Search

Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide variety of developmental processes. Here we describe the cloning of three Wnt family members from the zebrafish, Danio rerio, which encode proteins with homology to murine Wnt-2, -4 and -5A/B. The expression patterns of the latter two zebrafish genes, designated ZfWnt4 and ZfWnt5 show considerable similarity with their homologues in other vertebrates; ZfWnt2, however, is expressed in the developing viscera in a pattern distinct from its closest murine homologue. In the light of the similarities and differences in the patterns of expression of these genes relative to their homologues in other vertebrates, we speculate on their possible functions.

Vertebrate cells routinely experience small plasma membrane lesions induced, for example, by mechanical stress. It is well known that especially muscle cells possess efficient membrane self-repair mechanisms, and their failure leads to death of the myofiber, depletion of muscle stem cells and myopathy. Membrane repair involves a well-coordinated interplay among many different proteins, including dysferlin, calpains, annexins and MG53. To study these processes, the cell membrane is damaged in a controlled fashion, by applying mechanical force (with a needle or beads), by electroporation or by using focused laser illumination. Laser damage is arguably the most convenient strategy, especially in combination with fluorescence microscopy imaging of the ensuing events. Here we have used a 405-nm laser to locally damage cell membranes of thin adherent cells (HeLa cells) as well as thick tissues (zebrafish). Damage formation was observed as a function of time by using super-resolution localization microscopy on samples expressing fusion proteins with photoactivatable mEosFP markers. We have also measured changes in calcium level and reactive oxygen species by using Fluo-4 and CellROX Deep Red fluorescent indicators.

Reporting in this issue of Developmental Cell, Kizil et al. (2012) identify the transcription factor Gata3 as an important player in regeneration of the zebrafish adult telencephalon. Other injured tissues like the heart and the fin also upregulate Gata3 in response to injury, suggesting that Gata3 is a general response to tissue damage.

Neurogenesis in the developing central nervous system consists of the induction and proliferation of neural progenitor cells and their subsequent differentiation into mature neurons. External as well as internal cues orchestrate neurogenesis in a precise temporal and spatial way. In the last 20 years, the zebrafish has proven to be an excellent model organism to study neurogenesis in the embryo. Recently, this vertebrate has also become a model for the investigation of adult neurogenesis and neural regeneration. Here, we summarize the contributions of zebrafish in neural development and adult neurogenesis.

4 páginas, 1 tabla.-- Uwe Strähle ... et al., Small fresh water fishes such as the zebrafish (Danio rerio) have become important model organisms for biomedical research. They currently represent the best vertebrate embryo models in which it is possible to derive quantitative data on gene expression, signaling events, and cell behavior in real time in the living animal. Relevant phenotypes in fish mutants are similar to those of other vertebrate models and human diseases. They can be analyzed in great detail and much faster than in mammals. In recent years, approximately 2500 genetically distinct fish lines have been generated by European research groups alone. Their potential, including their possible use by industry, is far from being exploited. To promote zebrafish research in Europe, EuFishBioMed was founded and won support by the EU COST programme (http://www.cost.esf.org/). The main objective of EuFishBioMed is to establish a platform of knowledge exchange for research on small fish models with a strong focus on widening its biomedical applications and an integration of European research efforts and resources. EuFishBioMed currently lists more than 300 member laboratories in Europe, offers funding for short-term laboratory visits, organizes and co-sponsors meetings and workshops, and has successfully lobbied for the establishment of a European Zebrafish Resource Centre. To maintain this network in the future, beyond the funding period of the COST Action, we are currently establishing the European Society for Fish Models in Biology and Medicine, Our network is financed by the EU COST Action Eu- FishBioMed (BM 0804) from 2009 to 2013. The European Zebrafish Resource Center is partially funded by the Klaus Tschira Foundation (Project No. 00.170.2010) and KIT. US, RG, and LB-C are supported by the European Union’s 7th Framework Programme (EC Grant Agreement HEALTH-F4- 2010-242048, ZF-HEALTH).

Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O6-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.

SummaryMuscle cells have a remarkable capability to repair plasma membrane lesions. Mutations in dysferlin (dysf) are known to elicit a progressive myopathy in humans, probably due to impaired sarcolemmal repair. We show here that loss of Dysf and annexin A6 (Anxa6) function lead to myopathy in zebrafish. By use of high-resolution imaging of myofibers in intact animals, we reveal sequential phases in sarcolemmal repair. Initially, membrane vesicles enriched in Dysf together with cytoplasmic Anxa6 form a tight patch at the lesion independently of one another. In the subsequent steps, annexin A2a (Anxa2a) followed by annexin A1a (Anxa1a) accumulate at the patch; the recruitment of these annexins depends on Dysf and Anxa6. Thus, sarcolemmal repair relies on the ordered assembly of a protein-membrane scaffold. Moreover, we provide several lines of evidence that the membrane for sarcolemmal repair is derived from a specialized plasma membrane compartment.Video Abstract

Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sop(fixe) mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.

Genes encoding enzymes involved in gluconeogenesis are activated in liver shortly after birth by the synergistic effect of glucagon and glucocorticoids. This induction is achieved by the synergistic action of hormone responsive and liver-specific enhancer elements. In the case of glucocorticoids, this enhancer is composed of a glucocorticoid-response element (GRE) and a number of cell-specific hepatocyte nuclear factor 3 (HNF-3) binding sites. The GRE binds the ligand-activated glucocorticoid receptor (GR) which is ubiquitously expressed and the HNF-3 element binds a cell-specific protein factor. To further understand the role of cell-specific glucocorticoid signalling in the perinatal period and earlier during development we have studied the expression of the mouse GR gene. The gene has been cloned and fully characterized. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected back to day 9.5 of mouse development. The role of GR during mouse development has been further analysed by disruption of the GR gene in vivo by homologous recombination in mouse embryonic stem cells.

International audience; Dystrophin, the protein defective in Duchenne muscular dystrophy (DMD), plays a critical role in the formation and maintenance of the neuromuscular junction. In addition to dystrophin, activation of internal promoters of the DMD gene leads to the production of several short products. Among these, Dp71, which consists of the C-terminal domain of dystrophin, is the most abundant product of the gene in non-muscle tissues and brain. In this report, we compare the temporal and regional expression patterns of dystrophin and Dp71 at different stages of embryonic development and during retinal differentiation in zebrafish. The Dp71 transcripts are the earliest to be expressed at 9-10 h post-fertilization (hpf) in the axial mesoderm. As development proceeds, intense Dp71 staining is observed in the notochord, the developing brain, the marginal regions of the somites and the eye primordium. At the completion of retinal differentiation, Dp71 is expressed in the ganglion and inner nuclear layers. Transcripts encoding dystrophin have a slightly later onset of expression, 13-14 hpf, and remain restricted to the transverse myosepta through all the developmental stages examined. The complementary patterns of expression of dystrophin and Dp71 suggest that these two proteins exert different functions during embryonic development in zebrafish.

Numb is an adaptor protein implicated in diverse basic cellular processes. Using the yeast-two hybrid system we isolated a novel Numb interactor in zebrafish called NBP which is an ortholog of human renal tumor suppressor Kank. NBP interacts with the PTB domain of Numb through a region well conserved among vertebrate Kanks containing the NGGY sequence. Similar NBP and Numb morphant phenotype such as impaired convergence and extension movements during gastrulation, neurulation and epidermis defects and enhanced phenotypic aberrations in double morphants suggest that the genes interact genetically. We demonstrate that the expression of NBP undergoes quantitative and qualitative changes during embryogenesis and that the protein accumulates at the cell periphery to sites of cell–cell contact during gastrulation and later in development it concentrates at the basal poles of differentiated cells. These findings imply a possible role of NBP in establishing and maintaining cell adhesion and tissue integrity.

Background The modern coelacanth (Latimeria) is the extant taxon of a basal sarcopterygian lineage and sister group to tetrapods. Apart from certain apomorphic traits, its morphology is characterized by a high degree of retention of ancestral vertebrate structures and little morphological change. An insight into the molecular evolution that may explain the unchanged character of Latimeria morphology requires the analysis of the expression patterns of developmental regulator genes and their cis-regulatory modules (CRMs). Results We describe the comparative and functional analysis of the sonic hedgehog (shh) genomic region of Latimeria menadoensis. Several putative enhancers in the Latimeria shh locus have been identified by comparisons to sarcopterygian and actinopterygian extant species. Specific sequence conservation with all known actinopterygian enhancer elements has been detected. However, these elements are selectively missing in more recently diverged actinopterygian and sarcopterygian species. The functionality of the putative Latimeria enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos. Conclusions Latimeria shh CRMs represent the ancestral set of enhancers that have emerged before the split of lobe-finned and ray-finned fishes. In contrast to lineage-specific losses and differentiations in more derived lineages, Latimeria shh enhancers reveal low levels of sequence diversification. High overall sequence conservation of shh conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving Latimeria genome.

The telencephalon of the adult zebrafish is highly proliferative: Dividing cells are found along the entire ventricular zone and in the parenchyma. Here, we investigated the relation of proliferating cells in the telencephalic parenchyma to the oligodendrocyte lineage. We find at least three different cell types of the oligodendrocyte lineage (olig2-and sox10-positive) in the parenchyma of the telencephalon: Proliferating progenitors (PCNA-positive), including a subpopulation of slowly dividing progenitors (long term label-retaining), as well as mature oligodendrocytes (Mbp-positive) and presumptive quiescent OPCs (neither Mbp-positive nor proliferating). Furthermore, in the ventricular zone (in and ventral to the RMS), two different subpopulations of olig2-positive cell populations are present. Since these ventricular olig2-positive cells do not express the oligodendrocyte marker sox10, it is not clear whether these cells indeed belong to the oligodendrocyte lineage. Taken together, we detected at least five different classes of olig2-positive cells in the telencephalon of the adult zebrafish.

In the zebrafish spinal cord, two classes of neurons develop from the lateral floor plate: Kolmer-Agduhr′ (KA′) and V3 interneurons. We show here that the differentiation of the correct number of KA′ cells depends on the activity of the homeobox transcription factor Nkx2.9. This factor acts in concert with Nkx2.2a and Nkx2.2b. These factors are also required for the expression of the zinc-finger transcription factor Gata2 in the lateral floor plate. In turn, Gata2 is necessary for expression of the basic helix-loop-helix transcription factor Tal2 that acts upstream of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) in KA′ cells. Expression of the transcription factor Sim1, which marks the V3 interneurons in the lateral floor plate, depends also on the three Nkx2 factors. sim1 expression does not require, however, gata2 and tal2. KA′ cells of the lateral floor plate and the KA′ cells located more dorsally in the spinal cord share expression of transcription factors. The functional connections between the different regulatory genes, however, differ in the two GABAergic cell types: although gata2 and tal2 are expressed in KA′ cells, they are dispensable for gad67 expression in these cells. Instead, olig2 and gata3 are required for the differentiation of gad67-expressing KA′ cells. This suggests that the layout of regulatory networks is crucially dependent on the lineage that differs between KA′ and KA′ cells.

International audience; The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia-like cells, actively proliferating cells, and label-retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow-dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100beta), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA-NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow-cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA-positive cells in fast domains further display a morphology distinct from radial glia and co-express PSA-NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA-NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ.

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals. A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out. Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas. In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.

In this paper, an automated process to extract experiment-specific parameters out of microscope images of zebrafish embryos is presented and applied to experiments consisting of toxicological treated zebrafish embryos. The treatments consist of a dilution series of several compounds. A custom built graphical user interface allows an easy labeling and browsing of the image data. Subsequently image-specific features are extracted for each image based on image processing algorithms. By means of feature selection, the most significant features are determined and a classification divides the images in two classes. Out of the classification results dose-response curves as well as frequently used general indicators of substance's acute toxicity can be automatically calculated. Exemplary the median lethal dose is determined. The presented approach was designed for real high-throughput screening including data handling and the results are stored in a long-time data storage and prepared to be processed on a cluster computing system being build up in the KIT. It provides the possibility to test any amount of chemical substances in highthroughput and is, in combination with new screening microscopes, able to manage ten thousands of risk tests required e.g. in the REACH framework or for drug discovery.

International audience; The cis-regulatory regions of many developmental regulators and transcription factors are believed to be highly conserved in the genomes of vertebrate species, suggesting specific regulatory mechanisms for these gene classes. We functionally characterized five notochord enhancers, whose sequence is highly conserved, and systematically mutated two of them. Two subregions were identified to be essential for expression in the notochord of the zebrafish embryo. Synthetic enhancers containing the two essential regions in front of a TATA-box drive expression in the notochord while concatemerization of the subregions alone is not sufficient, indicating that the combination of the two sequence elements is required for notochord expression. Both regions are present in the five functionally characterized notochord enhancers. However, the position, the distance and relative orientation of the two sequence motifs can vary substantially within the enhancer sequences. This suggests that the regulatory grammar itself does not dictate the high evolutionary conservation between these orthologous cis-regulatory sequences. Rather, it represents a less well-conserved layer of sequence organization within these sequences.

The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish slothu45 mutant, in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly, we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly.

The signaling molecule Sonic hedgehog (Shh) is required for differentiation of the vertebrate retina. In the developing zebrafish retina, shh expression is initiated at the ventronasal region, from where it spreads as a wave through the retina. To investigate the molecular mechanism underlying this coordinated expression of shh, we mapped the cis-regulatory region and identified a novel regulatory sequence in the first intron of the shh locus. This sequence contains binding sites for the transcription factors Erm and Pea3 that are known transducers of Fgf signaling. Mutation of the binding sites or knockdown of Pea3 and Erm abolishes transgene expression, indicating that Fgf signaling regulates shh expression in the retina. We provide evidence that Fgf3 and -8 control initiation of expression, while Fgf19 is crucial for the propagation of transgene expression through the retina. Inhibitor experiments indicate a continued requirement of FGF and Hedgehog (Hh) signaling for transgene expression after initiation at the ventronasal aspect of the retina. We propose a model, in which Fgf3 and -8 initiate expression and Fgf19 and Shh signals cooperate subsequently to promote establishment of expression throughout the retina.

Genes coding for transcription factors and developmental regulators have a high likelihood to harbour cis-regulatory regions that are structurally conserved among orthologous genes in the vertebrate lineage. These regions can span up to several hundred basepairs with 70 and more percent sequence identity between fish and mammals. Even though this conservation is an efficient tool to discover cis-regulatory regions, we know little about why these specific genes maintain such highly conserved regulatory sequences. Here, we summarise work of the past few years on the regulatory modules of the sonic hedgehog and neurogenin1 genes. We will discuss the high sequence conservation of the regulatory elements in the context of models of enhancer evolution. Our data suggest that conservation of sequence does not necessarily imply a conserved function in other vertebrates.

Background Little is known about the control of the development of vertebrate unpaired appendages such as the caudal fin, one of the key morphological specializations of fishes. Recent analysis of lamprey and dogshark median fins suggests the co-option of some molecular mechanisms between paired and median in Chondrichthyes. However, the extent to which the molecular mechanisms patterning paired and median fins are shared remains unknown. Results Here we provide molecular description of the initial ontogeny of the median fins in zebrafish and present several independent lines of evidence that Sonic hedgehog signaling emanating from the embryonic midline is essential for establishment and outgrowth of the caudal fin primordium. However, gene expression analysis shows that the primordium of the adult caudal fin does not harbor a Sonic hedgehog-expressing domain equivalent to the Shh secreting zone of polarizing activity (ZPA) of paired appendages. Conclusion Our results suggest that Hedgehog proteins can regulate skeletal appendage outgrowth independent of a ZPA and demonstrates an unexpected mechanism for mediating Shh signals in a median fin primordium. The median fins evolved before paired fins in early craniates, thus the patterning of the median fins may be an ancestral mechanism that controls the outgrowth of skeletogenic appendages in vertebrates.

Investigation of the ar-C midline enhancer of sonic hedgehog orthologs and paralogs from distantly related vertebrate lineages identified lineage-specific motif changes; exchanging motifs between paralog enhancers resulted in the reversal of enhancer specificity., Background Cis-regulatory modules of developmental genes are targets of evolutionary changes that underlie the morphologic diversity of animals. Little is known about the 'grammar' of interactions between transcription factors and cis-regulatory modules and therefore about the molecular mechanisms that underlie changes in these modules, particularly after gene and genome duplications. We investigated the ar-C midline enhancer of sonic hedgehog (shh) orthologs and paralogs from distantly related vertebrate lineages, from fish to human, including the basal vertebrate Latimeria menadoensis. Results We demonstrate that the sonic hedgehog a (shha) paralogs sonic hedgehog b (tiggy winkle hedgehog; shhb) genes of fishes have a modified ar-C enhancer, which specifies a diverged function at the embryonic midline. We have identified several conserved motifs that are indicative of putative transcription factor binding sites by local alignment of ar-C enhancers of numerous vertebrate sequences. To trace the evolutionary changes among paralog enhancers, phylogenomic reconstruction was carried out and lineage-specific motif changes were identified. The relation between motif composition and observed developmental differences was evaluated through transgenic functional analyses. Altering and exchanging motifs between paralog enhancers resulted in reversal of enhancer specificity in the floor plate and notochord. A model reconstructing enhancer divergence during vertebrate evolution was developed. Conclusion Our model suggests that the identified motifs of the ar-C enhancer function as binary switches that are responsible for specific activity between midline tissues, and that these motifs are adjusted during functional diversification of paralogs. The unraveled motif changes can also account for the complex interpretation of activator and repressor input signals within a single enhancer.

International audience; In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.

Our understanding of the cellular and molecular mechanisms underlying the adult neural stem cell state remains fragmentary. To provide new models on this issue, we searched for stem cells in the adult brain of the zebrafish. Using BrdU tracing and immunodetection of cell-type-specific markers, we demonstrate that the adult zebrafish telencephalon contains self-renewing progenitors, which show features of adult mammalian neural stem cells but distribute along the entire dorso-ventral extent of the telencephalic ventricular zone. These progenitors give rise to newborn neurons settling close to the ventricular zone within the telencephalon proper. They have no equivalent in mammals and therefore constitute a new model of adult telencephalic neural stem cells. In addition, progenitors from the ventral subpallium generate rapidly dividing progenitors and neuroblasts that reach the olfactory bulb (OB) via a rostral migratory stream and differentiate into GABAergic and TH-positive neurons. These ventral progenitors are comparable to the mammalian neural stem cells of the subependymal zone. Interestingly, dorsal and ventral progenitors in the adult telencephalon express a different combination of transcription factors than their embryonic counterparts. In the case of neurogenin1, this is due to the usage of different enhancer elements. Together, our results highlight the conserved and unique phylogenic and ontogenic features of adult neurogenesis in the zebrafish telencephalon and open the way to the identification of adult neural stem cell characters in cross-species comparative studies. © 2006 Elsevier Inc. All rights reserved.

International audience; Normal motility of the zebrafish embryo requires a large number of gene loci, many of which have human orthologues implicated in myasthenias and other myopathies. We have identified a mutation in the zebrafish that abolishes body motility. Embryos have narrower myofibrils and lack clusters of nicotinic acetylcholine receptors (nAChRs) on the surface of the somitic muscle. We mapped the mutation to the delta-subunit of the nAChR, showing this mutant to be a new allele of the previously named sofa potato (sop). The mutant allele carries a missense mutation in the extracellular domain altering the cysteine at position 150 to an arginine. The delta-subunit is expressed in all striated muscles in embryonic and early larval stages together with the alpha1, beta1, epsilon, and gamma-subunits of nAChR. In contrast to mammals that show switching from the gamma embryonic to the adult epsilon-subunit, the two subunits are coexpressed in zebrafish embryos. We, furthermore, demonstrated that the sop/delta-nAChR mutation is a suppressor of the myopathy caused by lack of Dystrophin. The myofiber detachment phenotype of Dystroglycan-deficient embryos was not suppressed, suggesting that Dystrophin and Dystroglycan play distinct roles in muscle formation and maintenance of muscle integrity.

International audience; Sequence conservation has been used to find genes and to pinpoint functional non-coding sequences such as transcriptional regulatory elements. In this article, we analysed the conservation of 104 experimentally validated murine enhancer sequences between the mouse and zebrafish genomes. Surprisingly, only 10.5% of the mouse enhancers have homologues in zebrafish. All of the genes with conserved cis-elements have regulatory functions during embryonic development, perhaps reflecting substantial structural constraints on the integration of spatio-temporal signalling cues during the formation of the vertebrate body.

International audience; The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces selective loss of dopaminergic neurons in the mammalian midbrain, eliciting symptoms characteristic of Parkinson's disease. By exploiting the advantages of zebrafish embryos, we report here that dopaminergic neurons in this species are specifically perturbed when exposed to MPTP. In contrast to mammals, the zebrafish does not possess a midbrain dopaminergic system. Instead, the main population of neurons expressing the dopamine transporter is located in the posterior tuberculum of the diencephalon. Exposure of embryos to MPTP led to a pronounced reduction in the number of dopaminergic cells in the diencephalon. This effect can be reversed by deprenyl, a specific inhibitor of monoamine oxidase B that catalyses the conversion of MPTP to its active metabolite, MPP+. Similarly, direct treatment of embryos with MPP+ abolished the diencephalic dopaminergic neurons. These larvae also demonstrated behavioural defects in swimming responses. Thus, dopaminergic neurons in the posterior tuberculum of the zebrafish may be homologous to the midbrain dopaminergic system of mammals. In addition, the mechanism behind the loss of dopaminergic neurons following pharmacological perturbation may be conserved among vertebrates and suggest that the zebrafish can be used as a convenient and economical system to study the pathogenesis of Parkinson's disease and for testing potential therapeutic strategies.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos,we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves.Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphénucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Acetylcholinesterase (AChE) hydrolyses acetylcholine (ACh) ensuring the fast clearance of released neurotransmitter at cholinergic synapses. Many studies led to the hypothesis that AChE and the closely related enzyme butyrylcholinesterase (BChE) may play other, non-hydrolytic roles during development. In this review, we compare data from in vivo studies performed on invertebrate and vertebrate genetic models. The loss of function of ache in these systems is responsible for the appearance of several phenotypes. In all aspects so far studied, the phenotypes can be explained by an excess of the undegraded substrate, ACh, leading to misfunction and pathological alterations. Thus, the lack of AChE catalytic activity in the mutants appears to be solely responsible for the observed phenotypes. None of them appears to require the postulated adhesive or other non-hydrolytic functions of AChE.

International audience

The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1(ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. Here, we show that the upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. Our data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6expression was crucial in neocortex evolution.

Situated at the ventral-most part of the vertebrate neural tube, the floor plate (FP) is an important signalling centre that controls the regional differentiation of neurons in the nervous system. It secretes guidance molecules that direct ventrally navigating axons crucial for the correct wiring of neuronal circuits. Although the function of the FP is well-conserved from fish to humans, discrepancies exists with respect to both the signalling system involved in FP induction, and the origin of the FP in various vertebrate species. Recent findings from the embryos of zebrafish, chicken and mouse provide insights that reconcile previous results and suggest common themes in vertebrate FP specification.

International audience; Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. Here, we address the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. First, we find that Fgf signalling is required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. Second, combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with our model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations.

Persistent ecotoxicants, such as dioxin and PCBs, are thought to pose one of the greatest threats to public and ecological health in the industrial world. These compounds cause a range of macroscopic malformations, particularly to the craniofacial apparatus and cardiovascular system during vertebrate development. However, little is known about microscopic effects, especially on the sensitive early life stages or on the molecular basis of developmental neurotoxicity. Using zebrafish (Danio rerio), we have explored neurological deficits caused by early-life exposure to environmentally relevant concentrations of dioxin. We show, using a quantitative stereological technique, that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) substantially reduces the capacity for embryonic brain development, causing a 30% reduction in total neuronal number in the 168-h larval brain. Using transgenic GFP-expressing zebrafish lines, we link this to decreased expression of key developmentally regulated genes, namely neurogenin and sonic hedgehog. This disruption of neuronal development provides the basis for understanding the neurotoxic effects of these compounds.

In zebrafish, development of the ventral neural tube depends on the Nodal-related signal Cyclops (Cyc). One-day-old cyc mutant embryos lack the medial floor plate (MFP). We show here that cells expressing MFP marker genes differentiate gradually in cyc mutant embryos in a delayed manner during the second day of development. This late differentiation is restricted to the hindbrain and spinal cord and depends on an intact Hedgehog (Hh) signalling pathway. Cells expressing MFP marker genes in cyc mutant embryos appear to be derived from lateral floor plate (LFP) cells as they coexpress LFP and MFP marker genes. This finding suggests that the correct temporal development of the MFP is required for the distinction of LFP and MFP cells in wild-type embryos.

Previous reports have linked Neurogenin 1 with the general promotion of neurogenesis and gliogenesis. A new study in zebrafish, supported by recent findings in Drosophila, has now challenged this idea by demonstrating that the activity of Neurogenin 1 is limited to activation of development of two classes of sensory neurons.

Homology searches between DNA sequences of evolutionary distant species (phylogenetic footprinting) offer a fast detection method for regulatory sequences. Because of the small size of their genomes, tetraodontid species such as the Japanese pufferfish and green spotted pufferfish have become attractive models for comparative genomics. A disadvantage of the tetraodontid species is, however, that they cannot be bred and manipulated routinely under laboratory conditions, so these species are less attractive for developmental and genetic analysis. In contrast, an increasing arsenal of transgene techniques with the developmental model species zebrafish and medaka are being used for functional analysis of cis regulatory sequences. The main disadvantage is the much larger genome. While comparison between many loci proved the suitability of phylogenetic footprinting using fish and mammalian sequences, fast rate of change in enhancer structure and gene duplication within teleosts may obscure detection of homologies. Here we discuss the contribution and potentials provided by different teleost models for the detection and functional analysis of conserved cis-regulatory elements.

The neurotransmitter acetylcholine (ACh) has a crucial role in central and neuromuscular synapses of the cholinergic system. After release into the synaptic cleft, ACh is rapidly degraded by acetylcholinesterase (AChE). We have identified a mutation in the ache gene of the zebrafish, which abolishes ACh hydrolysis in homozygous animals completely. Embryos are initially motile but subsequently develop paralysis. Mutant embryos show defects in muscle fiber formation and innervation, and primary sensory neurons die prematurely. The neuromuscular phenotype in ache mutants is suppressed by a homozygous loss-of-function allele of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR), indicating that the impairment of neuromuscular development is mediated by activation of nAChR in the mutant. Here we provide genetic evidence for non-classical functions of AChE in vertebrate development.

Endoderm originates from a large endomesodermal field requiring Nodal signalling. The mechanisms that ensure segregation of endoderm from mesoderm are not fully understood. We first show that the timing and dose of Nodal activation are crucial for endoderm formation and the endoderm versus mesoderm fate choice, because sustained Nodal signalling is required to ensure endoderm formation but transient signalling is sufficient for mesoderm formation. In zebrafish, downstream of Nodal signals, three genes encoding transcription factors (faust, bonnie and clyde and the recently identified gene casanova) are required for endoderm formation and differentiation. However their positions within the pathway are not completely established. In the present work, we show that casanova is the earliest specification marker for endodermal cells and that its expression requires bonnie and clyde. Furthermore, we have analysed the molecular activities of casanova on endoderm formation and found that it can induce endodermal markers and repress mesodermal markers during gastrulation, as well as change the fate of marginal blastomeres to endoderm. Overexpression of casanova also restores endoderm markers in the absence of Nodal signalling. In addition, casanova efficiently restores later endodermal differentiation in these mutants, but this process requires, in addition, a partial activation of Nodal signalling.

Purpose: Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity. Materials and Methods: Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances. Results and Discussion: The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects asconfirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract). This effect might be related to different bioavailability of chemicals. Conclusions: The apparent difference between the exposure scenarios is an important aspect that needs to be addressed when conducting analyses of alterations in the expression level of mRNA., RWTH University Aachen abbr. for Rheinisch-Westfälische Technische Hochschule University Aachen

A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs(1) and recruits general transcription factors to initiate transcription(2). The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle(3-6). Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition(7). This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression(8), and determined the positions of H3K4me3-marked promoter-associated nucleosomes(9). Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (11) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.

International audience; Dystrophin, the protein responsible for Duchenne Muscular Dystrophy (DMD), plays a critical role in the maintenance of the muscle membrane integrity. There are several forms of dystrophin derived from the DMD gene by alternative promoter usage. In addition to full-length dystrophin (Dp427), four shorter transcripts have been identified: Dp260, Dp140, Dp116 and Dp71. The functional role played by the different products of the DMD gene is not yet determined. To get insight into the function of dystrophin and related products, we have investigated the presence of dystrophin in zebrafish. This choice takes advantage of large-scale mutagenesis screens in zebrafish, which have led to the identification of several mutants with motility defects. The identification and characterization of the genes affected by these mutations is likely to provide relevant information for the understanding of the molecular mechanisms of muscle development and function. Two cDNA clones encoding the homologues of dystrophin and Dp71 in zebrafish were identified and characterized. Both transcripts exhibit a high degree of sequence homology with the dystrophin and Dp71 proteins described in higher vertebrates. In addition, three alternative spliced transcripts that occur at the C-terminal end of the zebrafish DMD gene have been identified. These transcripts exhibit different patterns of tissue expression. We have also determined the chromosomal localization of dystrophin on the radiation hybrid map of the zebrafish genome. Our results indicate that the dystrophin gene is localized to linkage group one. Altogether, these results give new insights on the physiological role played by dystrophin and related proteins, and provide new tools for the identification of mutated genes associated with muscle defects in zebrafish.

The BMP3 related anti-dorsalizing morphogenetic protein (ADMP) has been proposed to function in the organizer of chick and Xenopus embryos. We report here the cloning and expression pattern of a zebrafish admp gene. The gene is expressed in involuting cells of the embryonic shield, but not in the non-involuting forerunner cells. During gastrulation, admp transcripts are detected in the posterior prechordal plate, in the notochord primordium and in cells of the dorsal blastoderm margin. Expression is also detectable in the neuroectoderm overlying the posterior prechordal plate. Expression persists in the tail bud until the end of somitogenesis while expression in other areas disappears during early somitogenesis stages.

casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene,10J3, which is expressed in the endoderm. Thecas phenotype is rescued by overexpression of 10J3and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role forcas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of casactivity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals.

Iroquois homeoproteins are prepatterning factors that positively regulate proneural genes and control neurogenesis. We have identified a zebrafish Iroquois gene, irx1, which is highly homologous to Xenopus Xiro1, Gallus c-Irx1 and mouse Irx1. Expression of irx1 was initially detected at the bud stage. By 16 h post-fertilization (hpf), irx1 expression was exclusively limited to the prospective midbrain and hindbrain. By 24 hpf, irx1 expression was clearly detected in the acousticovestibual ganglia, tectum, tegmentum, cerebellum and rhombomere 1 but not in rhombomere 2 or mid-hindbrain boundary.

General transcription factors TFIIA, B, D, E, F, H, and RNA polymerase II (Pol II) are required for accurate initiation of Pol II transcription. The TATA binding protein (TBP), a subunit of TFIID, is responsible for recognition of the TATA box, a core element shared by a category of class II promoters [1]. Recently, novel TBP-like factors (TLFs) have been described in metazoan organisms [2]. In spite of the numerous in vitro studies describing the general role of TBP in RNA polymerase II (Pol lI) transcription initiation, the precise function of TBP and the newly described TLF is poorly understood in vivo. We inhibited TBP and TLF function in zebrafish embryos to study the role of these factors during zygotic transcription. A dominant-negative variant of TLF mRNA and a TBP morpholino antisense oligo was used to block either TLF or TBP function. Both TBP- or TLF-blocked embryos developed normally until the midblastula stage; however, they then failed to gastrulate. Several zygotic regulatory genes were downregulated by a block in either TBP or TLF function, while others were differentially affected. These results suggest that TBP is not universally required for Pol II transcription in vertebrates and that there is a differential requirement for TBP and TLF during early embryogenesis.

Publisher Summary Recently, the zebrafish has become a popular system used to study vertebrate development, as it is accessible to such manipulations by both genetic and molecular means, thereby combining to a certain extent the potentials of other vertebrate systems such as mouse and Xenopus in one embryo. This chapter discusses the strategies to perturb development by molecular means with particular focus on the approaches to increase or decrease the activity of gene products by RNA and DNA microinjection methods. In addition, some treatments and reagents have been described that activate or inhibit specific proteins on a post-translational level. Although the description of administration methods and reagents refers largely to the applications in the zebrafish embryo, the experiments described also include the studies carried out in Xenopus to allow a better appreciation of the potential of molecular approaches to perturbing the embryonic development. The chapter also introduces inducible systems used in other organisms and demonstrates their potential applicability in zebrafish.