Biomedical subjects

Franck Bourrat

Publications and source records attributed to Franck Bourrat.

14 recordsLinked to original sources

PubMed2006

Medaka simplet (FAM53B) belongs to a family of novel vertebrate genes controlling cell proliferation.

The identification of genes that regulate proliferation is of great importance to developmental biology, regenerative medicine and cancer research. Using an in situ screen on a cortical structure of the medaka fish brain, we identified the simplet gene (smp), which is homologous to the human FAM53B gene. smp was expressed in actively proliferating cells of the CNS throughout embryogenesis. It belongs to a family of vertebrate-specific genes with no characterized biochemical domains. We showed that FAM53B bound 14-3-3 chaperones, as well as SKIIP proteins, adaptor proteins connecting DNA-binding proteins to modulators of transcription. smp inactivation with morpholinos led to delayed epiboly and reduced embryonic size. Absence of Smp activity did not induce apoptosis, but resulted in a reduced cell proliferation rate and enlarged blastomeres. Moreover, smp was shown to control the expression of the pluripotency-associated oct4/pou5f1 gene. We propose that smp is a novel vertebrate-specific gene needed for cell proliferation and that it is probably associated with the maintenance of a pluripotent state.

14-3-3 Proteins↗

PubMed2006

Morphological and gene expression similarities suggest that the ascidian neural gland may be osmoregulatory and homologous to vertebrate peri-ventricular organs.

Summary The central nervous system (cerebral ganglion) of adult ascidians is linked to the neural gland complex (NGC), which consists of a dorsal tubercle, a ciliated duct and a neural gland. The function of the NGC has been the subject of much debate. The recent publication of the complete genomic sequence of Ciona intestinalis provides new opportunities to examine the presence and distribution of protein families in this basal chordate. We focus here on the ascidian neuropeptide G-protein-coupled receptors (GPCRs), the vertebrate homologues of which are involved in homeostasis. In situ hybridization revealed that five Ciona GPCRs [vasopressin receptor, somatostatin receptor, CRH (corticotropin-releasing hormone) receptor, angiotensin receptor and tachykinin receptor] are expressed in the NGC of adult ascidians. These findings, together with histological and ultrastructural data, provide evidence to support a role for the ascidian NGC in maintaining ionic homeostasis. We further speculate about the potential similarities between the ascidian NGC and the vertebrate choroid plexus, a neural peri-ventricular organ.

Animals↗

PubMed2005

Cell proliferation in the developing and adult hindbrain and midbrain of trout and medaka (teleosts): a segmental approach.

We report here the histogenesis of the brainstem of the trout (Salmo trutta fario) and the medaka (Oryzias latipes) chosen as examples of teleosts with slow and fast growth, respectively. Our results reveal that the sequence of formation of brain structures is rather similar in the teleosts species examined so far, but some interspecific differences do exist in terms of brainstem maturation at particular developmental stages, such as the end of the gastrulation and hatching periods. This sequence includes the subdivision of the brainstem in different transverse segments and longitudinal zones, where morphologically discernible boundaries are observed along the caudorostral and ventrodorsal axis. The boundary formation and subsequent subdivision of the trout and medaka brainstems, together with the proliferation pattern observed by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), support a segmental model throughout the brainstem. The spatiotemporal pattern of PCNA immunoreactivity is similar in the mesencephalon and rhombencephalon of the two teleosts species studied, although proliferation centers are less clearly defined in the medaka. Moreover, the segmental appearance of the brainstem, as revealed by PCNA immunohistochemistry, is blurred earlier in the medaka than in the trout. Thus, the trout brain appears a suitable model for morphogenetic studies because it allows more gradual survey of the changes throughout development.

Age Factors↗

PubMed2005

Expression domains suggest cell-cycle independent roles of growth-arrest molecules in the adult brain of the medaka, Oryzias latipes.

Teleost fish are unique for their enormous potential to produce new neurons in the adult brain. Nevertheless, the regulation of this adult neurogenesis remains to be characterized. Does it resort to the same molecular mechanisms as those at play in embryonic development? Here, we analyse the expression of the neurogenic gene Ol-DeltaA in the brain of medaka (Oryzias latipes) embryos and adults. To determine the relationships between neurogenic and growth-arrest genes in the adult brain, we compare the expression domains of Ol-DeltaA with those of Ol-KIP and Ol-Gadd45gamma, two well-characterized genes involved in cell-cycle arrest and growth inhibition. While it is widely assumed that genes controlling cell-cycle exit show restricted expression domains next to proliferating cells (in the sites of prospective cell differentiation), we observe highly particular expression domains of Ol-KIP and Ol-Gadd45gamma not associated to proliferating areas of the adult brain, suggesting locally different and cell-cycle independent roles of these molecules in the adult brain.

Animals↗

PubMed2005

MEPD: a resource for medaka gene expression patterns.

The Medaka Expression Pattern Database (MEPD) is a database for gene expression patterns determined by in situ hybridization in the small freshwater fish medaka (Oryzias latipes). Data have been collected from various research groups and MEPD is developing into a central expression pattern depository within the medaka community. Gene expression patterns are described by images and terms of a detailed medaka anatomy ontology of over 4000 terms, which we have developed for this purpose and submitted to Open Biological Ontologies. Sequences have been annotated via BLAST match results and using Gene Ontology terms. These new features will facilitate data analyses using bioinformatics approaches and allow cross-species comparisons of gene expression patterns. Presently, MEPD has 19,757 entries, for 1024 of them the expression pattern has been determined.

Animals↗

PubMed2005

A modular cis-regulatory system controls isoform-specific pitx expression in ascidian stomodaeum.

Pituitary homeobox (pitx) genes have been identified in vertebrates as critical molecular determinants of various craniofacial ontogenetic processes including pituitary organogenesis. Accordingly, a prominent conserved feature of pitx genes in chordates is their early expression in the anterior neural boundary (ANB) and oral ectoderm, also known as the stomodaeum. Here we used the ascidian model species Ciona intestinalis to investigate pitx gene organization and cis-regulatory logic during early stages of oral development. Two distinct Ci-pitx mRNA variants were found to be expressed in mutually exclusive embryonic domains. Ci-pitx and vertebrate pitx2 genes display remarkably similar exon usage and organization, suggesting ancestry of the pitx transcriptional unit and regulation in chordates. We next combined phylogenetic footprinting, transient transgenesis, and confocal imaging methods to study the Ci-pitx cis-regulatory system, with special emphasis on the regulation of isoform-specific ANB/stomodaeal expression. Among 10 conserved noncoding sequences (CNSs) interspersed in C. intestinalis and Ciona savignyi pitx loci, we identified two separate cis-regulatory modules (CRMs) that drive ANB/stomodaeal expression in complementary spatiotemporal patterns. We discuss the developmental relevance of these data that provide an entry point to investigate the gene regulatory networks (GRNs) that position and shape oral structures in chordates.

Animals↗

PubMed2005

An automated in situ hybridization screen in the Medaka to identify unknown neural genes.

Despite the fact that a large body of factors that play important roles in development are known, there are still large gaps in understanding the genetic pathways that govern these processes. To find previously unknown genes that are expressed during embryonic development, we optimized and performed an automated whole-mount in situ hybridization screen on medaka embryos at the end of somitogenesis. Partial cDNA sequences were compared against public databases and identified according to similarities found to other genes and gene products. Among 321 isolated genes showing specific expression in the central nervous system in at least one of five stages of development, 55.14% represented genes whose functions are already documented (in fish or other model organisms). Additionally, 16.51% were identified as conserved unknown genes or genes with unknown function. We provide new data on eight of these genes that presented a restricted expression pattern that allowed for formulating testable hypotheses on their developmental roles, and that were homologous to mammalian molecules of unknown function. Thus, gene expression screening in medaka is an efficient tool for isolating new regulators of embryonic development, and can complement genome-sequencing projects that are producing a high number of genes without ascribed functions.

Aging↗

PubMed2004

Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45gamma during early embryogenesis.

Numerous studies, mostly performed on mammalian cell cultures, have implicated the Gadd45 family of small acidic proteins in cell cycle control (arrest and/or engagement in the apoptotic pathway). We report here the cloning, detailled expression pattern and functional characterisation in embryonic development of Ol-Gadd45gamma, the Oryzias latipes ortholog of mammalian Gadd45gamma. Its expression pattern, notably in the developing brain (optic tectum) strongly suggests that it is involved in cell cycle exit. Gain-of-function experiments (through mRNA injection) slowed down early development, and produced embryos clearly reduced in size, while morpholino knockdowns resulted in small embryos over-sensitive to DNA damage (UV irradiation). We further demonstrated that, following Ol-Gadd45gamma overexpression, cells are proliferation-arrested before both G1/S and G2/M cell cycle checkpoints, while in the MO-Ol-Gadd45 loss-of-function experiments cells are engaged in apoptosis rather than prevented from proliferating. These results show that Ol-Gadd45gamma is likely to play an important role in coordinating cell fate decisions during neurogenesis; they also demonstrate that the medakafish is a promising model to analyse in vivo the developmental control of the cell cycle.

Amino Acid Sequence↗

PubMed2004

Cloning and developmental expression patterns of Dlx2, Lhx7 and Lhx9 in the medaka fish (Oryzias latipes).

We have isolated three homeodomain and LIM-homeodomain developmental transcription factors from the medaka fish (Oryzias latipes): OlDlx2, OlLhx7, and OlLhx9, and we have studied their expression patterns in the developing and adult brain. This analysis showed that OlDlx2 and OlLhx7 (together with OlNkx2.1b) delineate the subpallial divisions of the medaka telencephalon, and that OlLhx9 exhibits a typical and specific topology of expression in the pallium and diencephalic neuromeres. The expression patterns of these three genes, when compared in details with those of their tetrapod homologs, reveal both commonalities and differences in the basic organization of the developing teleost and vertebrate forebrain.

Amino Acid Sequence↗

PubMed2004

Effects of microcystin-LR on development of medaka fish embryos (Oryzias latipes).

Chronic and subchronic toxicity from exposure to microcystins, cyclic hepatotoxic heptapeptides from cyanobacteria, receives increasing attention as a public human health biohazard. So far, the effects of microcystin on fish have been studied mainly in adults, rather than during early life stages. Limitations of direct ambient exposure experiments to fish egg have resulted from the difficult access of microcystin through the egg chorion. Using a microinjection technology, we have introduced microcystin-LR (MC-LR) directly into one-cell stage embryos or into the vitellus of late neurula embryos (stage 19) or into the vitellus of stage 25 embryos of medaka (Oryzias latipes) at the onset of the liver anlage. Microinjection (100 pl; stage 1 or 2 nl; stage 19 or 25) of MC-LR resulted in a dose dependent mortality of embryos. Survival rates were reduced up to 90% with microcystin concentrations of 10 or 1 microg/ml (corresponding to 1-20 pg or 0.1-2 pg of toxin injected), injected either at stages 1, 19 or 25. Also, a dose dependent advanced embryonic hatching processing was observed; hatching being brought forward from 2 or 3 days compared to controls in most of the microcystin injected groups. In agreement with the known hepatotoxic effects of microcystin, injected embryos consistently displayed hepatobiliary abnormalities such as liver hypertrophy and hepatic hemorrhage, also evidenced in post-hatching juveniles. Thus, the methodology presented in this paper should be valuable tool to analyze the effects of toxins on the development of aquatic vertebrate embryos.

Animals↗

PubMed2002

Pitx genes in Tunicates provide new molecular insight into the evolutionary origin of pituitary.

We have initiated a project aimed at documenting molecular and cellular changes underlying the emergence of the hypothalamo-hypophyseal axis in Chordates. Considering the phylogenetic position of Tunicates and the 'pan-hypophyseal' expression pattern of Pitx genes in Vertebrate pituitary, we searched for a Pitx-related homeobox gene in the ascidian Ciona intestinalis, and identified Ci-Pitx (ona intestinalis uitary homeobo gene). We also isolated Cs-Pitx and Bs-Pitx, the Ci-Pitx respective counterparts of Ciona savignyi and Botryllus schlosseri, two other Tunicate species. Ci-Pitx mRNA encodes a putative protein exhibiting the diagnostic K50-Paired-class homeodomain and a conserved C-terminal Aristaless domain. Embryonic expression pattern of Ci-Pitx revealed a conserved expression domain in the anterior neural ridge and subsequently in the pharyngeal primordium, defined in Vertebrates as the stomodeal ectomere, which encompasses the presumptive pituitary territory. This shows that expression at early steps of pituitary development is a feature of Pitx-related genes that was already present in the last common ancestor of Chordates.

Amino Acid Sequence↗

PubMed2002

Development of the pancreas in medaka.

In the present work, pancreatic organogenesis has been studied in the medaka (Oryzias latipes), a teleost fish with several advantages as an experimental system in developmental biology. We demonstrated that the pancreas develops from three primordia budding from the dorsal and ventral faces of the gut epithelium. Such buds then fuse to form a single endocrine islet surrounded by exocrine tissue. Interestingly, the endocrine tissue forms only from the dorsal bud. We next analyzed a collection of medakas that had been hybridized with cDNAs derived from an anterior brain library. We found new clones expressed in the pancreatic region demonstrating that the medaka can be used to define new genes expressed in the pancreatic region that follow a specific spatial and temporal pattern of expression.

Animals↗

PubMed2002

I-SceI meganuclease mediates highly efficient transgenesis in fish.

The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.

Animals↗

PubMed2002

Expression pattern in the antennae of a newly isolated lepidopteran Gq protein alpha subunit cDNA.

From the antennae of the moth Mamestra brassicae, we have identified a lepidopteran G protein alpha subunit belonging to the Gq family, through immunological detection in crude antennal extract and antennal primary cell cultures, followed by molecular cloning. The complete cDNA sequence (1540 bp) contains an open reading frame encoding a protein of 353 amino acids. This deduced sequence possesses all of the characteristics of the Gq family and shares a very high degree of amino-acid sequence identity with vertebrate (80% with mouse or human Gqalpha) and invertebrate subunits (varying between 60 and 87% for Gqalpha from organisms as diverse as sponge and Drosophila). The expression pattern of the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction. These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this G protein alpha subunit may be involved in the olfaction transduction process through interaction with G-protein-coupled receptors, stimulating the phospholipase C mediated second messenger pathway.

Amino Acid Sequence↗