Dynamics of spicule production in the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis> during in vitro cell culture and seasonal development in the field

To characterize the formation of silica spicules, the dynamics of spiculogenesis of an intertidal marine sponge Hymeniacidon perlevis (Montagu 1818) (Porifera: Demospongiae) were investigated by measuring the gene expression of silicatein (the enzyme responsible for spicule silicification) and the dimensional changes of spicules during the developmental process of individual sponges and in cell cultures of primmorphs of archaeocyte-dominant cell populations. The different developmental stages of spicules were documented by time-lapse microscopy and observed by transmission electron microscopy during a 1-month culture period. During its annual life cycle, H. perlevis has four different developmental stages: dormancy, resuscitation, bloom, and decline. Field-grown individual sponge samples at different stages were collected over 7 months (March to September 2005). The dimensions of the silica spicules from these samples were microscopically measured and statistically analyzed. This analysis and the material properties of the spicules allowed them to be classified into four groups representing the different developmental stages of spiculogenesis. Silicatein expression in the bloom stage was more than 100 times higher than that in the other stages and was correlated with the spicule developmental stage. The trend of spicule formation in field-grown sponges was consistent with the trend in cell culture. A new parameter, the maturation degree (MD) of spicules (defined as the ratio of actual to theoretical silica deposition of mature spicules), was introduced to quantify spicule development. Silica spiculogenesis during H. perlevis development was delineated by comparing MD and silicatein expression.     

The choanoderm of Sycettusa hastifera (Calcarea,Porifera) is able to generate new individuals

One of the main characteristics of sponges is their capacity for cell dedifferentiation. This capability can allow an impressive amount of asexual reproduction in these animals, because they are able to develop new individuals from just a few somatic cells. Studies of dedifferentiation, however, have focused mainly on sponges of the class Demospongiae. Therefore, we investigated here whether individuals of three different species of Calcarea are able to reconstitute new individuals following artificial fragmentation. We observed that fragmentation releases clumps of choanoderm able to initiate somatic embryogenesis. In Borojevia brasiliensis (asconoid aquiferous system, subclass Calcinea) and Paraleucilla magna (leuconoid aquiferous system, subclass Calcaronea), these clumps started to develop, but they did not pass through the first developmental phases. In Sycettusa hastifera (syconoid aquiferous system, subclass Calcaronea), the choanoderm was reorganized into primmorphs that fused to each other and formed an exopinacoderm. The first primmorphs’ spicules were triactines. Despite a large mortality rate, the primmorphs developed into olynthus stages. The somatic embryogenesis and the metamorphosis of the olynthus were similar to those observed during the sexual development of this and other calcareous sponge species. Our results show that in S. hastifera, and perhaps in other syconoid calcareous sponges, somatic embryogenesis occurs mainly from choanocytes, at least in vitro. However, primmorph development does not follow the same pattern observed in post‐metamorphic sexual development, as in that case diactines are always the first spicules to be synthesized in calcaronean species.     

Marine sponges and their microbial symbionts: love and other relationships

Many marine sponges harbour dense and diverse microbial communities of considerable ecological and biotechnological importance. While the past decade has seen tremendous advances in our understanding of the phylogenetic diversity of sponge-associated microorganisms (more than 25 bacterial phyla have now been reported from sponges), it is only in the past 3-4 years that the in situ activity and function of these microbes has become a major research focus. Already the rewards of this new emphasis are evident, with genomics and experimental approaches yielding novel insights into symbiont function. Key steps in the nitrogen cycle [denitrification, anaerobic ammonium oxidation (Anammox)] have recently been demonstrated in sponges for the first time, with diverse bacteria - including the sponge-associated candidate phylum 'Poribacteria'- being implicated in these processes. In this minireview we examine recent major developments in the microbiology of sponges, and identify several research areas (e.g. biology of viruses in sponges, effects of environmental stress) that we believe are deserving of increased attention.     

Developmental timing: let-7 function conserved through evolution

Expression of the heterochronic microRNA let-7 is tightly correlated with the onset of adult development in many animals, suggesting that it functions as an evolutionarily conserved developmental timer. This hypothesis has now been confirmed by studies in Drosophila.     

Reprogramming and epigenesis

The fact that the nucleus of a differentiated somatic cell can be reprogrammed in order to sustain embryonic development is now well established. Experiments of somatic cell nuclear transfer (cloning) have proved that a foreign nucleus introduced into an enucleated oocyte can give rise to physiologically normal offsprings, with a normal lifespan. Such evidence of genome expression plasticity is also observed experimentally with heterokaryons, created by the fusion or the nuclear transfer between two somatic cells, where differentiated nuclei are able to express genes characteristic of the host cell. However, the epigenetic mechanisms that permit nuclear plasticity remain poorly understood. In this paper we present the main evidences showing important modifications of the large scale organisation of chromosomal domains and of the DNA methylation pattern upon nuclear transfer and during the first cleavages. These modifications of epigenetic marks, brought by an intimate contact between the chromatin and the recipient oocyte cytoplasmic factors, appear essential for further development. They are established over the first cell cycles of development. The onset of embryonic genome activation and the first cellular differentiation events that occur over the implantation period are two additional check-points of reprogramming that appear to be also highly dependent on epigenetic alterations. Beyond those stages, defective placental functions might be directly responsible for the fetal and postnatal physiopathologies frequently observed in cloned animals. No direct link between preimplantation reprogramming defaults, placental dysfunctions and low development to term has been established yet. The epigenetics studies which are now used to characterise loci specific and probably genotype dependent alterations in cloned animals of different species will provide invaluable help to define the role of epigenesis in the achievement of a developmental program.     

Developmental stress increases reproductive success in male zebra finches

There is increasing evidence that exposure to stress during development can have sustained effects on animal phenotype and performance across life-history stages. For example, developmental stress has been shown to decrease the quality of sexually selected traits (e.g. bird song), and therefore is thought to decrease reproductive success. However, animals exposed to developmental stress may compensate for poor quality sexually selected traits by pursuing alternative reproductive tactics. Here, we examine the effects of developmental stress on adult male reproductive investment and success in the zebra finch (Taeniopygia guttata). We tested the hypothesis that males exposed to developmental stress sire fewer offspring through extra-pair copulations (EPCs), but invest more in parental care. To test this hypothesis, we fed nestlings corticosterone (CORT; the dominant avian stress hormone) during the nestling period and measured their adult reproductive success using common garden breeding experiments. We found that nestlings reared by CORT-fed fathers received more parental care compared with nestlings reared by control fathers. Consequently, males fed CORT during development reared nestlings in better condition compared with control males. Contrary to the prediction that developmental stress decreases male reproductive success, we found that CORT-fed males also sired more offspring and were less likely to rear non-genetic offspring compared with control males, and thus had greater overall reproductive success. These data are the first to demonstrate that developmental stress can have a positive effect on fitness via changes in reproductive success and provide support for an adaptive role of developmental stress in shaping animal phenotype.     

Fatty Acids and Other Lipids of Marine Sponges

At the present, the structure of lipids and fatty acids (FA) of almost 250 species of marine sponges has been studied. It was shown that sponges are leaders in FA diversity among aquatic animals. The great majority of the works on research of FA structure in sponges was focused on representatives of the class Demospongiae and was carried out on organisms from tropical and subtropical areas, whereas sponges from the boreal region remain poorly studied.     

Wnt signaling and induction in the sponge aquiferous system: evidence for an ancient origin of the organizer

The importance of polarity-the possession of a primary body axis-is evident in the functional features of animals, such as feeding, and therefore must have arisen simultaneously with the evolution of multicellular animal body plans. Sponges are thought to represent the most ancient extant lineage of multicellular animals and whereas adult sponges do not possess obvious polarity, they are useful study organisms in which to examine the origin and evolution of body polarity. We tested the effect of pharmacological agents known to disrupt the polarity of a wide variety of animals on sponge organization during development. Lithium chloride and alsterpaullone, which mimic canonical Wnt signaling in other animals, caused formation of ectopic oscula and disrupted the ability of the sponge to feed. Transplanted oscula were able to attach to and induce canal reorganization in host sponges suggesting that the osulum has inductive capabilities. This work suggests that canonical Wnt signaling is responsible for setting up the aquiferous system, which acts as an organizing center polarizing the sponge.     

Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos

Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the “Technique for Animal Knockout system by Electroporation (TAKE),” has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.     

Studies on the mechanisms of histidase development in rat skin and liver. II. Alterations in enzyme levels and synthetic rates during development.

The divergent postnatal developmental courses of histidase catalytic activity in rat liver and skin of the same animals, viz., a progressive rise in hepatic enzyme activity and a simultaneous decline in epidermal activity, has been shown to be accompanied by corresponding alterations in the quantities of the same enzyme protein. These tissue specific developmental changes in enzyme amount have been shown, furthermore, to be a result of selective parallel alterations in histidase synthetic rates. Rates of [³H]leucine incorporation into histidase, isolated by immunoprecipitation, relative to incorporation rates into total soluble tissue protein, increase in liver, while simultaneously decreasing in skin of the same animals during postnatal development.     

Biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach

Marine invertebrate animals such as sponges, gorgonians, tunicates and bryozoans are sources of biomedicinally relevant natural products, a small but growing number of which are advancing through clinical trials. Most metazoan and anthozoan species harbour commensal microorganisms that include prokaryotic bacteria, cyanobacteria (blue-green algae), eukaryotic microalgae, and fungi within host tissues where they reside as extra- and intra-cellular symbionts. In some sponges these associated microbes may constitute as much as 40% of the holobiont volume. There is now abundant evidence to suggest that a significant portion of the bioactive metabolites thought originally to be products of the source animal are often synthesized by their symbiotic microbiota. Several anti-cancer metabolites from marine sponges that have progressed to pre-clinical or clinical-trial phases, such as discodermolide, halichondrin B and bryostatin 1, are thought to be products derived from their microbiotic consortia. Freshwater and marine cyanobacteria are well recognised for producing numerous and structurally diverse bioactive and cytotoxic secondary metabolites suited to drug discovery. Sea sponges often contain dominant taxa-specific populations of cyanobacteria, and it is these phytosymbionts (= photosymbionts) that are considered to be the true biogenic source of a number of pharmacologically active polyketides and nonribosomally synthesized peptides produced within the sponge. Accordingly, new collections can be pre-screened in the field for the presence of phytobionts and, together with metagenomic screening using degenerate PCR primers to identify key polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes, afford a biodiscovery rationale based on the therapeutic prospects of phytochemical selection. Additionally, new cloning and biosynthetic expression strategies may provide a sustainable method for the supply of new pharmaceuticals derived from the uncultured phytosymbionts of marine organisms.     

A snapshot of the population structure of Branchiostoma lanceolatum in the Racou beach, France, during its spawning season

A methodology for inducing spawning in captivity of the lancelet Branchiostoma lanceolatum has been developed recently with animals collected at the Racou beach, in the southern coast of France. An increasing amount of laboratories around the world are now working on the evolution of developmental mechanisms (Evo-Devo) using amphioxus collected in this site. Thus, today, the development of new aquaculture techniques for keeping amphioxus in captivity is needed and the study of the natural conditions at which amphioxus is exposed in the Racou beach during their spawning season becomes necessary. We have investigated the amphioxus distribution, size frequency, and population structure in the Racou beach during its natural spawning season using multivariate methods (redundancy analysis and multiple regression). We found a clear preference of amphioxus for sandy sites, something that seems to be a general behaviour of different amphioxus species around the world. We have also estimated the amphioxus growth rate and we show how the animals are preferentially localized in shallow waters during April and June.     

The role of transgenic animals in the analysis of various biological aspects of normal and pathologic states

The introduction of foreign genes into the germ line of mammals has been a practical reality now for a number of years. This form of experimentation allows the creation of lines of animals tailor-made to answer specific molecular genetic questions. Manipulation of the mammalian embryos has been enormously important in developmental biology in recent years and that experience has brought about the possibility of new experiments allowing the molecular analysis of many biological processes. The methodologies involved in constructing transgenic animals have been published extensively in a number of comprehensive reviews. In typical experiments, pronuclear stage (one cell) embryos are collected after fertilization, but prior to the onset of cleavage. Exogenous cloned linearized DNA is injected into one of the two pronuclei by means of a finely drawn injection pipet. The manipulated embryo is transferred into the oviduct or ovarian bursal space of a surrogate mother previously mated with a sterile male. Alternative methods include retroviral transfection of cleavage stage embryos or insertion of genetically engineered embryo-derived embryonal stem cells into blastocysts. Offspring from these procedures are screened by standard molecular analyses to determine presence of the foreign genetic material. The present report explores the application of this methodology to a specific set of problems: (i) regulation of gene expression in vivo, (ii) the establishment of disease models for the study of pathogenesis, (iii) the use of exogenous genetic elements to correct specific genetic defects, (iv) the role of insertional mutagenesis in disruption of normal development, (v) analysis of genetic ablation, (iv) the use of transgenic animals to modulate carcinogens.     

Metamorphosis and fish vision

Many species of fish exhibit metamorphosis in which dramatic external transformations occur as a consequence of coordinated changes in gene expression within an organism. Because postembryonic development and change appears to be the rule rather than the exception in teleost fish species, we view metamorphosis as one of many developmental strategies in fish which have continued plasticity as a common theme. Metamorphic changes are manifested in the visual system by modification of photoreceptor peak sensitivity, rod photoreceptor cell addition, and retinal reorganization. These changes correspond to significant changes in the natural habitat of the animal and in its visual capabilities as demonstrated behaviorally. Thyroxine is the main metamorphic hormone as has also been found in amphibia. The sequence of metamorphic events occur in all teleosts, but they are compressed in time in direct developing animals suggesting that such animals might prove useful for understanding the evolution of metamorphosis in fish. It seems likely that rod photoreceptors may have evolved in conjunction with the change from larval to juvenile stage through metamorphosis in indirect developing fishes. During evolution, the contraction and/or loss of the larval stage has resulted in earlier appearance of rod photoreceptors during development although they always arise later than cone photoreceptors. This ontogenetic developmental sequence supports Walls's (1942) proposal that cones are phylogenetically older than rods and suggests that rods may have evolved several times.     

Problems of Coloniality,Modularity, and Individuality in Sponges and Special Features of Their Morphogeneses During Growth and Asexual Reproduction

A comparative analysis of the organization of sponges has been carried out to clarify problems of their coloniality, individuality, and modularity. The morphological, physiological, morphogenetic, and immunological aspects of the problem have been analyzed. The followers of the hypothesis of colonial organization of sponges interpret the process of “new zooid” formation as an “incomplete asexual reproduction.” A comparative analysis of morphogeneses in sponges during growth processes and asexual reproduction has clearly shown them to be different. A rearrangement (remodeling) of structures accompanied by disorganization and reorganization of tissues in neighboring elements of aquiferous system is the basis of growth. Migration of polypotent and secretory cells into the core of bud development is the major mechanism of budding. The formation of new aquiferous units (aquiferous modules) does not represent an “incomplete asexual reproduction.” Thus, the terms “colony” and “zooid” cannot be applied to the sponges. A morphologically separate sponge, irrespective of its level of organization (ascon, sycon, or leucon) and the number of oscula (aquiferous modules) should be considered as an individual.