1 initiates morphogenesis by inducing transformation of the patch of actin filaments to a structure that delivers vesicles to the growing tip, and a few hours later orients the spindle and cytokinetic plate.

Emergence of symmetry breaking in fucoid zygotes

Fucoid zygotes are model cells for the study of symmetry breaking in plants. After fertilization, their initial spherical symmetry reduces to an axial symmetry, even in the absence of any external cue. This indicates that zygotes have an intrinsic ability to break symmetry in a way that is solely dependent on their internal biochemical and/or biophysical state. In our opinion, symmetry breaking is a self-organized process. It arises around the fucoid zygotes from the ion dynamics through channels (voltage-dependent calcium channels and a potassium leak) and outside the membrane (electrodiffusion owing to slower calcium diffusion compared with potassium). The robustness of this self-organized process and its lability ensure its relevance in plants where symmetry breaking is correlated with transcellular ion currents.

Cytokinesis in brown algae: studies of asymmetric division in fucoid zygotes.

The mechanism of cytokinesis was investigated during the first asymmetric division in fucoid zygotes. A plate of actin assembled midway between daughter nuclei where microtubules interdigitated and defined the cytokinetic plane. A membrane was then deposited in islands throughout the cytokinetic plane; the islands eventually fused into a continuous partition membrane and cell plate material was deposited in the intermembrane space. All of these structures matured from the center of the cell outward (centrifugal maturation). Pharmacological agents were used to investigate the roles of microtubules, actin, and secretion in cytokinesis. The findings indicate a mechanism of cytokinesis that may be unique to the brown algae.

Polarity establishment requires dynamic actin in fucoid zygotes.

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 microM 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.

Asymmetric division in fucoid zygotes is positioned by telophase nuclei.

The relative contributions of cell polarity and nuclear position in specifying the plane of asymmetric division in fucoid zygotes were investigated. In zygotes developing normally, telophase nuclei were positioned parallel to the polar growth axis, and the division plane bisected both axes. To assess division plane specification, the colinearity of the nuclear and growth axes was uncoupled by treatment with pharmacological agents. Spatial correlations between the growth axis, telophase nuclei, and the division plane were analyzed in the treated zygotes. In all cases, cytokinesis was oriented transverse to the telophase mitotic array and was less well aligned with the growth axis. Telophase nuclei also played a predominant role in positioning the division plane in polyspermic zygotes. Microtubules from the telophase nuclei interdigitated throughout the plane of subsequent cytokinesis, and we speculate that they specify the division plane. Morphological markers of the division plane were not observed before telophase; the earliest division marker detected was a plate of actin that assembled in the zone of microtubule overlap late in telophase. These findings are consistent with division plane specification at cytoplast boundaries.

Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

SUMMARY Regulation of microtubule (MT) arrays and embryo-genesis by cytoplasmic pH (pHc) was investigated in zygotes of the brown alga Pelvetia compressa (J. Agardh) De Toni. pHc was clamped to (set to) acidic values using a weak acid, propionic acid (PA), and to alkaline values using a weak base, methylamine (MA). Acidification of pHc from the normal value of 7.4–7.5 to about 7.0 caused disruption of microtubule arrays. The nucleating activity was delocalized from the centrosomes and dispersed over the nuclear envelope, the number of MTs decreased, and MTs failed to extend into the cell cortex. Alkalinization to about pH 8.0 also caused dispersal of nucleating activity, but distinct centrosomes remained. MTs coursed in various directions following MA treatment, giving the array a disorganized appearance. Two MT-dependent processes, rhizoid morphogenesis and cell division, were found to be perturbed by small changes in pHc.

Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

A S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes.

S/M checkpoints prevent various aspects of cell division when DNA has not been replicated. Such checkpoints are stringent in yeast and animal somatic cells but are usually partial or not present in animal embryos. Because little is known about S/M checkpoints in plant cells and embryos, we have investigated the effect of aphidicolin, a specific inhibitor of DNA polymerases (alpha) and (delta), on cell division and morphogenesis in Fucus and Pelvetia zygotes. Both DNA replication and cell division were inhibited by aphidicolin, indicating the presence, in fucoid zygotes, of a S/M checkpoint. This checkpoint prevents chromatin condensation, spindle formation, centrosomal alignment with the growth axis and cytokinesis but has no effect on germination or rhizoid elongation. This S/M checkpoint also prevents tyrosine dephosphorylation of cyclin-dependent kinase-like proteins at the onset of mitosis. The kinase activity is restored in extracts upon incubation with cdc25A phosphatase. When added in S phase, olomoucine, a specific inhibitor of cyclin-dependent kinases, has similar effects as aphidicolin on cell division although alignment of the centrosomal axis still occurs. We propose a model involving the inactivation of CDK-like proteins to account for the S/M DNA replication checkpoint in fucoid zygotes and embryos.

4 Symmetry Breaking in the Zygotes of the Fucoid Algae: Controversies and Recent Progress

Despite its many advantages as an experimental system for the study of the epigenesis of polarity, it is obvious that the fucoid zygote also presents many problems. The development of polarity proceeds largely independently of direct gene action and thus may be considered a problem in cellular physiology. Ca2+ appears to play an important role in the process, but the optical properties of the zygotes (opacity and autofluorescence) hamper the use of modern methods of visualizing the distribution of Ca2+ and other ions. Likewise, other approaches, such as injection of fluorescent-labeled G-actin, in order to study the dynamics of actin filaments, are subject to the same limitations. It may be that the application of two-photon microscopy will enable experimenters to avoid some of these problems. This technique uses excitation wavelengths that are twice the wavelength of maximum absorption by fluorophores, and sufficient photon density for absorption is achieved only in a thin section. The fucoid zygotes are considerably more transparent to longer wavelengths, so attenuation of the exciting light and autofluorescence should be significantly reduced. Perhaps we will then be able to see further into these opaque cells. Another problem concerns the use of different species and genera. This may be unavoidable; for example, those of us who are land-locked tend to rely on Pelvetia, as it travels and stores better than the various species of Fucus and is less seasonal. Our colleagues fortunate enough to work near the ocean prefer to use the species that are locally available. Nevertheless, it is important to be careful about cross-genus and cross-species generalizations. While it is unlikely, based on what we know, that there are fundamental differences in physiological mechanisms among species, there may be small but still important differences in details. Obviously, investigators should directly compare results in more than one species whenever possible. The area of greatest disagreement, perhaps, concerns the mechanism of polarity formation, as opposed to its overt manifestation, germination. Are Ca2+ and actin involved or not? Assuming Ca2+ is involved, is the source internal or external? One basis for the different findings may be the differences in the strength of the polarizing signal provided to the zygotes. Clearly, the cells have powerful mechanisms for amplifying a faint asymmetry and developing an axis in response to an external signal. Furthermore, the fucoids generally develop in the intertidal zone and thus must be adapted to meeting the challenge of a widely varying external environment. They may have alternate mechanisms for responding to unilateral light. We have adopted the approach of presenting the cells with a fairly weak light signal--the minimum required to induce a considerable degree of organization of a population of zygotes. We then determine the effects of various inhibitors on photopolarization. One advantage of this approach is that it has allowed us to find treatments that increase the sensitivity of the zygotes to light, something that would not be possible if the untreated controls were fully polarized. Some of the differences between our results and those of others may be related to their use of a stronger light stimulus. It may be that if given a strong stimulus, a sufficient trace is left in the cells so that they can organize an axis when an inhibitor is removed. Careful consideration of this point may help to reconcile apparently contradictory findings. Despite these difficulties, the fucoid zygotes are likely to continue to be an important experimental system. Technology, including the development of more specific inhibitory reagents, may allow some of the shortcomings of the system to be overcome, and careful consideration of experimental conditions may resolve some of the points of disagreement.

Induction of Polarity in Fucoid Zygotes.

Early events 'in the vegetative development of higher land plants are difficult to investigate because the young embryo is encased in the ovarian tissue of the previous gametophytic and sporophytic generations. Recently, genetic approaches have succeeded in identifying mutations that alter early development (Jurgens, 1995; see also Laux and Jurgens, 1997, in this issue), but cellular and physiological analyses of higher plant embryogenesis remain tedious. Brown algae of the family Fucales (comprising the genera Fucus and Pelvetia) are, however, very tractable for investigating early plant embryogenesis, especially the establishment of developmental polarity. These marine organisms grow attached to rocks in the intertidal zone and are typically 0.25 to 0.5 m in height at maturity. Zygotes, eggs, and sperm are shed from fronds of mature plants in the laboratory 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 synchronously through early development. In the Fucales, mature eggs are radially symmetric, and cell polarization does not occur until after fertilization. The primary developmental axis is organized early in the first cell cycle and defines the growth axis of the young embryo. Unlike higher plants, in which positional cues are often conveyed 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 by the application of externa1 vectors such as unidirectional light (Jaffe, 1968). A few hours after polarization, localized growth occurs at one pole of the nascent axis, as shown in Figure 1. The first zygotic division is an invariant, asymmetric division, with the cell plate oriented transverse to the growth axis. The smaller rhizoid cell contains the growing apex and is the precursor to the holdfast of the alga; the larger thallus cell gives rise to the stipe and fronds of the mature plant (Figure 1). After the initial division of the zygote, the rhizoid cell elongates and repeatedly divides transverse to the growth axis, whereas the thallus cell undergoes proliferative divisions, each of which is transverse to the previous division.

Fucoid Zygotes Germinate from Their Darkest Regions, Not Their Brightest Ones.

Fucoid Zygotes Germinate from Their Darkest Regions, Not Their Brightest Ones.

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.

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.

To shape a cell: an inquiry into the causes of morphogenesis of microorganisms

We recognize organisms first and foremost by their forms, but how they grow and shape themselves still largely passes understanding. The objective of this article is to survey what has been learned of morphogenesis of walled eucaryotic microorganisms as a set of problems in cellular heredity, biochemistry, physiology, and organization. Despite the diversity of microbial forms and habits, some common principles can be discerned. (i) That the form of each organism represents the expression of a genetic program is almost universally taken for granted. However, reflection on the findings with morphologically aberrant mutants suggests that the metaphor of a genetic program is misleading. Cellular form is generated by a web of interacting chemical and physical processes, whose every strand is woven of multiple gene products. The relationship between genes and form is indirect and cumulative; therefore, morphogenesis must be addressed as a problem not of molecular genetics but of cellular physiology. (ii) The shape of walled cells is determined by the manner in which the wall is laid down during growth and development. Turgor pressure commonly, perhaps always, supplies the driving force for surface enlargement. Cells yield to this scalar force by localized, controlled wall synthesis; their forms represent variations on the theme of local compliance with global force. (iii) Growth and division in bacteria display most immediately the interplay of hydrostatic pressure, localized wall synthesis, and structural constraints. Koch's surface stress theory provides a comprehensive and quantitative framework for understanding bacterial shapes. (iv) In the larger and more versatile eucaryotic cells, expansion is mediated by the secretion of vesicles. Secretion and ancillary processes, such as cytoplasmic transport, are spatially organized on the micrometer scale. The diversity of vectorial physiology and of the forms it generates is illustrated by examples: apical growth of fungal hyphae, bud formation in yeasts, germination of fucoid zygotes, and development of cells of Nitella, Closterium, and other unicellular algae. (v) Unicellular organisms, no less than embryos, have a remarkable capacity to impose spatial order upon themselves with or without the help of directional cues. Self-organization is reviewed here from two perspectives: the theoretical exploration of morphogens, gradients, and fields, and experimental study of polarization in Fucus cells, extension of hyphal tips, and pattern formation in ciliates. Here is the heart of the matter, yet self-organization remains nearly as mysterious as it was a century ago, a subject in search of a paradigm.

To shape a cell: an inquiry into the causes of morphogenesis of microorganisms.

We recognize organisms first and foremost by their forms, but how they grow and shape themselves still largely passes understanding. The objective of this article is to survey what has been learned of morphogenesis of walled eucaryotic microorganisms as a set of problems in cellular heredity, biochemistry, physiology, and organization. Despite the diversity of microbial forms and habits, some common principles can be discerned. (i) That the form of each organism represents the expression of a genetic program is almost universally taken for granted. However, reflection on the findings with morphologically aberrant mutants suggests that the metaphor of a genetic program is misleading. Cellular form is generated by a web of interacting chemical and physical processes, whose every strand is woven of multiple gene products. The relationship between genes and form is indirect and cumulative; therefore, morphogenesis must be addressed as a problem not of molecular genetics but of cellular physiology. (ii) The shape of walled cells is determined by the manner in which the wall is laid down during growth and development. Turgor pressure commonly, perhaps always, supplies the driving force for surface enlargement. Cells yield to this scalar force by localized, controlled wall synthesis; their forms represent variations on the theme of local compliance with global force. (iii) Growth and division in bacteria display most immediately the interplay of hydrostatic pressure, localized wall synthesis, and structural constraints. Koch's surface stress theory provides a comprehensive and quantitative framework for understanding bacterial shapes. (iv) In the larger and more versatile eucaryotic cells, expansion is mediated by the secretion of vesicles. Secretion and ancillary processes, such as cytoplasmic transport, are spatially organized on the micrometer scale. The diversity of vectorial physiology and of the forms it generates is illustrated by examples: apical growth of fungal hyphae, bud formation in yeasts, germination of fucoid zygotes, and development of cells of Nitella, Closterium, and other unicellular algae. (v) Unicellular organisms, no less than embryos, have a remarkable capacity to impose spatial order upon themselves with or without the help of directional cues. Self-organization is reviewed here from two perspectives: the theoretical exploration of morphogens, gradients, and fields, and experimental study of polarization in Fucus cells, extension of hyphal tips, and pattern formation in ciliates. Here is the heart of the matter, yet self-organization remains nearly as mysterious as it was a century ago, a subject in search of a paradigm.

Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae

Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes (K.R. Robinson and L.F. Jaffe, 1975, Science 187, 70-72; K.R. Robinson and R. Cone, 1980, Science 207, 77-78), but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.

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.