An electron microscope study of primary epidermis formation in freshwater planarian embryos

Early development in freshwater planarians is generally considered to be highly modified to the point of being unique. A careful examination by TEM, however, suggests that the primary epidermis (Skaer, 1965) is formed in a rather regular manner but is partially inverted with respect to the definitive body axes. After formation of the yolk cell syncytium, the blastomeres enclosed within it increase in number in the central area. Some of these blastomeres then move peripherally as a group and fuse to form another syncytium, the primordium of the primary epidermis. This primordium contacts the surface of the yolk-cell syncytium at the place where the primordium will subsequently flow out. The primordium spreads to the opposite pole through the spaces among the syncytial and non-syncytial yolk cell masses.     

Dedifferentiation and redifferentiation of the penis of the freshwater planarian Bdellocephala brunnea

Following decapitation of Bdellocephala brunnea Ijima et Kaburaki and subsequent repeated removal of regenerating head-blastemas, the copulatory apparatus degenerated within 40–60 days. The copulatory apparatus disintegrated into cell clusters that further divided into separate neoblast-like cells that, in turn, eventually transformed into ordinary parenchymal cells by 60 days after decapitation. When the head of worms with a degenerate copulatory apparatus was allowed to regenerate by discontinuing excision of the head blastema, a new copulatory apparatus differentiated, ostensibly from the dedifferentiated cells. The neoblast-like cells appear to participate in the redifferentiation of the penis and of other parts of the copulatory apparatus.     

Formation of malformed pharynx and neoplasia in the planarian Bdellocephala brunnea following treatment with a carcinogen

The effect of benzo(a)pyrene on regeneration of the pharynx in Bdellocephala brunnea Ijima et Kaburaki was studied by inserting a 5-µg crystal of this carcinogen into the pharyngeal cavity of worms from which the pharynx had been extirpated 2 d previously. Multiple pharynges formed in the regenerates, and neoplasia was induced in a few cases. The malformed pharynges appeared as protrusions overlapping the anterior part of the main pharynx. Electron microscopy of the pharynx in normal, intact worms shows eight layers comprising inner and outer ciliated epithelia, muscle, and glands.     

Detection and changes in levels of testosterone during spermatogenesis in the freshwater planarian Bdellocephala brunnea

It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.     

Separation of planarian neoblasts based on density gradient centrifugation

For highly purified preparations of neoblasts, density gradient centrifugation in Percoll solutions (Pertoft et al., 1978) was applied to cell suspensions obtained by disintegrating Dugesia polychroa (Schmidt) in culture medium contained in a Dounce homogenizer (tolerance: 50 µm; one animal 12 mm in length per ml). To reduce the high viscosity caused by mucus, 0.00063% (w/v) of dithiothreitol was added during disintegration and purification. Based on previous experiments (Schürmann & Peter, 1988), five media were compared.For prepurification, four washing steps (differential centrifugations at 500 × g for 5 min each) were followed by subsequent filtration through a series of nylon gauzes (40, 30, 20 and 15 µm mesh size) and a final washing step. The resulting cell suspensions were then fractionated by isopycnic centrifugation (500 × g, 45 min) in one continuous (1.018–1.121 g ml^–1) or one of seven different discontinuous Percoll gradients (Schürmann, 1993). The best yield and highest purity of neoblasts in one fraction was obtained with a four step gradient (1.03–1.09 g ml^–1): the neoblasts (purity: 91%) were concentrated in one sharp band at the boundary between the densities 1.05 and 1.07 g ml^–1. The spherical cells (diameter from 10 to 13 µm in vivo) stained as typical neoblasts (Pedersen, 1959).Primary cultures were obtained with all media. The medium developed by Teshirogi and Tohya (1988) and its isotonic modification (Schürmann, 1993) proved best, resulting in 86% of viable cells without signs of differentiation after 17 days of culture at 18 °C, with still 46% being left after 31 days. Earlier reports state that isolated neoblasts only survive for 4 days (Betchaku, 1967) and total planarian cell suspensions only 2–3 weeks (Teshirogi & Tohya, 1988).     

The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

Differential expression of parental genomes during formation of embryonic organs in the early development of the fernMarsilea quadrifolia

This paper reports some experimental results related to the expression of parental genomes during the development of the water fernMarsilea quadrifolia L., a plant with graduated heteroblastic development. We found a non-random segregation of the original chromatids of the paternal genome, as shown by autoradiography of embryo sections after fertilization of eggs with [³H]thymidin-tagged sperm. From the 16-cell stage on, label is mainly concentrated in the sectors where apical cells will differentiate, and later in or near the apical areas of the young organs. Similar results had been found previously inMarsilea vestita. We also observed aberrations in primary organ development after fertilization with sperm treated with actinomycin, ethidium bromide, hydroxyurea, bromodeoxyuridin, or chlorambucil. The growth of the first organs was quite normal, indicating that they develop under the control of the maternal genome. The subsequent organs displayed abnormal development, indicating that they are under the control of both parental genomes.     

Regenerative and reproductive capacities of the fissiparous planarian Dugesia tahitiensis

A pilot study was performed to assess the regenerative capacities of Dugesia tahitiensis Gourbault, 1977, an exclusively fissiparous planarian species. Animals measuring 9.5–12.5 mm in length were used. Head regeneration rate determined by the appearance of eye spots (Brødsted, 1969: 29–46) was extremely high: at 23 °C, it took 43–59 h to regenerate clearly discernible eye spots in 28 specimens. For comparison, 3.8 days were reported for the regeneration of eye spots in 11.9–12.7 mm long D. tigrina (Girard) at 24 °C (Mead, 1985). As all posterior fragments regenerated a head, irrespective of the cutting level, D. tahitiensis seems to match the Phagocata velata (Stinger) type with a head frequency of 100% at every level (Teshirogi et al., 1977; cf. also Brøndsted, 1969:30).     

Gene expression and pattern formation during early embryonic development in amphibians

Temporal and spatial gene expression and inductive interactions control the establishment of the body plan during embryogenesis in invertebrates and vertebrates. The best-studied vertebrate model system is the amphibian embryo. Seventy-five years after the famous organizer experiment of Hans Spemann and Hilde Mangold in 1924 our knowledge of the molecular mechanisms of the multi-step formation of embryonic axis has substantially improved. Although in the 30s and 40s the interest of many laboratories was focussed on neural induction (determination of the central nervous system), only crude factors from so-called heterogeneous inducers (liver, bone marrow, etc.,) could be isolated by the traditional biochemical techniques available at this time. An important breakthrough was the characterization and purification of a mesoderm inducing factor, the so-called vegetalizing factor (homologous to Activin) in highly purified from chicken embryos. Much later after the introduction of molecular techniques Vgl and Activin (both belonging to the TGF-β family) and FGFs could be identified as important factors for mesoderm formation. It was in the 90s that secreted neuralizing factors (chordin, noggin, follistatin and cerberus) could be detected, which are expressed at the dorsal side of the early embryo including the Spemann organizer. In contrast to the classical view, these proteins act as antagonists to factors like BMP-4 localized on the ventral side. Of special interest was the fact that inDrosophila sog, homologous to chordin, determines the ventral side, whiledpp, homologous toBMP-4, participates in the formation of the dorsal side. These data of evolutionary conserved genes in both invertebrates and vertebrates support the view that they are descendents of common ancestors, the urbilateralia, living around 300 million years ago. The expression of those genes coding for secreted proteins is closely related to inductive interactions between cells and germ layers. Recently it was shown that planar signals are not sufficient to generate a specific anterior/posterior pattern during the primary steps of neural induction, i.e., formation of the central nervous system in amphibians.     

Viral contamination of a mosquito cell line,Aedes albopictus,associated with syncytium formation

Summary Viral contamination associated with syncytium formation in two sublines of Singh’sAedes albopictus cell cultures was investigated. Electron microscopy of the syncytia revealed the presence of five different types of virus-like particles, which morphologically resembled the parvo-, picorna-, toga-, and orbi-, and bacterial viruses. When a virusfree subline of theA. albopictus cells (SL3) was inoculated with extracts of the syncytium-formingA. albopictus cells, the parvo-, toga-, and orbi-type viral agents were consistently observed. Among these three agents, the togavirus-type agent is most likely responsible for the syncytium induction. Serological examination of the infected cell extract indicated that at least one of three virus-like agents, presumably the togavirus-type agent, was related to Chikungunya, O’nyong-nyong, and Western equine encephalomyelitis viruses (alphaviruses of the Togaviridae), but separable from these. Supported, in part, by USPHS-NIH General Research Grant No. PH S 5 SO1 RRO5679-06 from U.S. Pubic Health Service, Washington, D.C., to Boyce Thompson Institute.     

非洲爪蟾早期胚胎发育中的Wnt信号

非洲爪蟾是脊椎动物胚胎发育研究中的几种重要模式生物之一,为揭示早期胚胎发育中的分子调控机制做出了显著的贡献.其中一个重要的发现就是细胞信号通路在胚胎发育中起到非常关键的调控作用.本文简单介绍Wnt信号在爪蟾早期胚胎发育不同时期的几种调控作用.     

Changes in carbohydrates and lipids during embryonic development ofAntheraea mylitta (Lepidoptera)

Changes in carbohydrate and lipid metabolism during embryonic development inAntheraea mylitta were studied. While carbohydrates were metabolized during early embryogenesis, lipids were catabolised at the later stages. A significant increase in both total carbohydrates and glycogen on days 5 and 6 suggested the concurrent occurrence of both gluconeogenesis and glycogenesis. As the development of the embryo proceeds, both lipids and carbohydrates were utilised, resulting in the increase in the concentration of citrate, pyruvate and lactate.     

Analysis of pattern formation during embryonic development of Hydractinia echinata

Summary Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.     

Studies on the speciation of the European freshwater planarians Polycelis nigra and Polycelis tenuis based on the analysis of enzyme variation by means of iso-electric focusing

Polycelis nigra Ehrenberg and Polycelis tenuis Ijima differ morphologically and karyologically. No difference, however, was found in the isozyme pattern of malate dehydrogenase and tetrazolium oxidase, indicating a close relationship. Most sibling species differ at half of the loci.It could be deduced that the reproductive behaviour of a single population of Polycelis nigra in a Dutch pond was not panmictic. Two genetically different strains retained their identities during two years of observation. If pseudogamy occurs in this diploid planarian, the presence of heterozygous specimens indicates the absence of a true meiosis.The iso-electrofocusing technique by which these population-genetical studies were carried out, also lends itself to a comparison of overall protein banding patterns. The membrane proteins especially are conservative. The sodium dodecyl sulphate extracted proteins of Polycelis nigra-tenuis, Planaria torva and Phagocata vitta were very similar, while their water soluble proteins were not. This technique may be of great help in taxonomic studies of the higher taxa.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Paraquat-induced oxidative stress response during amphibian early embryonic development

In addition to the endogenous production of reactive oxygen species (ROS) as a result of normal development, amphibian external development often forces embryos to deal with oxidative stress-producing agents present in the environment. Embryos should therefore develop protective systems to reduce ROS toxicity and achieve successful development. The present work was aimed to characterize the effects produced by the widespread-used ROS-generator pesticide Paraquat during early embryonic development in the toad Chaunus arenarum, as well as to get insights into the defense response elicited by amphibian embryos. The approach consisted in generating a sharp and brief oxidative stress condition early during embryonic development to stimulate the cellular mechanisms involved in ROS-antioxidant response. Results revealed that Paraquat-treatment reduced the ability of embryos to develop normally, leading to arrests of development and severe malformations such as tail abnormalities, abdominal edema, reduced head development and curved dorsal structures. Although Paraquat effects were morphologically evident from gastrula stage on, alterations such as chromatin condensation were observed even at blastula stage by histological examinations. Regarding detoxifying enzymes, a significant induction of Mn-superoxide dismutase activity was detected at stages beyond gastrula in embryos surviving Paraquat treatment, suggesting a major role of this enzyme in the antioxidant response during early embryonic development.     

Distribution,abundance, and size of rhabdoids in Dugesia polychroa. (Turbellaria: Tricladida)

The ontogenetic changes in the distribution, abundance, and size of rhabdoids were examined in an undisturbed laboratory population of Dugesia polychroa. Irrespective of triclad age, rhabdoids in the epidermis and parenchyma were more abundant on the dorsal than the ventral side of the body. No significant differences were found in the abundance of epidermal or parenchymal rhabdoids among the anterior, medial, and posterior regions of the body. Rhabdoid number and size changed significantly with triclad age, with a marked depression coinciding with the onset of cocoon production. Rhabdoid discharge was correlated with physical and/or physiological disturbance and occurred in the absence of any overt environmental disturbance. Simple allometric relationships were observed between rhabdoid size and number on one hand and body plan area on the other. Different allometric trends were observed from field-collected individuals compared to the undisturbed laboratory population. The potential function of rhabdoids in the Tricladida is discussed in light of these findings.