Franck Bourrat

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.

Evidence for neural stem cells in the medaka optic tectum proliferation zones

Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. [ 1999 ]: J Comp Neurol 413:385–404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid‐binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency‐associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio‐temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates.

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.

Expression of the medaka (Oryzias latipes) Ol-Rx3 paired-like gene in two diencephalic derivatives, the eye and the hypothalamus

Here we report the expression pattern of the homeobox Ol-Rx3 gene, a medaka gene homologous to the mouse, Xenopus, zebrafish and Drosophila Rx genes. Ol-Rx3 starts to be expressed, at late gastrula stages, in the presumptive territories of the anterior brain. Subsequently, transcripts are localised in an antero-ventral region of the prosencephalon and in the primordia of the optic vesicles. During organogenesis, distribution of Ol-Rx3 transcripts are gradually restricted to the floor of the diencephalon, the prospective territory of the hypothalamus and the neurohypophysis. During late development and in adult, Ol-Rx3 expression is maintained in hypothalamic nuclei bordering the third ventricle. In the optic vesicles, Ol-Rx3 expression is temporarily switched off when the eye cup morphogenesis is complete, but it is turned on again in the inner nuclear layer of the retina. Thus, the early expression pattern of Ol-Rx3 is in agreement with a conserved role in the specification of the ventral forebrain and eye field. Putative functions linked to late expression domains are discussed in light of the different hypothesis concerning the involvement of vertebrate Rx genes in the maintenance of particular cell fate.

Distribution of the orphan nuclear receptor Nurr1 in medaka (Oryzias latipes): cues to the definition of homologous cell groups in the vertebrate brain.

The orphan nuclear receptor Nurr1 has been extensively studied in mammals and shown to contribute to the differentiation of several cell phenotypes in the nervous and endocrine systems. In this study, the gene homologous to the mammalian Nurr1 (NR4A2) was isolated in the teleost fish medaka (Oryzias latipes), and the distribution of its transcripts was analyzed within brains of embryos and adults. Nurr1 has a widespread distribution in the medaka brain. Large amounts of Nurr1 transcripts were found in the intermediate nucleus of the ventral telencephalon, preoptic magnocellular nucleus, ventral habenula, nucleus of the periventricular posterior tuberculum, and nuclei of glossopharyngeal and vagus nerves. To search for homologous cell groups between teleost fish and tetrapods brains, the co‐localization of Nurr1 and tyrosine hydroxylase (TH) transcripts was analyzed. Neither Nurr1 nor TH expression was detected in the ventral midbrain, but both transcripts were present in the periventricular nucleus of the posterior tuberculum. This observation supports the hypothesis that this nucleus is homologous to dopaminergic mesencephalic nuclei of mammals. The presence of Nurr1 in the preoptic magnocellular nucleus of medaka and paraventricular hypothalamic nucleus of mammals reinforces the hypothesis of homology between these areas. TH and Nurr1 transcripts are also co‐localized, among others, in the nucleus of the paraventricular organ and nucleus of the vagus nerve. This work suggests that the differentiating role of Nurr1 in the central nervous system is conserved in gnathostomes. J. Comp. Neurol. 431:276–292, 2001.

An in situ screen for genes controlling cell proliferation in the optic tectum of the medaka (Oryzias latipes).

The optic tectum is a dorsal, prominent and well corticalised structure of the fish brain. It grows according to a pattern exceptional in the vertebrate central nervous system, by addition of radial columns of cells at its periphery. We took advantage of this peculiar feature to readily identify genes differentially expressed in the tectal proliferative (marginal) vs. post-mitotic (central) zones. Out of 500 medaka cDNA clones screened by WMISH, more than 100 were expressed in one or the other of these zones. Unexpectedly, we also identified a small class of genes expressed between these two zones. All the characterised genes of this class encode down regulators of the cell cycle. Therefore, such a screening strategy allows in particular cases to raise testable hypotheses on the involvement of genes in the control of the cell cycle, in addition to characterising unknown genes with patterned expression related to cell proliferation.

Phylogenomic analysis and expression patterns of large Maf genes in Xenopus tropicalis provide new insights into the functional evolution of the gene family in osteichthyans.

We have performed an exhaustive characterization of the large Maf family of basic leucine zipper transcription factors in vertebrates using the genome data available, and studied the embryonic expression patterns of the four paralogous genes thus identified in Xenopus tropicalis. Our phylogenetic analysis shows that, in osteichthyans, the large Maf family contains four orthology classes, MafA, MafB, c-Maf and Nrl, which have emerged in vertebrates prior to the split between actinopterygians and sarcopterygians. It leads to the unambiguous assignment of the Xenopus laevis XLmaf gene, previously considered a MafA orthologue, to the Nrl class, the identification of the amphibian MafA and c-Maf orthologues and the identification of the zebrafish Nrl gene. The four X. tropicalis paralogues display partially redundant but nevertheless distinct expression patterns in the somites, developing hindbrain, pronephros, ventral blood island and lens. Comparisons with the data available in the mouse, chick and zebrafish show that these large Maf expression territories are highly conserved among osteichthyans but also highlight a number of differences in the timing of large Maf gene expression, the precise extent of some labelled territories and the combinations of paralogues transcribed in some organs. In particular, the availability of robust phylogenies leads to a reinterpretation of previous expression pattern comparisons, suggesting an important part for function shuffling within the gene family in the developing lens. These data highlight the importance of exhaustive characterizations of gene families for comparative analyses of the genetic mechanisms, which control developmental processes in vertebrates.

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

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.

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.

Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45γ 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.