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Nervous systems of bilaterian animals generally consist of two cell types: neurons and glial cells. Despite accumulating data about the many important functions glial cells serve in bilaterian nervous systems, the evolutionary origin of this abundant cell type remains unclear. Current hypotheses regarding glial evolution are mostly based on data from model bilaterians. Non-bilaterian animals have been largely overlooked in glial studies and have been subjected only to morphological analysis. Here, we provide a comprehensive overview of conservation of the bilateral gliogenic genetic repertoire of non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, and Porifera). We overview molecular and functional features of bilaterian glial cell types and discuss their possible evolutionary history. We then examine which glial features are present in non-bilaterians. Of these, cnidarians show the highest degree of gliogenic program conservation and may therefore be crucial to answer questions about glial evolution., (© 2024. The Author(s).)

Medusozoans, the Cnidarian subphylum, have multiple life stages including sessile polyps and free-swimming medusae or jellyfish, which are typically bell-shaped gelatinous zooplanktons that exhibit diverse morphologies. Despite having a relatively complex body structure with well-developed muscles and nervous systems, the adult medusa stage maintains a high regenerative ability that enables organ regeneration as well as whole body reconstitution from the part of the body. This remarkable regeneration potential of jellyfish has long been acknowledged in different species; however, recent studies have begun dissecting the exact processes underpinning regeneration events. In this article, we introduce the current understanding of regeneration mechanisms in medusae, particularly focusing on cellular behaviors during regeneration such as wound healing, blastema formation by stem/progenitor cells or cell fate plasticity, and the organism-level patterning that restores radial symmetry. We also discuss putative molecular mechanisms involved in regeneration processes and introduce a variety of novel model jellyfish species in the effort to understand common principles and diverse mechanisms underlying the regeneration of complex organs and the entire body.

In Cnidaria, modes of gastrulation to produce the two body layers vary greatly between species. In the hydrozoan species Clytia hemisphaerica gastrulation involves unipolar ingression of presumptive endoderm cells from an oral domain of the blastula, followed by migration of these cells to fill the blastocoel with concomitant narrowing of the gastrula and elongation along the oral-aboral axis. We developed a 2D computational boundary model capable of simulating the morphogenetic changes during embryonic development from early blastula stage to the end of gastrulation. Cells are modeled as polygons with elastic membranes and cytoplasm, colliding and adhering to other cells, and capable of forming filopodia. With this model we could simulate compaction of the embryo preceding gastrulation, bottle cell formation, ingression, and intercalation between cells of the ingressing presumptive endoderm. We show that embryo elongation is dependent on the number of endodermal cells, low endodermal cell-cell adhesion, and planar cell polarity (PCP). When the strength of PCP is reduced in our model, resultant embryo morphologies closely resemble those reported previously following morpholino-mediated knockdown of the core PCP proteins Strabismus and Frizzled. Based on our results, we postulate that cellular processes of apical constriction, compaction, ingression, and then reduced cell-cell adhesion and mediolateral intercalation in the presumptive endoderm, are required and when combined, sufficient for Clytia gastrulation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.

Cnidarians possess remarkable powers of regeneration, but the cellular and molecular mechanisms underlying this capability are unclear. Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation, forming a blastema at the oral pole within 24 hr. This process is necessary for head regeneration. Knocking down Piwi1, Vasa, Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation. EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1(+) stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area. Surprisingly, no blastema developed at the aboral pole after stolon removal. Instead, polyps transformed into stolons and then budded polyps. Hence, distinct mechanisms act to regenerate different body parts in Hydractinia. This model, where stem cell behavior can be monitored in vivo at single cell resolution, offers new insights for regenerative biology.

Understanding how new cell types arise is critical for understanding the evolution of organismal complexity. Questions of this nature, however, can be difficult to answer due to the challenge associated with defining the identity of a truly novel cell. Cnidarians (anemones, jellies, and their allies) provide a unique opportunity to investigate the molecular regulation and development of cell-novelty because they possess a cell that is unique to the cnidarian lineage and that also has a very well-characterized phenotype: the cnidocyte (stinging cell). Because cnidocytes are thought to differentiate from the cell lineage that also gives rise to neurons, cnidocytes can be expected to express many of the same genes expressed in their neural "sister" cells. Conversely, only cnidocytes posses a cnidocyst (the explosive organelle that gives cnidocytes their sting); therefore, those genes or gene-regulatory relationships required for the development of the cnidocyst can be expected to be expressed uniquely (or in unique combination) in cnidocytes. This system provides an important opportunity to: (1) construct the gene-regulatory network (GRN) underlying the differentiation of cnidocytes, (2) assess the relative contributions of both conserved and derived genes in the cnidocyte GRN, and (3) test hypotheses about the role of novel regulatory relationships in the generation of novel cell types. In this review, we summarize common challenges to studying the evolution of novelty, introduce the utility of cnidocyte differentiation in the model cnidarian, Nematostella vectensis, as a means of overcoming these challenges, and describe an experimental approach that leverages comparative tissue-specific transcriptomics to generate hypotheses about the GRNs underlying the acquisition of the cnidocyte identity., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Background: Wound healing is the first stage of a series of cellular events that are necessary to initiate a regenerative response. Defective wound healing can block regeneration even in animals with a high regenerative capacity. Understanding how signals generated during wound healing promote regeneration of lost structures is highly important, considering that virtually all animals have the ability to heal but many lack the ability to regenerate missing structures. Cnidarians are the phylogenetic sister taxa to bilaterians and are highly regenerative animals. To gain a greater understanding of how early animals generate a regenerative response, we examined the cellular and molecular components involved during wound healing in the anthozoan cnidarian Nematostella vectensis., Results: Pharmacological inhibition of extracellular signal-regulated kinases (ERK) signaling blocks regeneration and wound healing in Nematostella. We characterized early and late wound healing events through genome-wide microarray analysis, quantitative PCR, and in situ hybridization to identify potential wound healing targets. We identified a number of genes directly related to the wound healing response in other animals (metalloproteinases, growth factors, transcription factors) and suggest that glycoproteins (mucins and uromodulin) play a key role in early wound healing events. This study also identified a novel cnidarian-specific gene, for a thiamine biosynthesis enzyme (vitamin B synthesis), that may have been incorporated into the genome by lateral gene transfer from bacteria and now functions during wound healing. Lastly, we suggest that ERK signaling is a shared element of the early wound response for animals with a high regenerative capacity., Conclusions: This research describes the temporal events involved during Nematostella wound healing, and provides a foundation for comparative analysis with other regenerative and non-regenerative species. We have shown that the same genes that heal puncture wounds are also activated after oral-aboral bisection, indicating a clear link with the initiation of regenerative healing. This study demonstrates the strength of using a forward approach (microarray) to characterize a developmental phenomenon (wound healing) at a phylogenetically important crossroad of animal evolution (cnidarian-bilaterian ancestor). Accumulation of data on the early wound healing events across numerous systems may provide clues as to why some animals have limited regenerative abilities.

Coral bleaching occurs when there is a breakdown of the symbiosis between cnidarian hosts and resident Symbiodinium spp. Multiple mechanisms for the bleaching process have been identified, including apoptosis and autophagy, and most previous work has focused on the Symbiodinium cell as the initiator of the bleaching cascade. In this work we show that it is possible for host cells to initiate apoptosis that can contribute to death of the Symbiodinium cell. First we found that colchicine, which results in apoptosis in other animals, causes cell death in the model anemone Aiptasia sp. but not in cultured Symbiodinium CCMP-830 cells or in cells freshly isolated from host Aiptasia (at least within the time frame of our study). In contrast, when symbiotic Aiptasia were incubated in colchicine, cell death in the resident Symbiodinium cells was observed, suggesting a host effect on symbiont mortality. Using live-cell confocal imaging of macerated symbiotic host cell isolates, we identified a pattern where the initiation of host cell death was followed by mortality of the resident Symbiodinium cells. This same pattern was observed in symbiotic host cells that were subjected to temperature stress. This research suggests that mortality of symbionts during temperature-induced bleaching can be initiated in part by host cell apoptosis.

The symbiotic relationship between cnidarians and their dinoflagellate symbionts, Symbiodinium spp, which underpins the formation of tropical coral reefs, can be destabilized by rapid changes to environmental conditions. Although some studies have concluded that a breakdown in the symbiosis begins with increased reactive oxygen species (ROS) generation within the symbiont due to a decoupling of photosynthesis, others have reported the release of viable symbionts via a variety of host cell derived mechanisms. We explored an alternative model focused upon changes in host cnidarian mitochondrial integrity in response to thermal stress. Mitochondria are often likened to being batteries of the cell, providing energy in the form of ATP, and controlling cellular pathway activation and ROS generation. The overall morphology of host mitochondria was compared to that of associated symbionts under an experimental thermal stress using confocal and electron microscopy. The results demonstrate that hyperthermic stress induces the degradation of cnidarian host mitochondria that is independent of symbiont cellular deterioration. The potential sites of host mitochondrial disruption were also assessed by measuring changes in the expression of genes associated with electron transport and ATP synthesis using quantitative RT-PCR. The primary site of degradation appeared to be downstream of complex III of the electron transport chain with a significant reduction in host cytochrome c and ATP synthase expression. The consequences of reduced expression could limit the capacity of the host to mitigate ROS generation and maintain both organelle integrity and cellular energy supplies. The disruption of host mitochondria, cellular homeostasis, and subsequent cell death irrespective of symbiont integrity highlights the importance of the host response to thermal stress and in symbiosis dysfunction that has substantial implications for understanding how coral reefs will survive in the face of climate change.

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

The evolutionary origin of stem cell pluripotency is an unresolved question. In mammals, pluripotency is limited to early embryos and is induced and maintained by a small number of key transcription factors, of which the POU domain protein Oct4 is considered central. Clonal invertebrates, by contrast, possess pluripotent stem cells throughout their life, but the molecular mechanisms that control their pluripotency are poorly defined. To address this problem, we analyzed the expression pattern and function of Polynem (Pln), a POU domain gene from the marine cnidarian Hydractinia echinata. We show that Pln is expressed in the embryo and adult stem cells of the animal and that ectopic expression in epithelial cells induces stem cell neoplasms and loss of epithelial tissue. Neoplasm cells downregulated the transgene but expressed the endogenous Pln gene and also Nanos, Vasa, Piwi and Myc, which are all known cnidarian stem cell markers. Retinoic acid treatment caused downregulation of Pln and the differentiation of neoplasm cells to neurosensory and epithelial cells. Pln downregulation by RNAi led to differentiation. Collectively, our results suggest an ancient role of POU proteins as key regulators of animal stem cells.

The kinase Mos, which activates intracellularly the MAP kinase pathway, is a key regulator of animal oocyte meiotic maturation. In vertebrate and echinoderm models, Mos RNA translation upon oocyte hormonal stimulation mediates "cytostatic" arrest of the egg after meiosis, as well as diverse earlier events [1-5]. Our phylogenetic survey has revealed that MOS genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges. We demonstrated MAP kinase-mediated cytostatic activity for Mos orthologs from Pleurobrachia (ctenophore) and Clytia (cnidarian) by RNA injection into Xenopus blastomeres. Analyses of endogenous Mos in Clytia with morpholino antisense oligonucleotides and pharmacological inhibition demonstrated that Mos/MAP kinase function in postmeiotic arrest is conserved. They also revealed additional roles in spindle formation and positioning, strongly reminiscent of observations in starfish, mouse, and Xenopus. Unusually, cnidarians were found to possess multiple Mos paralogs. In Clytia, one of two maternally expressed paralogs accounted for the majority MAP kinase activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles. Our findings indicate that Mos appeared early during animal evolution as an oocyte-expressed kinase and functioned ancestrally in regulating core specializations of female meiosis.

Cnidarian bleaching is a breakdown in the mutualistic symbiosis between host Cnidarians, such as reef building corals, and their unicellular photosynthetic dinoflagellate symbionts. Bleaching is caused by a variety of environmental stressors, most notably elevated temperatures associated with global climate change in conjunction with high solar radiation, and it is a major contributor to coral death and reef degradation. This review examines the underlying cellular events that lead to symbiosis dysfunction and cause bleaching, emphasizing that, to date, we have only some pieces of a complex cellular jigsaw puzzle. Reactive oxygen species (ROS), generated by damage to both photosynthetic and mitochondrial membranes, is shown to play a central role in both injury to the partners and to inter-partner communication of a stress response. Evidence is presented that suggests that bleaching is a host innate immune response to a compromised symbiont, much like innate immune responses in other host-microbe interactions. Finally, the elimination or exit of the symbiont from host tissues is described through a variety of mechanisms including exocytosis, host cell detachment and host cell apoptosis.

Proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is the most prevalent parasitic infection affecting commercial channel catfish (Ictalurus punctatus) aquaculture. There are currently no effective chemotherapeutic or biological control measures for PGD, which often peaks during the spring and fall when water temperatures are between 16-25 degrees C. The current diagnostic techniques of gross examination of gill clip wet mounts and histopathology are subject to false-negatives during the early stages of infection, and the quantifiable nature of end-point polymerase chain reaction (PCR) is subjective. Consequently, a rapid and more sensitive quantitative real-time PCR assay was developed for the detection of H. ictaluri during the early stages of infection in channel catfish. A 23 base-pair TaqMan probe was designed based on previously published H. ictaluri PCR protocols. The sensitivity of the assay was the equivalent of a single H. ictaluri actinospore, and in a pond challenge study, quantitative real-time PCR proved to be more sensitive than gross examination, microscopic examination of gill clip wet mounts, and histopathologic examination of gill tissue sections. Future applications of this assay will focus on developing methodologies to be used in conjunction with current pond-monitoring protocols to evaluate potential treatments and better manage this significant seasonal disease.

Little is known about the viability of myxozoan actinospore stages after harvest from laboratory cultures of infected oligochaete worms. The viability and reactivity of actinospores of three myxozoan species was evaluated after short-term storage at 4 degrees C and 12 degrees C. Two methods of determining actinospore viability were compared: differential fluorescent staining and direct microscopic observation of morphological indicators of spore integrity. Spore reactivity was quantified by measuring polar filament discharge rates in a micro-assay with fish mucus substrate and mechanical stimulation by vibration. The age-dependent viability of the three species showed clear differences. Myxobolus cerebralis actinospores had the shortest effective life span whereas Henneguya nuesslini actinospores survived significantly longer. Storage at lower temperatures yielded higher viability in all species. Myxobolus pseudodispar actinospores were significantly robust up to 12 degrees C when assessed by staining, but showed similar viability characteristics as H. nuesslini when analyzed morphologically. Evaluation of spore viability by fluorescent staining correlated with morphological assessment, although fewer viable actinospores were usually detected microscopically. Polar filament discharge activity of morphologically intact actinospores did not significantly decrease until the third day of storage compared to freshly harvested samples. The results indicate that durability and reactivity trends during storage of actinospores differ among myxozoan species.

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.

Fossilized embryos from the late Neoproterozoic and earliest Phanerozoic have caused much excitement because they preserve the earliest stages of embryology of animals that represent the initial diversification of metazoans. However, the potential of this material has not been fully realized because of reliance on traditional, non-destructive methods that allow analysis of exposed surfaces only, and destructive methods that preserve only a single two-dimensional view of the interior of the specimen. Here, we have applied synchrotron-radiation X-ray tomographic microscopy (SRXTM), obtaining complete three-dimensional recordings at submicrometre resolution. The embryos are preserved by early diagenetic impregnation and encrustation with calcium phosphate, and differences in X-ray attenuation provide information about the distribution of these two diagenetic phases. Three-dimensional visualization of blastomere arrangement and diagenetic cement in cleavage embryos resolves outstanding questions about their nature, including the identity of the columnar blastomeres. The anterior and posterior anatomy of embryos of the bilaterian worm-like Markuelia confirms its position as a scalidophoran, providing new insights into body-plan assembly among constituent phyla. The structure of the developing germ band in another bilaterian, Pseudooides, indicates a unique mode of germ-band development. SRXTM provides a method of non-invasive analysis that rivals the resolution achieved even by destructive methods, probing the very limits of fossilization and providing insight into embryology during the emergence of metazoan phyla.

The phylum Placozoa is represented by a single known species, Trichoplax adhaerens, a tiny marine organism that represents the most simple metazoan bauplan. Because of the latter, placozoans were originally considered the most basal metazoan phylum. A misinterpretation of the life cycle at the turn of the century and some more recent molecular phylogenetic analyses have placed Trichoplax as a derived species within the Cnidaria. The latter hypothesis assumes that the primitive organization of the Placozoa is the result of secondary reduction. Here we compare the molecular morphology of the predicted 16S rDNA structure and the mitochondrial genome between Trichoplax and representatives of all four cnidarian classes. Trichoplax shares a circular mtDNA molecule as a plesiomorphy with all other metazoans except for the derived cnidarian classes Hydrozoa, Scyphozoa, and Cubozoa. The predicted secondary structure of the 16S rRNA molecule differs substantially between Trichoplax and cnidarians, particularly with respect to the number and length of stem and loop regions. The new molecular morphological characters provide compelling evidence that Trichoplax is not a derived (medusozoan) cnidarian. Furthermore, it was found that the mitochondrial genome in Cubozoa consists of four linear molecules instead of a single circular molecule or two linear molecules, suggesting that the cubozoans may represent the most derived cnidarian group.

Programmed cell death is not only known as a mechanism mediating tissue destruction, but also as an organismic tool for body shaping and regulation of morphological events during development. Here we report the tight and vital link of the most prominent form of programmed cell death, apoptosis, to one of the oldest, most basic, and most radical developmental processes, the metamorphosis of the marine hydrozoon Hydractinia echinata. Apoptosis, represented by DNA fragmentation, appears very early during metamorphosis, approximately 20 minutes post induction. It is then executed in a very distinct spatial and temporal pattern, including the removal or phagocytosis of a large number of larval cells prior to the appearance of stolons and tentacles. Our data indicate a developmental program striving to reduce all body parts that are no longer necessary, before reaching a distinct turning point, when the development of adult features is initiated. During these events, morphogenesis of basal and apical structures correlates with recycling of that particular larval region, indicated by the presence of apoptosis. Based on these data, the necessity of apoptosis for normal development of adult patterns is inferred and a fundamental association of apoptosis with developmental processes can be stated.

Conditions for the primary culture of branching scleractinian coral (Acropora micropthalma and Pocillopora damicornis) cells were established with a calcium-free seawater cell dissociation method. Cells were isolated and cultured in supplemented Dulbecco's modified Eagle media with heat-inactivated fetal bovine serum, antibiotics, and sterile seawater. Among the isolated cell types, large (60-100 microm) multicellular endothelial isolates (MEIs) were seen in high numbers. These isolates were observed to continually spin for up to 300 h without media change. The following parameters were optimized: media, serum, light, trace elements, and growth factor supplements. Rotations per minute were calculated to determine MEI motility in relation to size. Finally, analyses of external and internal structures were conducted with scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Additional coral species, Montipora digitata, Stylophora pistillata, Seriatopora hystrix and Porites sp. were also cultured to determine the applicability of isolation techniques. The relatively long survival time of MEIs in primary culture makes them ideal candidates for in vitro studies examining coral disease processes (e.g., mode of infection and intracellular effects of disease-causing agents) as well as aspects of general coral growth and health (e.g., trace element requirements and transfer of products between host cell and zooxanthellae).

Beroë muscle fibers are single cells which may be 20-40 micrometer in diameter in mature specimens. Longitudinal muscles may be 6 cm or more long. There is no striation pattern and the muscles were observed to contract in a tonic fashion when stretched. They are innervated by a nerve net, and external recording revealed what are probably nerve net impulses. Intracellular stimulation of the muscles themselves was found to initiate large propagating action potentials which were recorded intracellularly. The action potentials were insensitive to tetrodotoxin (10(-5) g/ml), tetraethylammonium ions (50 mM), MnCl2 (25 mM), and low concentrations of verapamil (2 X 10(-6) g/ml). Full-size action potentials were recorded in sodium- or calcium-deficient salines, but were small and graded in salines deficient in both sodium and calcium. Cable analysis yielded mean values for lambda (1.95 mm), Ri (154 omega cm), Rm (9,253 omega cm2), and tau m (13.9 ms). The conduction velocity depended primarily on fiber diameter and maximum rate of rise of the action potential and could be predicted from the theoretical analysis of Hunter et al. (1975 Prog. Biophys. Mol. Biol. 30: 99-144). The calculated membrane capacity (less than microF/cm2) indicates little infolding of the surface membrane, a conclusion which is in agreement with anatomical studies.

No significant differences in macro- and micromorphology were found between the parasitic stolon and free-living polyps of Polypodium sp. obtained from infected eggs of the North American acipenseriform fish Polyodon spathula and corresponding developmental stages of Polypodium hydriforme Ussov, parasitic in the Volga sterlet (Acipenser ruthenus). Therefore, both the American and the European forms of Polypodium belong to the species P. hydriforme Ussov.

Developing oocytes of the cnidarian Bunodosoma cavernata are located within the mesoglea of the mesenteries of the gastrovascular cavity. The cortex of the more mature vitellogenic oocytes contains numerous, electron-dense, membrane-bound, cortical granules. The surface of these oocytes possesses prominent radially projecting structures termed cytospines. Each cytospine has a core of microfilaments, 50-70 A in diameter, that extends basally as a rootlet through the cortical layer. During spawning, ova lacking any extraneous investments are released from the enclosing gastrodermis. As a consequence of fertilization or events associated with the earliest stages of development the ova undergo a massive cortical reaction. This reaction, which occurs during or just after release of the ova, involves extensive reorganization of the cortical layer. The cortical granule membranes and egg surface membrane fuse and vesiculate resulting in the massive discharge of granule contents. This event is accompanied by the loss of vesicular remnants of cortical ooplasm and the disruption of cytospine organization. Light and electron microscope comparisons of unreacted and reacted eggs show that the reaction results in a significant decrease in egg diameter with the oolemma of the reacted egg reorganizing in a position centripetal to its original location.

Neurosecretory granules of Hydra littoralis were isolated by differential centrifugation. Excised midsegments of hydra that were exposed to the fraction containing neurosecretory granules developed additional heads at any site, as they regenerated. Hence neurosecretory granules may contain a factot that regulates or stimulates growth in normal and regenerating hydra.

Sections of the stenotele type of nematocyst of Chlorohydra hadleyi have revealed that the stenotele, upon firing, completely everts its stylets and spines and the long, thin tubule, much as the eversion of the tubule of the nematocyst of the jewel anemone (Picken, 1953; Robson, 1953). Alternative mechanisms for supplying the energy necessary to forcefully discharge the stenotele contents are discussed as well as the possible significance of several regions containing highly ordered periodic structure. The origin of nematocysts as kinetosomal derivatives is discussed as a possibility suggested by the symmetry of the stenotele contents and the structure, location, and function of the cnidocil.