The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Effect of exogenous factors on the induction of spicule formation in sea urchin embryonic cell cultures

The effect of exogenous factors on the realization of the spicule formation program in two sea urchin species, Strongylocentrotus intermedius and S. nudus, has been studied in primary embryonic cell cultures derived from the blastula and gastrula stages. It has been shown that the process of spicule formation depends on the type of substrate and the composition of the medium. An original finding is that calf or horse serum necessary for spicule formation in vitro can be replaced by a complex of factors including insulin, transferrin, and lectins. Methods allowing control over the growth and differentiation of marine invertebrate embryonic cells in vitro open prospects for their application to practical problems such as the establishment of cell cultures producing certain mineral structures.     

Long-term stimulation of trout head kidney cells with the cytokines MCSF, IL-2 and IL-6: gene expression dynamics

The production of salmonid leukocyte cell lines from primary cell cultures has been attempted on several occasions, however, to date only monocyte/macrophage like cell lines exist (e.g. RTS-11 and SHK-1 cells). With the increasing number of cytokines discovered in fish in recent years, many of which are growth factors for leukocytes, we now have the possibility of using these molecules to promote leukocyte development and differentiation in culture.We have generated stable cell lines transfected with a variety of plasmids expressing cytokines (Interleukin (IL)-2, IL-6 and Macrophage Colony Stimulating Factor (MCSF)), in order to produce conditioned media rich in these cytokines. The cytokine-conditioned media were used to assess their activity and ability to support the growth of primary head kidney (HK) leukocyte cultures. Here, we describe a series of experiments aimed to determine which cell population(s) of primary HK cultures is supported and will grow in conditioned media containing MCSF, IL-2 or IL-6. For a period of 5 weeks, cells were incubated at 22 °C and media were changed every 3-4 days. Samples were taken at different time points, from freshly isolated HK cells (T0), one week post-stimulation (1-WPS), 3-WPS and 5-WPS for RNA extraction. A variety of cell lineage markers (MCSF Receptor 2 (MCSFR2) for macrophages, CD4 and CD8a for T cells and IgM heavy chain for B cells) were then analysed by real-time qPCR to study the cell population dynamics as influenced by the different recombinant cytokines in the cultures. We show here that whilst MCSF appears to drive macrophage differentiation and maintenance, IL-2 and IL-6 seem to preferentially drive lymphocyte differentiation.     

Muscle cell culture as a tool in animal growth research

Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.     

Cell cultures derived from embryos and melanoma of poeciliid fish

In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed.     

Molecular Organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural Features and Evolution of Piscine 5S rRNA Genes and Nontranscribed Intergenic Spacers

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Fish stem cell cultures

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.     

鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。     

Studies on Calpain Expression during Differentiation of Rat Satellite Cells in Primary Cultures in the Presence of Heparin or a Mimic Compound

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.     

Cell cultures derived from embryos and melanoma of poeciliid fish

Summary In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed. Dedicated to Professor Dr. C. Kosswig on the occasion of his 75th birthday. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 103, Marburg, Zellenergetik und Zelldifferenzierung), and is part of the thesis of C. K.     

In search of determinants: gene expression during gonadal sex differentiation

The diversity of inputs that guide sexual fate during development is both intriguing and daunting. In the field of fish biology, the study of sex determination is of great importance. For example, in aquaculture, sexually dimorphic growth rates and overall size leads to one sex being more marketable than the other. Moreover, for breeding purposes it is important to maintain balanced sex ratios. Furthermore, sex determination is sensitive to environmental factors, such as temperature and contaminants, which can lead to skewed sex ratios, intersexes and sterility in wild or farmed fish. The gonad is typically the first organ to exhibit morphological signs of sexual dimorphism and therefore is likely to be the primary organ system whose fate is controlled by the sex determination cues in many fish species. Additionally, the sexual fate of the gonad has been shown to fully or partially control organismal sex differentiation. Thus, understanding the genetic regulation of gonadal sex differentiation is critical in studies of fish sex determination. This review summarizes recent knowledge of genes expressed during gonadal sex differentiation in gonochoristic teleost fish. Three species are discussed, which serve as excellent model systems for probing teleost sex differentiation: the Oreochromis niloticus, Oryzias latipes and Danio rerio. The similarities and differences between gonadal gene expression in these three species and in comparison to mammals suggest conserved roles during vertebrate gonadal sex differentiation. In the future, it will be essential to develop tools to assay the function of genes expressed during gonadal sex differentiation in fish.     

Serotonin-like immunoreactivity in the epidermal club cells of teleost fishes

Summary The present immunocytochemical study concerns the distribution of serotonin in the epidermis of three species of teleost fish. Serotonin-like immunoreactivity was found in the club cells of Heteropneustes fossilis and Carapus acus but not in those from the sea eel Conger conger. This study is the first immunocytochemical identification of serotonin in the club cells of teleost epidermis. By comparing data from the literature (Zaccone et al. 1986, 1987, 1988) regarding the occurrence of serotonin and GRP/bombesin in the exocrine sacciform gland cells of piscine skin, it is worthy mentioning here that the serotonin contained in the club cells of the species studied may have the ability to affect the pheromonal or other possible functions of these cells. The presence of serotonin in these systems has been correlated with the capacity of the exocrine glands of fish skin to secrete, ectopically, amine messengers in contrast to those produced eutopically i.e. in the neuron-paraneuron system in some vertebrates (Fujita et al. 1988).     

Alkaloid production in elicited cell suspension cultures of Erythrina americana Miller

A number of species of the genus Erythrina are rich in secondary metabolites, particularly phenolics and alkaloids that exhibit interesting anti-inflammatory, anti-plasmodial, bactericidal, curariform and fungicidal activities. Unfortunately, the isolation of these compounds through the extraction of organs and seeds of whole plants is becoming more difficult since the natural growing areas of many of the species, and particularly of Erythrina americana, are being urbanised. Plant tissue culture not only constitutes a viable method through which to preserve the species, but may also represent a constant and stable source of target alkaloids. Currently, however, in vitro systems, and especially cell suspension cultures, only accumulate low levels of the desired products. A number of strategies for increasing production have been proposed, the most successful of which involves elicitation of suspension cells with plant growth regulators. In this context, excellent results have been reported following elicitation of cell cultures derived from different species and genera with jasmonic acid and methyl jasmonate. The present paper provides a brief review of the novel approaches to the use of Erythrina alkaloids that have recently been described, and of the advances that have been made in the formation of tissue cultures of E. americana and their subsequent elicitation in attempts to augment alkaloid accumulation.     

Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation

A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic fibroblast growth factor (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell growth was examined. Cells grew well between 24 and 30 degrees C, but had a reduced growth rate below 18 degrees C. The growth rate of FEC cells in medium containing 15% FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the growth rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.     

Primary culture of insect midgut cells

This protocol describes the preparation of primary cell cultures from Lepidopteran midgut. These cultures have been used to identify factors that control midgut growth and differentiation, cell responses to these factors, effects of toxins on midgut growth, and the regulation of cell physiology. The protocol is divided into (1) procedures for cell collection, (2) composition of the culture, and (3) assay methods used for cell health, proliferation, and differentiation. Collection and setup require 4–6 h. Once established, a culture can survive several months at 25°C, be kept a year or longer at 4°C, or be frozen for indefinite storage.     

In vitro culture of <Emphasis Type="Italic">Taxus</Emphasis> sp.: strategies to increase cell growth and taxoid production

Interest in Taxus species has increased since paclitaxel, an anticancer drug, was isolated from the bark of Taxus brevifolia (Pacific yew) in the 1960’s. Great effort has been carried out to establish an efficient callus cultures of Taxus species. Culture media must be optimized for each Taxus species, and in general, there is no one method that guarantees success for Taxus cultures. The source of explant, culture medium composition and the growth regulators used appear to affect callus initiation and maintenance. Research effort has focused on obtaining a cell culture that exhibits good growth and a high rate of taxoid accumulation. In this sense, many strategies have been employed to stimulate taxoid production without affecting cell growth. In an attempt to scale-up cell culture, problems such us shear stress, oxygen supply and gas composition have been studied. A more detailed knowledge of the pathway and the fluxes of intermediates towards taxane accumulation will be key factors to obtain cell lines with increased taxane accumulation through metabolic engineering.     

The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions

The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.     

Parasite species coexistence and limiting similarity: a multiscale look at phylogenetic,functional and reproductive distances

The factors that control biodiversity have been the focus of numerous recent investigations; these include species interactions, speciation, environmental gradients and heterogeneity, all of these operating differently at each observation scale. We used a null model to examine the influence of two forces shaping the community structure of Dactylogyrus species parasitic on roach (Rutilus rutilus) gills: interspecific competition that might prevent the coexistence of the most similar species and environmental filters that might result in the most similar species coexisting together. The study was carried out on two sets of fish from two different localities in the Morava river basin (Czech Republic) to evaluate the consistency of the results across host populations, and at three different scales of observation to test for the scale dependence of assembly rules. In decreasing order, from largest to smallest, the three spatial scales investigated were: the individual fish, the individual gill arch and individual sections of each gill arch. The similarity between pairs of parasite species was measured using three different criteria: (1) phylogeny, (2) quantitative functional traits consisting of parasite size and morphometric measurements of the attachment organ’s sclerotized parts and (3) qualitative attributes of reproductive organs. First, our study reveals a strong conservatism of ecological characters for the nine Dactylogyrus parasite species, in particular regarding the attributes of their copulatory organs. Second, our study did not find any limitation of similarity among coexisting Dactylogyrus species due to interspecific competition, irrespective of the scale and the similarity measures considered. Conversely, our results support the niche filtering hypothesis, preventing the co-occurrence of species too dissimilar from one another. This process is particularly apparent at the scale of the individual fish and for functional traits associated with the hard parts of the parasites’ attachment organs. In both localities, the Dactylogyrus species that occur on the same fish individuals tend to have similar values for the haptor dimensions. Our study supports previous studies on monogenean parasite communities indicating a weak influence of competition as a structuring force, but it goes a step further by identifying environmental filtering as a key process shaping these communities.     

Species of Eimeria in Nonepithelial Sites1

In a few homoiothermic host species, Eimeria spp. in a schizogonic stage, a gamogonic stage, or as resting sporozoites infect nonepithelial tissues. However, epithelium serves as a critical site in the cycle of these and all other species known from warm-blooded hosts. Eimeria funduli, infecting at least four different killifishes, undergoes both schizogony and gamogony in non-epithelial hepatic and pancreatic cells and requires an invertebrate host to complete its cycle. Oocysts are not released from the living fish into the environment. The cycle for this species, or aspects of it, may exemplify those found in several piscine species infecting nonepithelial (and possibly epithelial) cells. Eimeriu funduli differs from most species of Eimeria infecting homoiotherms in other respects, some of which may also characterize traits for piscine and other poikilotherm eimerians in general. Endogenous development is affected by temperature and host-age, sporogony occurs in the host, and infections occur in several related fishes. The presence of an intermediate host in at least E. funduli and the presence of sporozoites in macrophages of several eimerians of homoiotherms, as well as other features, suggest a closer relationship between eimeriids and Lankesterella, Schellackia, and even the Haemosporina than previously assumed.