Mechanically Stable Ultrathin Layered Graphene Nanocomposites Alleviate Residual Interfacial Stresses: Implications for Nanoelectromechanical Systems.

Advanced nanoelectromechanical systems made from polymer dielectrics deposited onto 2D-nanomaterials such as graphene are increasingly popular as pressure and touch sensors, resonant sensors, and capacitive micromachined ultrasound transducers (CMUTs). However, durability and accuracy of layered nanocomposites depend on the mechanical stability of the interface between polymer and graphene layers. Here we used molecular dynamics computer simulations to investigate the interface between a sheet of graphene and a layer of parylene-C thermoplastic polymer during large numbers of high-frequency (MHz) cycles of bending relevant to the operating regime. We find that important interfacial sliding occurs almost immediately in usage conditions, resulting in more than 2% expansion of the membrane, a detrimental mechanism which requires repeated calibration to maintain CMUTs accuracy. This irreversible mechanism is caused by relaxation of residual internal stresses in the nanocomposite bilayer, leading to the emergence of self-equilibrated tension in the polymer and compression in the graphene. It arises as a result of deposition-polymerization processing conditions. Our findings demonstrate the need for particular care to be exercised in overcoming initial expansion. The selection of appropriate materials chemistry including low electrostatic interactions will also be key to their successful application as durable and reliable devices.

Hydractinia, a pioneering model for stem cell biology and reprogramming somatic cells to pluripotency.

Hydractinia, a representative marine colonial hydroid, was the first organism in the history of biology in which migratory precursors of germ cells were described and termed "stem cells" (Weismann, 1883). These stem cells, now known as interstitial cells (i-cells), are thought to remain pluripotent throughout their life. Using animals depleted of their own stem cells and repopulated with allogeneic mutant donor stem cells, it was shown that Hydractinia i-cells differentiate into any cell type including epithelial cells and germ cells that express germ line markers such as Vasa, Piwi and Nanos. In Hydra, i-cells also provide germ cells and somatic cells with the exception of epithelial cells. The latter derive from two subpopulations of differentiated epithelial cells with self-renewal capacity. In Hydractinia, forced expression of the Oct4-like transcription factor, Polynem (Pln), in epithelial cells transforms them into stem cells that develop neoplasms. I-cells express the Wnt-receptor Frizzled and are Wnt responsive. Activation of Wnt signaling induces the production of numerous nematocytes (stinging cells) and nerve cells. In parallel, supernumerary tentacles develop. I-cells also express Myc and Nanos. Their misexpression causes severe developmental defects. Hydractinia polyp buds arise from aggregating stem cells, in contrast to Hydra buds, which derive from evaginating epithelial cells. Wnt activation increases budding frequency and the emergence of ectopic head structures. The potential of stem cells to invade neighbors may have provided selection pressure for the evolution of allorecognition and histo-incompatibility. Hence, Hydractinia have now attained the position of a powerful model in stem cell research, axis formation and allorecognition.

Wnt signaling in hydroid development: formation of the primary body axis in embryogenesis and its subsequent patterning.

We have studied the role the canonical Wnt pathway plays in hydroid pattern formation during embryonic development and metamorphosis. Transcripts of Wnt and Tcf were asymmetrically deposited in the oocyte and subsequent developmental stages, marking the sites of first cleavage, posterior larval pole and the upcoming head of the metamorphosed polyp. To address the function of these genes, we activated downstream events of the Wnt pathway by pharmacologically blocking GSK-3beta. These treatments rendered the polar expression of Tcf ubiquitous and induced development of ectopic axes that contained head structures. These results allow concluding that Wnt signaling controls axis formation and regional tissue fates along it, determining one single axis terminus from which later the mouth and hypostome develop. Our data also indicate Wnt functions in axis formation and axial patterning as in higher metazoans, and thus point to an ancestral role of Wnt signaling in these processes in animal evolution.

An evolutionary conserved role of Wnt signaling in stem cell fate decision.

Wnt/Frizzled/ss-catenin-based signaling systems play diverse roles in metazoan development, being involved not only in the establishment of body axes in embryogenesis but also in regulating stem cell fate in mammalian post-embryonic development. We have studied the role the canonical Wnt cascade plays in stem cell fate determination in Hydractinia, a member of the ancient metazoan phylum Cnidaria, by analyzing two key molecules in this pathway, frizzled and ss-catenin, and blocking GSK-3. Generally, frizzled was expressed in cells able to divide but absent in post-mitotic, terminally differentiated cells such as nerve cells and nematocytes. Transcripts of frizzled were identified in all embryonic stages beginning with maternal transcripts in the oocyte. Following gastrulation and in the planula larva, frizzled expression concentrated in the central endodermal mass from which the first interstitial stem cells and their derivatives arise. In post-metamorphic development, high levels of frizzled transcripts were detected in interstitial stem cells. Activating downstream events of the Wnt-cascade in the post-metamorphic life phase by blocking GSK-3 with paullones induced recruitment of nematocytes and nerve cells from the pool of interstitial stem cells. Terminal differentiation was preceded by an initial burst of proliferation of frizzled-positive i-cells. In activated i-cells, ss-catenin appeared in the cytoplasm, later in the nucleus. It was subsequently again observed in the cytoplasm and eventually faded out during terminal differentiation. Our results suggest an ancient role of Wnt signaling in stem cell fate determination.

Totipotent migratory stem cells in a hydroid.

Hydroids, members of the most ancient eumetazoan phylum, the Cnidaria, harbor multipotent, migratory stem cells lodged in interstitial spaces of epithelial cells and are therefore referred to as interstitial cells or i-cells. According to traditional understanding, based on studies in Hydra, these i-cells give rise to several cell types such as stinging cells, nerve cells, and germ cells, but not to ectodermal and endodermal epithelial cells; these are considered to constitute separate cell lineages. We show here that, in Hydractinia, the developmental potential of these migratory stem cells is wider than previously anticipated. We eliminated the i-cells from subcloned wild-type animals and subsequently introduced i-cells from mutant clones and vice versa. The mutant donors and the wild-type recipients differed in their sex, growth pattern, and morphology. With time, the recipient underwent a complete conversion into the phenotype and genotype of the donor. Thus, under these experimental conditions the interstitial stem cells of Hydractinia exhibit totipotency.

Patterning a multi-headed mutant in Hydractinia: enhancement of head formation and its phenotypic normalization.

In a mutant strain of Hydractinia (Cnidaria: Hydrozoa), the polyps develop ectopic supernumerary tentacles and heads (hypostomes) after an initial phase of wild-type growth. In order to elucidate the molecular mechanisms implicated in the development of aberrant phenotypes, we tried to enhance or suppress the expressivity of this hypomorphic mutation by exposing subclones to factors supposedly influencing pattern formation. Upon iterated treatment with alsterpaullone, an inhibitor of GSK-3, the formation of additional, ectopic head structures and the budding of new polyps were dramatically accelerated and enhanced. The endogenous stolon-inducing factor (SIF) had opposite effects by reducing head forming potential while increasing stolon-forming potential. SIF could be used to rescue extremely aberrant phenotypes. In these mutant colonies, long polyps with multiple heads eventually detach from stolons and lose the ability to regenerate stolons. Upon exposure to SIF, such free-floating multi-headed polyps resumed production of stolons and acquired wild-type morphology. We conclude that a canonical WNT signaling cascade is involved in patterning the body axis of polyps and in the initiation of budding, and that SIF counteracts this signaling system.

Autoaggressive, multi-headed and other mutant phenotypes in Hydractinia echinata (Cnidaria: Hydrozoa).

In an inbreeding program conducted with the colonial hydroid Hydractinia echinata, each F1 mating produced up to 50% F2 offspring displaying an aberrant, clone-constant phenotype, hence referred to as mutant strain. In autoaggressive strains, in one or several areas of the colony autoreactive stolons direct their aggressive devices (stolon tips filled with cytotoxic stinging cells), normally used to kill allogeneic competitors for living space, towards neighboring stolons or polyps (hydranths) of their own colony. In these areas tumor-like masses of self-aggressive stolons were formed, in severe cases causing the death of the colony. Based on previous genetic studies, the interpretation proposed here attributes autoaggressive behavior to a mosaic-type alternative expression of arl (allorecognition) alleles in heterozygous individuals. Developmental mutant strains termed He-mh form supernumerary heads during regeneration and normal development as well. Common to all He-mh phenotypes isthe production of additional headsalong the bodycolumn of fully-grown polyps. The heads give rise to complete hydranths connected by a tube that derives from the gastric region of the original polyp and eventually transforms into a stolon. In bastol strains, polyps convert the basal region of their body column into a periderm-covered stolon from which the residual apical hydranth detaches. Colonies expressing both the He-mh and the bastol (bst) phenotype frequently lose detaching multi-headed hydranths and the colony disintegrates. The large number of mutant F2 offspring reveals high genetic variability in Hydractinia.

Arachidonic acid and the control of body pattern inHydra.

Repeated stimulation ofHydra magnipapillata with the diacylglycerol (DG) 1,2-sn-dioctanoylglycerol (diC8) induces an increase in positional value and eventually the development of ectopic heads. Upon stimulation, the polyps release [14C]-arachidonic acid from previously labelled endogenous sources. Arachidonic acid (AA) is not released into the external medium but remains within the animal, AA, linoleic acid and their lipoxygenase products were identified by gas chromatography-mass spectrometry. Several metabolites were found, most abundantly 12-HETE (hydroxy-eicosa-tetraenoic acid), 8-HETE, 9-HODE (hydroxy-octadecadienoic acid), and 13-HODE; this is the first evidence of their presence in coelenterates. Externally applied AA causes ectopic head formation, though less effectively than diC8. When administered simultaneously, (diC8) and AA, which both are known to activate protein kinase C (PKC), act synergistically in inducing ectopic head formation. Since released endogenous AA can spread in tissues, it may mediate a temporal and spatial extension of PKC activation and, hence, broaden the range in which positional value increases. However, in addition to the activation of PKC, the generation of AA metabolites appears to be essential for the induction of ectopic head formation, since not only a selective inhibitor of PKC, chelerythrine, but also an inhibitor of lipoxygenases, NDGA (nordihydroguaiaretic acid), significantly reduces the effectiveness of both AA and DG.

Pulses of ammonia and methylamine induce down-regulation of nematocyte and nerve cell populations in Hydrozoa (Hydra; Hydractinia).

Hydrozoa replace used-up nematocytes (cnidocytes) by proliferation and differentiation from interstitial stem cells (i cells). Repeated pulsed exposure ofHydra to elevated levels of unprotonated ammonia leads to successive loss of the various types of nematocytes: first of the stenoteles, then of the isorhizas and finally of the desmonemes. The loss is due to deficits in supply; the number of nematoblasts and differentiating intermediates is reduced. In the hydroidHydractinia the main process leading to numerical reduction was observed in vivo: mature nematocytes as well as precursors emigrate from their place of origin into the gastrovascular channels where they are removed by phagocytosis. This is a regular means by which these animals down-regulate an induced surplus of nematocytes. With lower effectiveness, pulses of methylamine, trimethylamine and glutamine also induce elimination of the nematocyte lineages. In the long term the population of nerve cells, which are permanently but slowly renewed from interstitial neuroblasts, decreases, too. After 2 months of daily repeated treatment the density of the Arg-Phe-amide-positive nerve cells was reduced to 50% of its normal level. Thus, ammonia induces down-regulation of all interstitial cell lineages. The temporal sequence of the ammonia-induced loss reflects the diverse rates with which the various i cell descendants normally are renewed.

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

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.

Signal transmission and covert prepattern in the metamorphosis of Hydractinia echinata (Hydrozoa).

Planulae are simply structured larvae lacking an overt longitudinal organization. In the course of a rapid metamorphosis, however, they transform into polyps, which display striking structural patterns. Metamorphosis takes place only in response to external stimuli. Surgical removal and transplantation of larval parts reveal that external stimuli, including artificial inducers such as cesium ions, tumor promoters and diacylglycerol, act on the anterior quarter of the larva where sensory cells containing Arg-Phe-amide-like peptides are located. The external stimuli initiate the release of an internal signal, which is transmitted to the posterior end causing the successive transformation of larval into adult tissue. The transformation front moves from the anterior to the posterior quarter in 60 min. The internal signal can be released or bypassed by a transitory lowering of the Mg2+ content of the seawater. By using this procedure, or by administering an extract containing the putative internal signal substance, each isolated part of the larva can be induced to metamorphose separately. Provided there is no time for regeneration after cutting before metamorphosis is initiated, the most anterior fragment forms only stolons, the most posterior fragment forms only a head. The overt pattern of the polyp is, therefore, generated under the influence of a covert anterior-posterior prepattern of the larva.

Quantitative analysis of an inhibitory gradient field in the hydrozoan stolon.

An inhibitory field which emanates from the mobile tips of elongating stolons of colonial hydroids has been identified and analyzed. It extends proximally with decreasing intensity for about 400-700 µm and ensures that branching sites occur at appropriate distances along the stolon. The local strengths of inhibition within the field have been measured with a new method which permits high temporal and spatial resolution. Kinetic studies reveal three characteristics. First, inhibition decays rather rapidly after removal of its source. The half life is about 30 min. Second, loss of inhibition immediately triggers initiation of future tip formation. Third, restoration and spreading of inhibition are slow processes which take 8-24 h to recover 90% of the original inhibitory levels.

Polar morphogenesis in early hydroid development: Action of caesium, of neurotransmitters and of an intrinsic head activator on pattern formation.

1) Polar pattern formation and morphogenesis during metamorphosis ofHydractinia echinata are pre-programmed by the spatial distribution of morphogenetic potencies along the body axis of the planula larva. This prepattern becomes manifest when parts of larvae are brought to metamorphosis immediately after isolation. The anterior region forms only stolons, the posterior only a head. The middle region can develop stolons as well as heads. The potencies for head and stolon formation establish gradients with opposite directions. 2) The origin of this pattern during embryogenesis and its expression in the course of metamorphosis can be influenced by chemical stimuli. Two alternative effects can be obtained: activation of head formation at the expense of stolonal growth (=oralization); hyperdevelopment of stolon tissue at the expense of head structures (=aboralization). 3) Caesium ions temporarily applied during cleavage result in larvae which complete metamorphosis with symptoms of aboralization. Cs+ ions applied during early metamorphosis (disc stage) exert an oralizing influence. The sensitive periods coincide with phases of high Na+-K+-ATPase activity. Ouabain abolishes the morphogenetic action of Cs+. 4) Strong symptoms of oralization or aboralization can also be evoked by exposing disc stages to neuropharmacological agents. Acetylcholine (carbachol) brings about oralization; serotonin causes aboralization. 5) In planulae a substance can already be traced which corresponds to the neurosecretory head activator known from adult organisms. Along with initiation of metamorphosis, head activator activity rises steeply and already approaches its final level in early metamorphosis. Activities extracted from metamorphosing larvae or various adult Cnidaria when applied to disc stages promote head formation without affecting stolonal growth. Dose response curves display optimum peaks. 6) The significance of pre-programming and of neuroid functions in polarized patterning are discussed.

[Interpersonal differentiation of the enzymatic pattern in the polymorphic hydroidHydractinia echinata]. / Interpersonale Differenzierung des Enzymaktivitäts-Musters bei dem polymorphen HydroidenHydractinia echinata.

Interpersonal differentiation between gasterozooids and gonozooids, inHydractinia echinata, is reflected by the pattern of extractable enzyme activities. With regard to their activity levels in the different hydranths the enzymes can be arranged in two groups. In the first group the specific activities are highest in gasterozooids and decline in the order gasterozooids>male gonozooids>female gonozooids. This group includes GAPDH, LDH, ICDH, and GPT. The activities of the second group are highest in female polyps and display the inverse sequence. This group comprises CS, GOT, and GLDH. When the GAPDH levels, taken as 100 pc each, are chosen as point of reference only this second sequence can be established and is now represented by MDH, ICDH, and G-6-PDH as well. 6-PGDH activity could not be determined in adult hydranths.According to the ratio of GAPDH/CS and the LDH level the gasterozooids prefer the anaerobic glycolytic pathway whereas in the sexual hydranths relatively more substrate is supplied at the disposal of the citrate cycle. Two metabolites of the citrate cycle, ketoglutarate and succinate, are known to promote the transformation of nutritive zooids into sexual zooids. The differences observed in the activities of GOT, GLDH, and ICDH, therefore, may be correlated not only with the production of gonocytes but also with the specific type of differentiation which in sexual hydranths is governed by a specific morphogen.

[Activities of enzymes of carbohydrate-metabolism and of Na+-K+-ATPase durign Embryogenesis and Metamorphosis ofHydractinia echinata (Hydrozoa)]. / Aktivitäten von Enzymen des Kohlenhydrat-Stoffwechsels und der Na+−K+-ATPase im Zuge der Embryonalentwicklung und Metamorphose vonHydractinia echinata (Hydrozoa).

1. In order to elaborate some general correlations between metabolic pathways and morphogenetic events the activity-profiles of the enzymes GAPDH, LDH, CE, MDH, GOT, G-6-PDH, and 6-PGDH have been determined in different developmental stages. Unusually high activities of these enzymes could be extracted from unfertilized eggs. In the course of embryogenesis and metamorphosis two metabolic patterns alternate repeatedly. The first pattern is characterized by a relatively low glycolytic potency (low GAPDH- and LDH-levels) connected with a relatively high oxidative capacity (low ratio of GAPDH/CE and rising O2-consumption). This pattern, which indicates the predominance of energy production, is realized during cleavage and during the phase of contraction at the onset of metamorphosis. The second metabolic pattern combines high glycolytic potency (high GAPDH- and LDH-levels) with a high ratio of GAPDH/CE. This predominance of anaeroblic metabolism is correlated with high activities of MDH and GOT. An important portion of the substrate-flux may be directed towards anabolic processes. This metabolic condition is found during gastrulation and during the middle phase of metamorphosis: stages in which differentiation is initiated. This repeated change in the main metabolic behavior is also reflected by the operation of the pentose-phosphate-cycle. The activities of G-6-PDH and 6-PGDH decrease during cleavage and during early metamorphosis and increase during the differentiation of the planula and of the primary polyp. In the fully developed polyp, however, the 6-PGDH-activity disappears whilst that of the G-6-PDH remains high. 2. The induction of metamorphosis which normally is brought about by a bacterial agent and artificially by a Cs+-pulse, is characterized by an enhanced activity of the Na+\t-K+-ATPase. The maximum activity could be measured in homogenates and in living larvae 2 hrs and 0.5\2-1.5 hrs respectively after the application of Cs+. This finding supports the hypothesis that cation carriers are involved in the larval response to inductive stimuli. The induced peak of activity, in early metamorphosis, is followed by a second peak occurring spontaneously 3-4 hrs later. Relatively high ouabain-sensitive as well as ouabaininsensitive ATPase activities could also be observed in homogenates of young embryos.

[Induction of metamorphosis in planulae : I. The bacterial inducer]. / Metamorphose-Induktion bei Planulalarven : I. Der bakterielle Induktor.

1. The metamorphosis of the planulae ofHydractinia echinata (Hydrozoa) is induced by certain marine, gramnegative bacteria which at the end of the exponential growth release a stimulating principle. 2. The stimulus is liberated by stationary cells previously cultivated at low population densities (up to 107 cells/ml) in a proper medium (e.g. extract of meat). Transfer into seawater lacking nutritive sources enhances the inductive capacity. 3. The concentration of the inducing agent normally surpasses the threshold level only in the close microenvironment of living cells. But when shocked by a drop in the osmotic pressure the bacteria discharge increased amounts which become traceable in the filtered cell-free medium. 4. Thus the inducer can be accumulated and isolated by a process of osmotic shock which does not affect the viability of the microbes. The principle belongs to a category of microbial substances which are subsumed under the comprehensive term "leakage"-products. 5. The active principle can be precipitated from the leakage solution with acetone and extracted with chloroform. The inducer seems to be an unstable, nondialyzable, polar lipid. 6. In order to evoke complete metamorphosis the isolated agent must be applied in a pulse-like fashion. Using the onset of metamorphosis as criterion for the velocity of reaction the dose-response curves display Michaelis-like saturation kinetics. At short pulses the percentages of induced metamorphoses yield a saturation curve as well. This indicates that an enzyme or carrier-system is involved in the larval response. 7. The inducing effect of the bacterial principle is antagonized by ouabain. Conversely, high doses of the isolated leakage material abolish the ouabain inhibition. The primary effect of the inducer, therefore, can be interpreted as stimulation of the active cation transport, especially of the Na+/K+-ATPase.

[Induction of metamorphosis in planulae : II. Induction by monovalent cations: The significance of the Gibbs-Donnan ratio and of the Na+/K+-ATPase]. / Metamorphose-Induktion bei Planulalarven : II. Induktion durch monovalente Kationen: Die Bedeutung des Gibbs-Donnan-Verhältnisses und der Na+/K+-ATPase.

1. The metamorphosis of the planulae ofHydractinia echinata (Hydrozoa) can be induced by a pulse-type (2-3 hrs) exposure to the ions of caesium, rubidium, lithium, and potassium. With mixtures of seawater with isoosmolar solutions of the chlorides of these ions the dose-response curves for Rb+-, Li+-, and K+-stimulation display optima peaks, the smallest effective range being shown by K+. The Cs+-curve is sigmoid shaped. The half-maximal doses are 6 mval Cs+, 50 mval Rb+, 80 mval Li+, and 130 mval K+ respectively. 2. Potassium-induced stimulation is an ouabain-insensitive event governed by the Gibbs-Donnan principle. With solutions containing only K+, Ca++, and Sucrose the optimal efficacy is always related to an invariable ratio of[Formula: see text], regardless of the actual concentrations employed. 3. The Cs+-induction is based upon active events which indicate the involvement of a carrier-like system. Indirect reaction kinetics taking the velocity of the larval response versus the dose as measure of cation uptake yield Michaelis-like saturation curves. Reduction of external calcium and sodium as well as application of the glutathione oxidizing agent diamide enhance the efficacy of Cs+. The shape of the response curve becomes closer to a rectangular hyperbola and the half-maximal concentration is shifted towards lower values. The inducing power of Cs+, Rb+, and Li+ (but not of K+) is abolished by ouabain (1 mM). These findings give evidence that the relevant ions act by stimulation of the Na+/K+-transport ATPase. 4. Thus, induction of metamorphosis by monovalent cations is due to alterations of membrane-bound functions. This applies to bacteria-induced metamorphosis as well.

Induction of metamorphosis by bacteria and by a lithium-pulse in the larvae ofHydractinia echinata (Hydrozoa).

The planulae ofHydractinia, the metamorphosis of which normally is induced by certain bacteria, will undergo transformation into polyps also when exposed to a lithiumpulse. The optimal concentration and incubation period for rapid and complete transformation have been determined at 24 mM Li+ and 2 hrs respectively. 96 mM K+ applied for 2 hrs will also result in some induction. The possible mode of action exerted by the Li-ion as compared with induction caused by bacteria is discussed.

The "polarizing inducer" in hydra: A reexamination of its properties and its origin.

In order to prove the gradient hypothesis an attempt was made to isolate and accumulate the "polarizing inducer" present in homogenates of hydra and assumed to be a neurosecretory product. By means of gel chromatography two fractions were obtained which brought about the development of supernumary apical structures (tentacles and hypostomes) thus exhibiting the symptoms attributed to this polarizing agent: a low molecular fraction with only modest effectiveness and a main fraction with strong animalizing ability. Increasing the concentration affected only the quantity but not the qualitative properties of the structures produced, a result inconsistent with the postulate of the gradient hypothesis. By analysing the chemical and biological nature of the main agent and by applying pure isolated toxins compelling evidence is given that the inducer in question is nothing but a component of the nematocyst toxins. This component, being heat-stable and trypsin-sensitive, elicites its animalizing effect in unspecific means by disturbing the normal pattern of morphallactic events. A side effect with interest in respect of graded tissue properties could be recorded: by the influence of the relevant toxin, growing together of regenerating animals occurs whereby predominantly apical primordia fuse with apical primordia, thus forming stable parabioses. This observation may indicate the significance of surface bound, contact establishing components in polar differentiation.