Anna Maria Fausto

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Early treatment with Lactobacillus delbrueckii strain induces an increase in intestinal T-cells and granulocytes and modulates immune-related genes of larval Dicentrarchus labrax (L.).

Lactobacillus delbrueckii ssp. delbrueckii (AS13B), isolated from the gut of adult Dicentrarchus labrax, was administered live to developing sea bass using rotifers and Artemia as live carriers. Immune-related gene transcripts were quantified in post-larvae at day 70 post-hatch (ph) and histology, electron microscopy and immunocytochemistry of the intestinal tissue were performed at day 74 ph. Since the probiotic was orally administered the studies were focused on intestinal immunity. In treated fish gut integrity was unaffected, while the density of T-cells and acidophilic granulocytes in the intestinal mucosa was significantly higher than in controls. Probiotic-induced increases in intestinal T-cells and total body TcR-beta transcripts are first reported in fish. Significantly lower IL-1beta transcripts and a trend towards lower IL-10, Cox-2 and TGF-beta transcription were found in the treated group. Evidence is provided that early feeding with probiotic-supplemented diet stimulated the larval gut immune system and lowered transcription of key pro-inflammatory genes.

Immunoglobulin protein and gene transcripts in ovarian follicles throughout oogenesis in the teleost Dicentrarchus labrax

Transfer of immunoglobulins (IgM-like) from the female to the teleost embryo has been demonstrated but mechanisms of uptake into and storage within the eggs remain to be clarified. The monoclonal antibody DLIg3 against Dicentrarchus labrax Ig light chain revealed an active role of both follicle cells and oocytes in the Ig uptake. The primordial follicular cells showed DLIg3 immunoreactivity even at a pre-vitellogenetic stage. Early vitellogenetic oocytes (lipid vesicle stages) had DLIg3 staining of pore canals, plasmalemma and outer cortex and of their follicular cells. In protein yolk granule oocytes, DLIg3 staining was also detected within vesicles of the outer-mid cortex and juxtanuclear yolk granules; therefore, a centripetal transport of Ig throughout oocyte development is apparently carried out. Immunoelectron microscopy confirmed the presence of Ig within thecal and granulosa cells (and in the interposed basement membrane) of pre-vitellogenic and vitellogenic follicles. Thus, the transport of Ig to the egg apparently occurs also by transcytosis across the follicle cells. Igs were localised in the pore canals surronding the microvilli and in vesicles of outer-mid cortex of vitellogenic oocytes. Reverse transcription/polymerase chain reaction with primers designed for the constant region of sea bass Ig light chain detected Ig mRNA in hydrated oocytes, a smaller content in released eggs and no signal in larvae at day two post-hatching. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.

Transcription of T cell-related genes in teleost fish, and the European sea bass (Dicentrarchus labrax) as a model

In recent years the cloning of genes coding for immuno-regulatory peptides, as well as the sequencing of genomes, provided fish immunologists with a growing amount of information on nucleotide sequences. Research is now also addressed in investigating the functional immunology counterpart of nucleotide sequence transcripts in various fish species. In this respect, studies on functional immunology of T cell activities are still at their beginning, and much work is needed to investigate T cell responses in teleost fish species. In this review we summarise the current knowledge on the group of genes coding for main T cell-related peptides in fish, and the expression levels of these genes in organs and tissues. Particular attention is paid to European sea bass (Dicentrarchus labrax), a marine species in which some information on functional immunology has been obtained, and we reassume here the expression of some T cell-related genes in basal conditions. In addition, we provide original data showing that T cells purified from the intestinal mucosa of sea bass with a specific mAb, express transcripts for TRβ, TRγ, CD8α, and RAG-1, thus showing similarities with intra-epithelial leucocytes of mammals.

Characterization of sex determination and sex differentiation genes in Latimeria.

Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique “living fossils”, could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.

Intestinal T cells of Dicentrarchus labrax (L.): gene expression and functional studies.

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-β, rag-1 and TCR-β gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T-cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-β and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T-cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles.

Lymphocyte differentiation in sea bass thymus: CD4 and CD8-α gene expression studies

Different developmental stages (from eggs to 1-year-old juveniles) of the teleost fish Dicentrarchus labrax (L.) were assayed for CD4 gene expression. RT-PCR revealed the appearance of CD4 transcripts in post-larvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-alpha mRNA. Real-time PCR with specific primers quantified CD4, CD8-alpha and TCR-beta transcripts in larvae and post-larvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-beta and CD8-alpha transcripts were significantly higher (P < 0.001) than in previous stages, as CD4 transcripts compared with 51 dph (P < 0.01). High levels of TCR-beta and CD8-alpha transcripts were found in the thymus, while CD4 transcripts were lower (P < 0.05 vs. TCR-beta). In situ hybridization identified CD4 mRNAs at 51 dph, localized in thymocytes of the outer and lateral zones of the thymic glands. From 75 dph on the signal was mainly detected in the outer region, drawing a cortex-medulla demarcation. Developmental expression of CD4 and CD8-alpha almost coincided. In each adult thymic lobe CD4(+) and CD8-alpha(+) thymocytes filled the cortex. The expression patterns of CD4 and CD8-alpha largely overlap, except in the medulla, where CD4(+) thymocytes were isolated, while CD8-alpha(+) ones mainly arranged in cords. These results provide new information about the thymic compartmentalization and lymphocyte differentiation pathways in a teleost, almost demonstrating that double negative thymocytes fill the cortex giving rise to further selection in the medulla.

Yolk granules are differentially acidified during embryo development in the stick insect Carausius morosus

Abstract. Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.

Characterization of the main egg chorion proteins of the whitefish Coregonus lavaretus L. (Osteichthyes, Salmonidae)

This study characterized the eggshell morphology and proteinaceous content of unfertilized eggs of the salmonid Coregonus lavaretus. Solubilization of the purified eggshell proteins was achieved in a buffer containing 0.6 M NaCl and 1 mM 2-mercaptoethanol followed by one- and two-dimensional gel electrophoresis. Glycoproteins were detected by altinity blotting analysis using the concanavalin-A-peroxidase method in one- and two-dimensional gels. The principal glycoproteins had a molecular mass ranging from 17 to 105 kDa. Purification of a few polypeptides in the 45–55 kDa range was achieved by preparative SDS-PAGE followed by electroelution.

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so‐called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.