Werner A. Müller

Das vegetative Nervensystem

Im zentralen Hohlengrau des dritten Ventrikels (siehe Abb. 121 u. 157), liegen eine Reihe von Ganglienzellengruppen, welche offenbar der Regulation wichtiger Lebensvorgange dienen: Die Aufrechterhaltung der Korpertemperatur auf ihrer normalen Hohe wird dadurch bewerkstelligt, das bei Abkuhlung eine vermehrte Warmeproduktion und das bei Erhohung der Korpertemperatur eine vermehrte Warmeabgabe durch Erweiterung der peripherischen Blutgefase und durch Schweisproduktion vom Zentrum aus angeregt wird. Ferner geschieht die Regulation der Wasserausscheidung durch den Harn und den Schweis unter dem Einflus dieser grauen Substanz und des damit zusammenhangenden Hinterlappens der Hypophyse, bei deren Erkrankung eine ubermasige Wasserausscheidung durch den Harn zustande kommt. Spritzt man dagegen den Saft einer normalen Hypophyse subcutan ein, so vermindert sich die Harnsekretion. Durch die zentrale Regulation der Wasserausscheidung wird erreicht, das der Wassergehalt des Blutes und der Gewebe auf normaler Hohe bleibt. Im Zusammenhang damit wird auch der Kochsalzgehalt des Blutes und der Gewebssafte in dem osmotischen Bereich einer physiologischen Kochsalzlosung gehalten. Sinkt der Wassergehalt des Blutes und der Gewebe unter ein gewisses Mas, so treten Osophaguskontraktionen auf, welche das Durstgefuhl zum Ausdruck bringen.

Metamorphose-Induktion bei Planulalarven

Summary1.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.Zusammenfassung1.Die Metamorphose der Planulae vonHydractinia echinata (Hydrozoa) wird durch bestimmte marine, gramnegative Bakterien ausgelöst, die gegen Ende des exponentiellen Wachstums ein stimulierendes Prinzip freisetzen.2.Der Reiz wird nur von stationären Zellen nach Anzucht bei niederer Populationsdichte (bis zu 107 Zellen/ml) in einem geeigneten Medium (z.B. Fleischextrakt) abgegeben. Übertragung der Mikroben in Nährstoff-freies Seewasser erhöht ihre Induktionskapazität.3.Die Konzentration des Induktors übertrifft normalerweise den Schwellenwert nur in der engsten Umgebung lebender Bakterien. Nur wenn die Mikroben durch einen plötzlichen Abfall des osmotischen Drucks geschockt werden, hinterlassen sie im filtrierten Medium nachweisbare Spuren.4.Entsprechend kann das Agens durch ein osmotisches Schockverfahren, das die Mikroben am Leben läßt, angereichert und isoliert werden. Der Induktor gehört zu einer Kategorie mikrobieller Substanzen, die unter dem Begriff “leakage” -Produkte zusammengefaßt werden.5.Das aktive Prinzip kann aus der leakage-Lösung mit Aceton gefällt und mit Chloroform extrahiert werden. Es scheint ein instabiles, nicht-dialysierbares, polares Lipid zu sein.6.Um eine vollzählige und vollständige Metamorphose zu erzielen, muß der isolierte Induktor in Art eines Pulses appliziert werden. Nimmt man den Beginn der Metamorphose als Maß der Reaktionsgeschwindigkeit, zeigen die Dosis-Wirkungskurven einen Michaelisartigen Sättigungsverlauf. Bei kurzen Pulsen ergeben die Prozentsätze induzierter Metamorphosen ebenfalls eine Sättigungskurve. Dies deutet die Beteiligung eines Enzyms oder Carrier-systems bei der larvalen Reaktion an.7.Der induzierende Effekt wird durch gleichzeitige Applikation von Ouabain gehemmt. Umgekehrt kann eine hohe Dosis an leakage-Material den Ouabainhlock überrollen. Die Primärwirkung des Induktors kann somit als Stimulation des aktiven Kationentransports, speziell als Stimulation der Na+/K+-ATPase interpretiert werden.

Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming

Abstract Metamorphosis of the planula larvae into polyps does not occur spontaneously but depends on the reception of external trigger stimuli. Artificially, metamorphosis can be initiated by a pulse-type application of Cs+ or tumor-promoting phorbol esters ( W. A. Muller (1985) Differentiation 29, 216–222 ). In the present study we examined the putative involvement of the phosphatidylinositol system in signal transduction. Planulae of Hydractinia echinata were preincubated with [3H]-inositol. Upon exposure of the larvae to Cs+ the label in inositol trisphosphate (InsP3) increased twofold as early as 15 sec after addition of Cs+. Within the first 60 sec the levels of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) were also elevated compared to the values in nonstimulated larvae. After 1 and 3 hr, respectively, of incubation with Cs+, only the label in InsP2 was increased. When applied to saponin-permeabilized larvae, InsP3 did not induce metamorphosis. But 1,2-dioctanoyl-rac-glycerol (diC8) was effective in inducing metamorphosis with a half-maximal effective concentration of 9 μM. The percentage of metamorphosed animals after the application of 5 μM diC8 (30 mM Cs+) was increased by the simultaneous application of 1 μM (0.1 μM) of the diacylglycerol kinase inhibitor R 59022. The results are interpreted as evidence for the involvement of the PI-signaling/diacylglycerol transduction system in the initiation of metamorphosis of planula larvae of H. echinata.

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

SummaryPlanulae 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.

Pattern formation in the immortal Hydra

Well-documented experimental studies with Hydra, done 10-20 years before Mozart was born, marked the dawn of modern developmental biology. Since those days, the immortal and perpetually embryonic hydra has been a classic model system, but despite its deceptively simple appearance, hydra has not yielded its secrets readily. Recent evidence points to a pivotal role of PI-PKC-type signal transduction pathways in morphogenesis: interference with these pathways results in polyps with multiple heads or feet. While molecular techniques are revealing genes involved in pattern realization, a new model of pattern regulation, based on competition for hormonal factors by autoregulatory receptors, emphasizes epigenetic interactions.

Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras

To analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system.

Arachidonic acid and the control of body pattern inHydra

SummaryRepeated 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.

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

SummaryDuring 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.

Cnidarian Interstitial Cells: The Dawn of Stem Cell Research

The first stem cells described in the biological literature were those of hydroid cnidarians; their detection by Weismann in 1883 gave rise to his germ line and “germ plasm” theory (with “germ plasm” meaning what is called genome today). Somatic cells preserving properties of eggs (called Stammzellen, i.e. stem cells, by him) were considered by him to be the cellular source of regeneration and reproduction. Weismann’s studies have been the foundation of modern cnidarian stem cell research. In the latter, hydroid stem cells have been referred to as interstitial cells (shortly i-cells), and have mostly been studied in two cnidarian genera: the freshwater polyp Hydra and the colonial marine hydroid Hydractinia. In these animals, i-cells constitute a complex system of multipotent (in Hydra) or totipotent (in Hydractinia) stem cells and their derivatives. I-cells’ potencies have been investigated by specific elimination of stem cells and reintroduction of i-cells from donors. The complement of stem cells confers potential immortality to the genetic individual. Cnidarians’ cells in general have an unmatched capability of re- and transdifferentiation. Isolated, fully differentiated striated muscle cells of hydroid medusae may resume features of multipotent stem cells and give rise to almost all cell types including germ cells. Reverse development of adult stages back into juveniles is a further manifestation of cnidarian developmental plasticity. Typical i-cells have not been described in other cnidarian groups. In these taxa the source of new nematocytes nerve and germ cells may be differentiated cells that preserve plasticity. Following a historical perspective we review recent advances in hydroid i-cell research, and discuss the potential of invertebrate stem cell work.

Regeneration in Hydrozoa: distal versus proximal transformation in Hydractinia

SummaryPolyps of mature colonies of Hydractinia echinata obey the “rule of distal transformation” by regenerating heads but not stolons. However, this rule is not valid for young polyps as these regenerate stolons from proximal cut ends. Also, small cell aggregates and even small fragments excised from full-grown polyps are capable of stolon formation. Aggregates produced from dissociated cells undergo either distal or proximal transformation depending on their size, speed of head regeneration in the donor used for dissociation and the positional derivation of the cells. The latent capability of stolon formation is released under conditions that cause loss of morphogens and depletion of their sources. However, internal regulative processes can also lead to gradual proximal transformation: regenerating segments of polyps sometimes form heads at both ends and the distal pattern is duplicated. Subsequently, all sets of proximal structures, including stolons, are intercalated. In contrast to distal transformation, proximal transformation is a process the velocity of which declines with the age and size of the cell community.