Cellular and intracellular pathways mediating the metamorphic stimulus in hydrozoan planulae

Summary Both the natural metamorphic stimulus (an unidentified bacterial product) and an artificial trigger of metamorphosis (Cs⁺) cause large calcium transients in planula cells of the hydrozoanMitrocomella polydiademata. When these transients are inhibited with calcium channel blockers, metamorphosis is also inhibited. All cells of theMitrocomella planula contain a calcium-specific photoprotein. The cells where the calcium transients occur during natural- and Cs⁺-induced metamorphosis have been visualized in normal and entoderm free planulae that lack ganglion cells, using a compound microscope coupled to an image intensifier and video camera. During bacteria- and Cs⁺-induced metamorphosis, groups of contiguous cells, occupying from about 10% to the entire visible surface of the planula, simultaneously exhibit calcium transients. When the cells that initiate a transient comprise only part of the planula surface, the calcium transient frequently propagates and can eventually involve every cell on the visible planula surface. There is no special site on the planula surface where calcium transients are more apt to be initiated. There is no indication that propagation of a flash in one direction is more likely than in another. The velocity of propagation is virtually the same in all directions. The only feature of the spatial distribution of bacteria- and Cs⁺-induced calcium transients that appears to be necessary for the induction of metamorphosis is that at least one transient must involve all of the surface cells of the planula. The spatial behavior of calcium transients is the same in entoderm free planulae (lacking ganglion cells) as in normal planulae. The propagation of these calcium transients most probably occurs via epithelial conduction. This metamorphic step involving calcium transients is probably the intercellular communication system that informs the cells of the planula that metamorphosis will commence.Metamorphosis inMitrocomella planulae can also be induced with phorbol esters. Calcium transients do not occur during phorbol ester-induced metamorphosis, indicating that they act at a different point in the metamorphic pathway. Calcium channel blockers do not inhibit phorbol ester-induced metamorphosis. Inhibitors of protein kinase-C, inhibit both phorbol ester-induced metamorphosis and Cs⁺- and bacteria-induced metamorphosis, but have no effect on the calcium transients induced by Cs⁺. This indicates that the calcium transient mediated step in the metamorphic pathway occurs prior to protein kinase-C activation. Calcium transients probably play a major role in activating protein kinase-C.     

Pattern of cell proliferation in embryogenesis and planula development ofHydractinia echinata predicts the postmetamorphic body pattern

Summary During embryogenesis and planula development of the colonial hydroidHydractinia echinata cell proliferation decreases in a distinct spatio-temporal pattern. Arrest in S-phase activity appears first in cells localized at the posterior and then subsequently at the anterior pole of the elongating embryo. These areas do not resume S-phase activity, even during the metamorphosis of the planula larva into the primary polyp. Tissue containing the quiescent cells gives rise to the terminal structures of the polyp. The posterior area of the larva becomes the hypostome and tentacles, while the anterior part of the larva develops into the basal plate and stolon tips. In mature planulae only a very few cells continue to proliferate. These cells are found in the middle part of the larva. Labelling experiments indicate that the prospective material of the postmetamorphic tentacles and stolon tips originates from cells which have exited from the cell cycle in embryogenesis or early in planula development. Precursor cells of the nematocytes which appear in the tentacles of the polyp following metamorphosis appear to have ceased cycling before the 38th hour of embryonic development. The vast majority of the cells that constitute the stolon tips of the primary polyp leave the cell cycle not later than 58 h after the beginning of development. We also report the identification of a cell type which differentiates in the polyp without passing through a post-metamorphic S-phase. The cell type appears to be neural in origin, based upon the identification of a neuropeptide of the FMRFamide type.     

eIF4E‐Binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs

Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap‐binding protein eIF4E, which is released from its repressor 4E‐BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E‐BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E‐BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E‐BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E‐BP and eIF4G. Mol. Reprod. Dev. 77: 83–91, 2010. © 2009 Wiley‐Liss, Inc.     

Post-embryonic larval development and metamorphosis of the hydroidEudendrium racemosum (Cavolini) (Hydrozoa,Cnidaria)

The morphology and histology of the planula larva ofEudendrium racemosum (Cavolini) and its metamorphosis into the primary polyp are described from light microscopic observations. The planula hatches as a differentiated gastrula. During the lecithotrophic larval period, large ectodermal mucous cells, embedded between epitheliomuscular cells, secrete a sticky slime. Two granulated cell types occur in the ectoderm that are interpreted as secretory and sensorynervous cells, but might also be representatives of only one cell type with a multiple function. The entoderm consists of yolk-storing gastrodermal cells, digestive gland cells, interstitial cells, cnidoblasts, and premature cnidocytes. The larva starts metamorphosis by affixing its blunt aboral pole to a substratum. While the planula flattens down, the mucous cells penetrate the mesolamella and migrate through the entoderm into the gastral cavity where they are lysed. Subsequently, interstitial cells, cnidoblasts, and premature cnidocytes migrate in the opposite direction, i.e. from entoderm to ectoderm. Then, the polypoid body organization, comprising head (hydranth), stem and foot, all covered by peridermal secretion, becomes recognisable. An oral constriction divides the hypostomal portion of the gastral cavity from the stomachic portion. Within the hypostomal entoderm, cells containing secretory granules differentiate. Following growth and the multiplication of tentacles, the head periderm disappears. A ring of gland cells differentiates at the hydranth's base. The positioning of cnidae in the tentacle ectoderm, penetration of the mouth opening and the multiplication of digestive gland cells enable the polyp to change from lecithotrophic to planktotrophic nutrition.     

Inductive activity of the posterior tip of planula in the marine hydroid <Emphasis Type="Italic">Dynamena pumila</Emphasis>

Activity of organizer regions is required for body plan formation in the developing organism. Transplanting a fragment of such a region to a host organism leads to the formation of a secondary body axis that consists of both the donor’s and the host’s tissues (Gerhart, 2001). The subject of this study, the White Sea hydroid cnidarian Dynamena pumila L. (Thecaphora, Sertulariidae), forms morphologically advanced colonies in the course of complex metamorphosis of the planula larva. To reveal an organizer region, a series of experiments has been performed in which small fragments of donor planula tissues were transplanted to embryos at the early and late gastrula stage, as well as to planulae. Only transplantations of a posterior tip fragment of a donor planula to a host planula of the same age led, in the course of metamorphosis, to the formation of a secondary shoot, which involved up to 50% of the host’s tissues. After transplantations of tissue fragments of the anterior tip and the middle of the planula body, the formation of any ectopic structures was never observed. It was concluded that the posterior tip of the planula has organizer properties in Dynamena.     

Injections of porcine sperm extracts trigger fertilization-like calcium oscillations in oocytes of a marine worm

The precise mechanisms by which sperm trigger calcium transients in eggs or oocytes during fertilization remain unknown. Based on time-lapse confocal microscopy, we show that intracellular injections of porcine sperm extracts cause the oocytes of a marine nemertean worm to undergo repetitive calcium oscillations resembling those obtained during normal fertilizations. Such findings are consistent with the view that fertilization involves a soluble sperm factor (SF) which is capable of eliciting calcium transients without binding to externally situated receptors on the oocyte plasmalemma. This study also describes for the first time the wave-like propagation patterns of SF-induced calcium transients that are generated in a heterologous combination of gametes obtained from different phyla of animals. Such cross-reactivity between distantly related taxa suggests that the intracellular signaling pathways triggered by sperm factors can be well conserved.     

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.     

Metamorphosis ofHydractinia echinata Insights into pattern formation in Hydroids

Summary Hydractinia echinata is a marine, colony-forming coelenterate. Fertilized eggs develop into freely swimming planula larvae, which undergo metamorphosis to a sessile (primary) polyp. Metamorphosis can be triggered by means of certain marine bacteria and by Cs⁺. Half a day after this treatment a larva will have developed into a polyp. The induction of metamorphosis can be prevented by addition of inhibitor I, a substance partially purified from tissue ofHydra. The larvae ofH. echinata also appear to contain this substance. Inhibitor I appliedafter the onset of metamorphosis blocks its continuation as long as it remains in the culture medium. Cs⁺ applied within the same period of time also blocks the continuation of metamorphosis. However, these two agents have opposite effects on the body pattern of the resultant polyps. The experiments indicate that application of Cs⁺ triggers the generation of the pre-pattern. Inhibitor I appears to be a factor of this prepattern. A model is proposed which describes the basic features of head and foot/stolon formation not only forHydractinia but also for other related hydroids.     

Reorganization of the nervous system during metamorphosis of a hydrozoan planula

Abstract. Laser scanning confocal microscopy is used to reveal the changes that occur in the RFamide-positive nerve net as a free-swimming, solid hydrozoan planula larva is transformed into a sessile, hollow, young polyp. Seven stages of development in Pennaria tiarella are described: planula competent to metamorphose, attaching planula, disc, pawn, crown, developing polyp, and developed primary polyp. The RFamide-positive nervous system undergoes dramatic reorganization during metamorphosis: (1) larval neurons degenerate; (2) new neurons differentiate and reform a nerve net; and (3) the overall distribution pattern of the nervous system changes. This study confirms earlier observations on RFamide-positive neurons of Hydractinia which also show the loss of these cells after the onset of metamorphosis.     

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.     

Prospective fate of early blastomeres in Hydractinia echinata (Cnidaria,Hydrozoa)

Summary Blastomeres of two-cell, four-cell, and eight-cell embryos of Hydractinia echinata were injected with horseradish-peroxidase (HRP) or fluorescein isothiocyanate (FITC)-dextran. The fate of the descendants of the injected blastomeres was followed until the planula larva had developed. The results obtained after HRP or FITC-dextran injection were essentially the same. Blastomeres are equivalent up to the four-cell stage, i.e. half-blastomeres produce half of the ectoderm of the planula larva and quarter-blastomeres give rise to one quarter of the larval ectoderm. During normal embryogenesis, the larval anterior-posterior axis corresponds to the animal-vegetal axis of the zygote. Thus, the labelled areas of larvae consisting of the progeny of injected half or quarter blastomeres normally stretch along the larval anterior-posterior axis. Normally, material giving rise to anterior or posterior larval parts, respectively, is separated at the third cleavage. Irrespective of the type of experiment, the progeny of injected blastomeres always contributed to endoderm formation, i.e. in larvae resulting from injected embryos the endoderm was more or less uniformly labelled. Application of vital stains locally to the exterior of zygotes and following these markers through first and second cleavage, produced evidence that in the vast majority of cases, the second cleavage is meridional. Offprint requests to: A. Schlawny     

A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in hamster eggs

Microinjection of cytosolic sperm extracts into unfertilized golden hamster eggs caused a series of increases in cytoplasmic free calcium, Ca2+i, and membrane hyperpolarizing responses, HRs. These HRs and Ca2+i transients are similar to those seen during in vitro fertilization of hamster eggs. The sperm factor that is responsible for causing these effects appears to be of high molecular weight and protein based. Injection of sperm factor activated eggs and mimicked fertilization in causing repetitive HRs in the presence of phorbol esters and in sensitizing the egg to calcium-induced calcium release. Since these effects cannot be mimicked by injecting G-protein agonists or calcium-containing solutions, it seems unlikely that a receptor-G-protein signalling system is involved at fertilization. These data instead suggest a novel signal transduction system operates during mammalian fertilization in which a protein factor is transferred from the sperm into the egg cytoplasm after gamete membrane fusion.     

Embryonic and larval development of the host sea anemones Entacmaea quadricolor and Heteractis crispa

Little information is available on the sexual reproductive biology of anemones that provide essential habitat for anemonefish. Here we provide the first information on the surface ultrastructural and morphological changes during development of the embryos and planula larvae of Entacmaea quadricolor and Heteractis crispa, using light and scanning electron microscopy. Newly spawned eggs of E. quadricolor and H. crispa averaged 794 microm and 589 microm diameter, respectively, and were covered by many spires of microvilli that were evenly distributed over the egg surface, except for a single bare patch. Eggs of both species contained abundant zooxanthellae when spawned, indicating vertical transmission of symbionts. Fertilization was external, and the resulting embryos displayed superficial cleavage. As development continued, individual blastomeres became readily distinguishable and a round-to-ovoid blastula was formed, which flattened with further divisions. The edges of the blastula thickened, creating a concave-convex dish-shaped gastrula. The outer margins of the gastrula appeared to roll inward, leading to the formation of an oral pore and a ciliated planula larva. Larval motility and directional movement were first observed 36 h after spawning. E. quadricolor larval survival remained high during the first 4 d after spawning, then decreased rapidly.     

The cleavage initiation site establishes the posterior pole of the hydrozoan embryo

Summary When the first cleavage of the hydrozoan egg is reversibly suppressed, two cleavage furrows frequently form simultaneously at the time of the second cleavage. If these two cleavage initiation sites are far enough apart, each one specifies a site of gastrulation, and the embryo that forms develops into a two tailed planula larva. When two tailed planulae are induced to metamorphose, they form a polyp with two stalks and hydranths.     

The effect of larval age on developmental changes in the polyp prepattern of a hydrozoan planula

The larvae of many marine organisms including hydrozoans are lecithotrophic and will not feed until after metamorphosis. In hydrozoans the aboral region of the planula becomes the holdfast and stolon, while the oral region becomes the stalk and hydranth that grows out of the holdfast following metamorphosis. If metamorphosis is delayed, the portion of the planula allocated to form holdfast and stolon shrinks and the region that forms the hydranth increases in size. Planulae also have the ability to regenerate their polyp prepattern. When the aboral region of the planula that does not normally form a hydranth is isolated and metamorphosis is delayed, it acquires the capacity to form a hydranth from the holdfast. A relatively high proportion of entodermal cells of young planulae engage in DNA synthesis (BrdU labeling index); as planulae age, the labeling index falls close to zero. When the polyp prepattern is modified during planula regeneration, entodermal cells are induced to engage in DNA synthesis. If DNA synthesis is inhibited in planulae, the polyp prepattern changes during regeneration and age-related developmental changes in planula are inhibited, suggesting that DNA synthesis is a necessary part of the pattern respecification process.     

Inhibition of metamorphosis by RFamide neuropeptides in planula larvae of Hydractinia echinata

The primitive nervous system in planula larvae of Hydractinia echinata (Cnidaria) has sensory neurons containing LWamide or RFamide neuropeptides. LWamides have been shown to induce metamorphosis of planula larvae into adult polyps. We report here that RFamides act antagonistically to LWamides. RFamides inhibit metamorphosis when applied to planula larvae during metamorphosis induction by treatment with LWamides (or other inducing agents such as CsCl ions, diacylglycerol and bacterial inducers). Our results show further that RFamides act downstream of LWamide release, presumably directly on target cells mediating metamorphosis. These observations support a model in which metamorphosis in H. echinata is regulated by sensory neurons secreting LWamides and RFamides in response to environmental cues.Edited by D. Tautz     

Embryonic development and metamorphosis of the scyphozoan <Emphasis Type="Italic">Aurelia</Emphasis>

We investigated the development of Aurelia (Cnidaria, Scyphozoa) during embryogenesis and metamorphosis into a polyp, using antibody markers combined with confocal and transmission electron microscopy. Early embryos form actively proliferating coeloblastulae. Invagination is observed during gastrulation. In the planula, (1) the ectoderm is pseudostratified with densely packed nuclei arranged in a superficial and a deep stratum, (2) the aboral pole consists of elongated ectodermal cells with basally located nuclei forming an apical organ, which is previously only known from anthozoan planulae, (3) endodermal cells are large and highly vacuolated, and (4) FMRFamide-immunoreactive nerve cells are found exclusively in the ectoderm of the aboral region. During metamorphosis into a polyp, cells in the planula endoderm, but not in the ectoderm, become strongly caspase 3 immunoreactive, suggesting that the planula endoderm, in part or in its entirety, undergoes apoptosis during metamorphosis. The polyp endoderm seems to be derived from the planula ectoderm in Aurelia, implicating the occurrence of “secondary” gastrulation during early metamorphosis.     

All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin

The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7A and 2.2 A, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hyroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with product, coleneteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.     

Autophagy is used as a survival program in unfertilized sea urchin eggs that are destined to die by apoptosis after inactivation of MAPK1/3 (ERK2/1)

A high MAPK1/3 (also known as ERK2/1, respectively) activity, preventing spontaneous activation, is essential to maintain cell cycle arrest of mature oocytes of mammals, frogs or invertebrates such as starfish. Mature oocytes would undergo a “suicide”-like cell death if not fertilized. We previously have reported that downregulation of MAPK1/3 in unfertilized sea urchin eggs induces a calcium-dependent entry into mitosis. We show here that this event is followed by a series of pseudo-mitotic cell cycles associated with transient Ca_i increases, preceding CASP3/caspase-3 activation and apoptosis. However, cell death was delayed after inhibition of the Ca_i transients or of cyclin-dependent kinases (CDK), with roscovitine. In these conditions, eggs enter an autophagy program as suggested by detection of processed LC3B by western blot, immunofluorescence and immunogold staining, visualization of autophagy vesicles by electron microscopy, and an increase in acidic vesicular organelles (AVOs). We found that bafilomycin A₁ or an association of leupeptin and pepstatin, which are widely used to study autophagy, may act upon calcium signaling or cell cycle events, respectively, and not only on autophagy events. Finally, inhibition of PtdIns 3-kinase with wortmannin or LY294002 powerfully stimulated cell death of unfertilized eggs, which suggests that this activity does not negatively regulate autophagy as is often reported, but rather stimulates survival in unfertilized eggs. We suggest that apoptosis of unfertilized eggs is the consequence of an aberrant short attempt of development that occurs if MAPK1/3 is inactivated, but these eggs can use autophagy as a survival program when the cell cycle is blocked.