Larval cells of sponge Halisarca dujardini (Demospongiae, Halisarcida). II. Some cell types marked by polyclonal antibodies

The recent morphological and experimental data concerning the involvement of flagellated cells in sponge larvae are contradictory and testify to or against the germinal layers inversion. A study of morphogenetic processes in sponges, in particular larval metamorphosis, is complicated by difficulties in identification and succession of certain cell types. It is possible to trace the destiny of flagellated and other larval cells by marking them with antibodies (AB) specified for each cell type. We separated larval and adult sponge cells of Halisarca dujardini in percoll density gradient and obtained polyclonal AB for the majority of these cell types. The protein pattern of larval flagellated cells differed significantly from that of other cell types. The major proteins of flagellated, collencyte-like and spherulous cells were used to raise the corresponding AB. Immunoblot showed all AB to be specific for certain proteins and suitable for immunofluorescence. The AB for flagellated cells reacted with the apical cytoplasm, but not with the flagellum, the AB for major protein of collencyte-like cells stained cytoplasm granules. The AB for spherulous cells of the adult sponge reacted with larval spherulous cells supposed to be of maternal origin. So, the method of cell marking with specific polyclonal AB can facilitate analysis of the layers inversion problem, as well as elucidate the degree of cell differentiation in larvae, their conformity to cells of the adult sponge or their provisional destiny.     

Metamorphosis of cinctoblastula larvae (Homoscleromorpha, porifera)

The metamorphosis of the cinctoblastula of Homoscleromorpha is studied in five species belonging to three genera. The different steps of metamorphosis are similar in all species. The metamorphosis occurs by the invagination and involution of either the anterior epithelium or the posterior epithelium of the larva. During metamorphosis, morphogenetic polymorphism was observed, which has an individual character and does not depend on either external or species specific factors. In the rhagon, the development of the aquiferous system occurs only by epithelial morphogenesis and subsequent differentiation of cells. Mesohylar cells derive from flagellated cells after ingression. The formation of pinacoderm and choanoderm occurs by the differentiation of the larval flagellated epithelium. This is possibly due to the conservation of cell junctions in the external surface of the larval flagellated cells and of the basement membrane in their internal surface. The main difference in homoscleromorph metamorphosis compared with Demospongiae is the persistence of the flagellated epithelium throughout this process and even in the adult since exo- and endopinacoderm remain flagellated. The antero-posterior axis of the larva corresponds to the baso-apical axis of the adult in Homoscleromorpha.     

Metamorphosis of coeloblastula performed by multipotential larval flagellated cells in the calcareous sponge Leucosolenia laxa

The calcareous sponge Leucosolenia laxa releases free-swimming hollow larvae called coeloblastulae that are the characteristic larvae of the subclass Calcinea. Although the coeloblastula is a major type of sponge larva, our knowledge about its development is scanty. Detailed electron microscopic studies on the metamorphosis of the coeloblastula revealed that the larva consists of four types of cells: flagellated cells, bottle cells, vesicular cells, and free cells in a central cavity. The flagellated cells, the principal cell type of the larva, are arranged in a pseudostratified layer around a large central cavity. The larval flagellated cells characteristically have glutinous granules that are used as internal markers during metamorphosis. After a free-swimming period the larva settles on the substratum, and settlement apparently triggers the initiation of metamorphosis. The larval flagellated cells soon lose their flagellum and begin the process of dedifferentiation. Then the larva becomes a mass of dedifferentiated cells in which many autophagosomes are found. Within 18 h after settlement, the cells at the surface of the cell mass differentiate to pinacocytes. The cells beneath the pinacoderm differentiate to scleroblasts that form triradiate spicules. Finally, the cells of the inner cell mass differentiate to choanocytes and are arranged in a choanoderm that surrounds a newly formed large gastral cavity. We found glutinous granules in these three principal cell types of juvenile sponges, thus indicating the multipotency of the flagellated cells of the coeloblastula.     

The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini

Sponge larval flagellated cells have been known to form the external layer of larva, but their subsequent fate and morphogenetic role are still unclear. It is actually impossible to follow flagellated cell developmental fate unless a specific marker is found. We used percoll density gradient fractionation to separate different larval cell types of Halisarca dujardini (Demospongiae, Halisarcida). A total of 5 fractions were obtained which together contained all cell types. Fraction 1 contained about 100% FC and its polypeptide composition was very different to that of the other fractions. Of all larval cell types, flagellated cells displayed the lowest in vitro aggregation capacity. We raised a polyclonal antibody against a 68 kDa protein expressed by larval flagellated cells. Its specificity was tested on total protein extract from adult sponges by Western blotting and proved to be suitable for immunofluorescence. By means of double immunofluorescence using both this polyclonal antibody and commercial anti-tubulin antibodies, we studied the distribution of the 68 kDa protein in larval flagellated cells and its fate at successive stages of metamorphosis. In juvenile sponges just after metamorphosis the choanocytes and the upper pinacoderm were labelled with both antibodies. In larval flagellated cells, the 68 kDa protein was found all over the cytoplasm appearing as granules, while in adult sponges, it was present in the apical part of choanocytes in the vicinity of collars. Direct participation of the larval flagellated cells in the development of definitive structures was demonstrated.     

Three‐dimensional fate mapping of larval tissues through metamorphosis in the glass sponge Oopsacas minuta

The tissue of glass sponges (Class Hexactinellida) is unique among metazoans in being largely syncytial, a state that arises during early embryogenesis when blastomeres fuse. In addition, hexactinellids are one of only two poriferan groups that already have clearly formed flagellated chambers as larvae. The fate of the larval chambers and of other tissues during metamorphosis is unknown. One species of hexactinellid, Oopsacas minuta, is found in submarine caves in the Mediterranean and is reproductive year round, which facilitates developmental studies; however, describing metamorphosis has been a challenge because the syncytial nature of the tissue makes it difficult to trace the fates using conventional cell tracking markers. We used three‐dimensional models to map the fate of larval tissues of O. minuta through metamorphosis and provide the first detailed account of larval tissue reorganization at metamorphosis of a glass sponge larva. Larvae settle on their anterior swimming pole or on one side. The multiciliated cells that formed a belt around the larva are discarded during the first stage of metamorphosis. We found that larval flagellated chambers are retained throughout metamorphosis and become the kernels of the first pumping chambers of the juvenile sponge. As larvae of O. minuta settle, larval chambers are enlarged by syncytial tissues containing yolk inclusions. Lipid inclusions at the basal attachment site gradually became smaller during the six weeks of our study. In O. minuta, the flagellated chambers that differentiate in the larva become the post‐metamorphic flagellated chambers, which corroborate the view that internalization of these chambers during embryogenesis is a process that resembles gastrulation processes in other animals.     

Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during <Emphasis Type="Italic">Xenopus</Emphasis> metamorphosis

Background

The thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.

Results

Here, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.

Conclusions

Our findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

     

Effects of calcium antagonists, calmodulin antagonists, and methylated xanthines on polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta

We have studied the ability of fertilized eggs of Ilyanassa obsoleta to undergo polar lobe formation and cytokinesis in the presence of Ca2+ antagonists (Ca2+ channel blockers, Ca2+ uptake inhibitors). Earlier work had suggested little need for exogenous Ca2+ during these cellular shape changes. Again it appears that exogenous Ca2+ probably is not required, based on cell ability to undergo the shape changes with no, or only minor, delay in the presence of 50 mM La3+ at pH 6.5, 10 mM concentrations of Ni2+ or Co2+, 1 mM Cd2+, and 100 microM concentrations of Mn2+, papaverine, verapamil, D600, or diltiazem. In nominally Ca2+-free seawater (containing approximately 10 microM Ca2+) (CFSW), there still is no effect of Cd2+ (up to 100 microM), Ni2+, Co2+, Mn2+, or diltiazem; however, papaverine, verapamil, and D600 in CFSW cause longer delays in the shape changes than they do in the presence of normal levels of Ca2+ (SW). In 10-50 microM nifedipine, shape changes are progressively delayed to the same extent in both SW and CFSW, but more so in CFSW at concentrations above 50 microM nifedipine. Among calmodulin antagonists, trifluoperazine up to 100 microM was without effect, but chlorpromazine at 25-100 microM and calmidazolium at 50-100 microM caused substantial, concentration-dependent delays in the starting times for the shape changes. Methylxanthines caused a substantial speed-up in the starting times for both polar lobe formation and cytokinesis. The most effective of these, caffeine, at optimal concentrations of 0.7-10 mM in SW or CFSW caused shape changes to occur 12-15 min earlier than in controls undergoing a normal 50-min cycle. Caffeine is known to cause release of Ca2+ from muscle sarcoplasmic reticulum. A putative antagonist of intracellular Ca2+ mobilization, TMB-8, significantly inhibited the shape changes of the Ilyanassa cells, whereas a variety of inhibitors of exogenous Ca2+ uptake noted above did not inhibit. We conclude that Ca2+ may be necessary for polar lobe formation and cytokinesis in Ilyanassa cells, but that it may be released from intracellular, sequestered stores rather than derived from exogenous sources.     

Larval cells of sponge Halisarca dujardini (Demospongiae, Halisarcida). I. Separation of the cells and their aggregation properties

The majority of Demospongia members have a parenchymula larva, whose inner cells are similar to definite sponge cells. There are some contradictory opinions about the structure of larva in the marine sponge Halisarca dujardini: some authors deny the presence of inner cells, while other investigators consider this larva as a real parenchymula. We defined the larval cell types by cell separation in the percoll gradient and analysed their morphology and behaviour. The results showed the existence of 6 cell types in the parenchymula larva of H. dujardini, including 2 types of external flagellated cells. Each cell category differs in its morphology and aggregation ability.     

Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-alpha, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-alpha, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-kappaB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections.     

Role of flagella in adhesion of Pseudomonas fluorescens to tendon slices.

Tendon slices were used as model surfaces to investigate the role of flagella in the adhesion of Pseudomonas fluorescens to meat. The slices were introduced into a specially designed flow chamber, which was then filled with a suspension of the organism, and the tendon surface was observed at a x640 magnification. The same events that occur during the colonization of glass surfaces (apical adhesion of cells with rotation around the contact point, longitudinal adhesion, detachment of apically and longitudinally adherent cells) were also observed on tendon. Mechanical removal of the flagella resulted in no change in the contact angles with 0.1 M saline or alpha-bromonaphthalene, in the electrophoretic mobility, or in the adhesion of the organism to hydrophobic and ion-exchange resins. In addition, cells from which flagella had been mechanically removed still adhered extensively to tendon. Nevertheless, under comparable conditions (bacterial concentration, contact time), flagellated cells adhered to tendon in larger numbers than did deflagellated cells. This was entirely due to the ability of the motile flagellated cells to reach tendon in greater numbers than deflagellated cells.     

Role of flagella in adhesion of Pseudomonas fluorescens to tendon slices

Tendon slices were used as model surfaces to investigate the role of flagella in the adhesion of Pseudomonas fluorescens to meat. The slices were introduced into a specially designed flow chamber, which was then filled with a suspension of the organism, and the tendon surface was observed at a x640 magnification. The same events that occur during the colonization of glass surfaces (apical adhesion of cells with rotation around the contact point, longitudinal adhesion, detachment of apically and longitudinally adherent cells) were also observed on tendon. Mechanical removal of the flagella resulted in no change in the contact angles with 0.1 M saline or alpha-bromonaphthalene, in the electrophoretic mobility, or in the adhesion of the organism to hydrophobic and ion-exchange resins. In addition, cells from which flagella had been mechanically removed still adhered extensively to tendon. Nevertheless, under comparable conditions (bacterial concentration, contact time), flagellated cells adhered to tendon in larger numbers than did deflagellated cells. This was entirely due to the ability of the motile flagellated cells to reach tendon in greater numbers than deflagellated cells.     

THE CYTOLOGICAL EFFECT OF ECDYSTERONE ON THE MIDGUT CELLS OF THE FLESH-FLY SARCOPHAGA BULLATA

Larvae of the flesh-fly, Sarcophaga bullata, were injected with the synthetic moulting hormone ecdysterone or saline at the beginning of the third and final larval instar. One group was left untreated. The ecdysterone-injected larvae showed an increase in number of secondary lysosomes in the midgut epithelial cells similar to that observed at the onset of metamorphosis, an event which would normally occur about 48 hr later in these larvae.     

Catecholamines modulate metamorphosis in the opisthobranch gastropod Phestilla sibogae

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a factor from their adult prey, the coral Porites compressa. Levels of endogenous catecholamines increase 6 to 9 days after fertilization, when larvae become competent for metamorphosis. Six- to nine-day larvae, treated with the catecholamine precursor L-DOPA (0.01 mM for 0.5 h), were assayed for metamorphosis in response to coral inducer and for catecholamine content by high-performance liquid chromatography. L-DOPA treatment caused 20- to 50-fold increases in dopamine, with proportionally greater increases in younger larvae, so that L-DOPA-treated larvae of all ages contained similar levels of dopamine. A much smaller (about twofold) increase in norepinephrine occurred in all larvae. The treatment significantly potentiated the frequency of metamorphosis of 7- to 9-d larvae at low concentrations of inducer. In addition, L-DOPA treatment at 9 d increased aldehyde-induced fluorescence in cells that were also labeled in the controls, and revealed additional cells. However, all labeled cells were consistent with the locations of cells showing tyrosine-hydroxylase-like immunoreactivity. Catecholamines are likely to modulate metamorphosis in P. sibogae, but rising levels of catecholamines around the time of competence are insufficient alone to account for sensitivity to inducer in competent larvae.     

The development of inducer and effector immune suppressor cell function in Xenopus laevis, the South African clawed toad: the effect of metamorphosis

Thymic capacity to induce suppression of antibody production by immunized Xenopus laevis toadlet spleen fragments was tested in co-cultures for different developmental stages (Nieuwkoop, P.D. and J. Faber: Normal Table of Xenopus laevis (Daudin) (North Holland, Amsterdam) 1967). While thymuses of stages 52-54 (premetamorphosis) induced suppression, those of stages 58-62 (metamorphosis) did not. This capacity returned in metamorphic climax (stages 63-65). Tests of lectin-induced suppressor function in spleens of different developmental stages exposed the same pattern of compromised activity during metamorphosis. To test whether larval thymuses could effect suppression, rather than just induce it, antigen-activated thymuses from the different stages were co-cultured with immunized toadlet spleen fragments which had been suppressor-depleted by cyclophosphamide. Only thymuses from premetamorphic larvae suppressed. Thus, when thymic capacity to induce suppression returned in metamorphic climax, it was adult-like: it lacked effector suppressor cells.     

Adsorption kinetics of laterally and polarly flagellated Vibrio.

The adsorption of laterally and polarly flagellated bacteria to chitin was measured, and from the data obtained, a modified Langmuir adsorption isotherm was derived. Results indicated that the adsorption of laterally flagellated Vibrio parahaemolyticus follows the Langmuir adsorption isotherm, a type of adsorption referred to as surface saturation kinetics, when conditions are favorable for the production of lateral flagella. When conditions were not favorable for the production of lateral flagella, bacterial adsorption did not follow the Langmuir adsorption isotherm; instead, proportional adsorption kinetics were observed. The adsorption of some polarly flagellated bacteria exhibited surface saturation kinetics. However, the binding index (the product of the number of binding sites and bacterial affinity to the surface) of polarly flagellated bacteria differed significantly from that of laterally flagellated bacteria, suggesting that polarly flagellated bacteria adsorb to chitin by a different mechanism from that used by the laterally flagellated bacteria. From the results of dual-label adsorption competition experiments, in which polarly flagellated V. cholerae competed with increasing concentrations of laterally flagellated V. parahaemolyticus, it was observed that laterally flagellated bacteria inhibited the adsorption of polarly flagellated bacteria. In contrast, polarly flagellated bacteria enhanced the adsorption of V. cholerae. In competition experiments, where V. parahaemolyticus competed against increasing concentrations of other bacteria, polarly flagellated bacteria enhanced V. parahaemolyticus adsorption significantly, whereas laterally flagellated bacteria only slightly enhanced the process. The direct correlation observed between surface saturation kinetics, the production of lateral flagella, and the ability of laterally flagellated bacteria to inhibit the adsorption of polarly flagellated bacteria suggests that lateral flagella represent a component of bacterial structure that is important in the adsorption of laterally flagellated bacteria to surfaces. A model for adsorption events of laterally flagellated bacteria is proposed, based on the evidence presented.     

Effects of cortisol on ion regulation in developing tilapia (Oreochromis mossambicus) larvae on seawater adaptation.

The yolk diameter of cortisol-treated tilapia (Oreochromis mossambicus) larvae, immersed in freshwater (FW) containing 10 mg L-1 cortisol from 48 h postfertilization to 12 d posthatching, was significantly larger than that of control larvae after 8 d of treatment, suggesting that inhibition on larval growth occurred only after a long-term treatment with cortisol. Tilapia embryos or larvae treated with 1-10 mg L-1 cortisol for 1-2 d and then transferred to 20-30 g L-1 seawater (SW) showed reduced cumulative larval mortality in SW compared with controls. Moreover, 4-5 d of cortisol treatments significantly diminished the degree of increase in larval body Na content after the transfer to SW. Significant effect of cortisol on body Na content of larvae occurred as early as 4-8 h after the transfer to SW, while no significant difference was found in the ouabain binding of yolk-sac epithelia between control and cortisol-treated larvae even 12 h after the transfer. Cortisol may be involved in the early phase of SW adaptation in developing larvae, and this mechanism may be achieved by other means than increasing the Na-K-ATPase of yolk-sac epithelia.     

Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina

Mitani, Michiko (National Institute of Genetics, Mishima, Japan), and Tetsuo Iino. Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina. J. Bacteriol. 90:1096-1101. 1965.-The arrangement of flagella was observed by dark-field and electron microscopy in three strains of Salmonella abortivoequina, namely, normal flagellar, curly flagellar, and paralyzed curly flagellar strains. With dark-field microscopy, bundled flagella could be seen in 5 to 10% of actively moving normal or curly mutant cells. Under the electron microscope, a great many bundled flagella were observed in the curly mutant strain, but in the normal strain most of the flagella were dissociated or the bundles were rather loose and irregular. Normal flagella seem to separate easily during the process of preparation, but not the curly ones. Single flagella were found to run parallel with each other and to form a bundle consisting of five or more flagella; the bundle was spirally gyrating, with the characteristic flagellar wave. It is thought that the bundle observed with the electron microscope corresponds to that observed under the dark-field microscope. Further, the marked decrease of bundle formation in the paralyzed curly mutant cells suggests that bundle formation is not caused by curly flagellar structure per se, but corresponds to the mode of locomotion of peritrichously flagellated bacteria.     

A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella.

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.     

Timing is everything: the effects of putative dopamine antagonists on metamorphosis vary with larval age and experimental duration in the prosobranch gastropod Crepidula fornicata

The signal transduction pathway through which excess potassium ion stimulates the larvae of many marine invertebrates to metamorphose is incompletely understood. Recent evidence suggests that dopamine plays important roles in the metamorphic pathway of Crepidula fornicata. Therefore, we asked whether blocking dopamine receptors might prevent excess potassium ion from stimulating metamorphosis in this species. Surprisingly, the effects of the three putative dopamine antagonists tested (all at 10 microM) varied with exposure duration and the age of competent larvae. Chlorpromazine, a nonspecific dopamine antagonist known to have a number of other pharmacological effects, blocked the inductive action of excess potassium ion during the initial 5-8-h exposure periods in most assays, particularly for younger or smaller competent larvae. However, chlorpromazine in the absence of excess potassium ion also stimulated metamorphosis, particularly over the next 18 h, and worked faster on older competent larvae than on younger competent larvae. The specific D(1) antagonist R(+)-Sch-23309 had similar effects, blocking potassium-stimulated metamorphosis in short-term exposures and stimulating metamorphosis in longer exposures, particularly for older competent larvae. Although the specific D(2) antagonist spiperone (SPIP) blocked the inductive effects of excess potassium ion in only 1 of 6 assays during the first 6 h of exposure, it blocked metamorphosis in 2 of the assays during 24-h exposures. Our results indicate that dopamine receptors are involved in the pathway through which excess potassium ion stimulates metamorphosis in C. fornicata. In addition, the largely latent inductive effects of chlorpromazine, an inhibitor of nitric oxide synthase, suggest that endogenous nitric oxide may play a natural role in inhibiting metamorphosis in this species. Overall, our results would then suggest that exposing larvae of C. fornicata to excess K(+) leads to a shutdown of nitric oxide synthesis via a dopaminergic pathway, a pathway that can be blocked by some dopamine antagonists. Alternatively, chlorpromazine might eventually be stimulating metamorphosis by elevating endogenous cyclic nucleotide (e.g., cAMP) concentrations, again acting downstream from the steps acted on directly by excess K(+).