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PubMed2003

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.

Animals↗
PubMed2006

Non destructive in vivo measurement of ethoxyresorufin biotransformation by zebrafish prolarva: development and application.

Biotransformation enzymatic activities, such as the cytochrome P450 one, have been used as biomarkers for pollution assessment for a long time. Nevertheless, such biochemical tools are destructive processes, because they are performed on fish liver or total larvae homogenates. Moreover, the adaptation of this bioassay to some fish larvae, like Danio rerio ones, is ineffective because it needs a lot of organisms. We thus developed an original, nondestructive method to detect the induction of a biotransformation activity in the prolarva of the fish, Danio rerio, exposed to chemicals. This methodology is based on the assessment of EROD activity, by measurement in the culture medium of the fluorescence increase due to the excretion of resorufin by fish during an ethoxyresorufin exposure. After exposure of fish embryos to known inducers (BaP and beta-naphtoflavone), the prolarvae were exposed to the substrate (ethoxyresorufin), and the kinetic of the fluorescence increase was measured. A dose-effect relationship was observed, with a three to five fold increase of EROD basal activity. This methodology also allowed us to compare between EROD activity induction by different environmental samples. The proposed methodology thus allows to perform a simple, sensitive, and reproducible microbiotest for the detection of sublethal concentrations of AhR chemical inducers in environmental samples.

Animals↗
PubMedDate not supplied

Chemical genetics: Drug screens in Zebrafish.

High throughput chemical genetic screens for compounds with specific biological activity in a whole organism are feasible using zebrafish embryos. At least two medium to large scale drug screens have been carried out to date, leading to the identification of compounds that disturb zebrafish development. Chemical genetics using zebrafish embryos may become an important step in the discovery of drugs and their targets.

Animals↗
PubMed2005

Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish.

The Lmo2 transcription factor, a T-cell oncoprotein, is required for both hematopoiesis and angiogenesis. To investigate the fate of lmo2-expressing cells and the transcriptional regulation of lmo2 in vivo, we generated stable transgenic zebrafish that express green fluorescent protein (EGFP) or DsRed under the control of an lmo2 promoter. A 2.5-kb fragment contains the cis-regulatory elements required to recapitulate endogenous lmo2 expression in embryonic hematopoietic and vascular tissues. We further characterized embryonic Lmo2+ cells through transplantation into vlad tepes (vlt), an erythropoietic mutant. These Lmo2+ primitive wave donor cells differentiated into circulating hematopoietic cells and extended the life span of vlt recipients, but did not demonstrate long-term repopulation of the erythroid lineage. Promoter analysis identified a 174-bp proximal promoter that was sufficient to recapitulate lmo2 expression. This element contains critical ETS-binding sites conserved between zebrafish and pufferfish. Furthermore, we show that ets1 is coexpressed with lmo2, and overexpression experiments indicate that ets1 can activate the lmo2 promoter through this element. Our studies elucidate the transcriptional regulation of this key transcription factor, and provide a transgenic system for the functional analysis of blood and blood vessels in zebrafish.

Animals↗
PubMed2003

Primordial germ-cell development: the zebrafish perspective.

Primordial germ cells follow a characteristic developmental path that is manifested in the specialized regulation of basic cell functions and behaviour. Recent studies in zebrafish have greatly enhanced our understanding of the mode of specification of primordial germ cells, cell-fate maintenance and the migration of these cells towards their target, the gonad, where they differentiate into gametes.

Animals↗
PubMed2003

Site-1 protease is required for cartilage development in zebrafish.

gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects.

Animals↗
PubMed2005

NXT2 is required for embryonic heart development in zebrafish.

BACKGROUND: NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes. RESULTS: A zebrafish mutant exhibiting specific heart defects during embryogenesis was generated by animal cloning-mediated retroviral insertions. Molecular analysis indicated that the mutant phenotype was caused by a disruption of NXT2. Whole-mount RNA in situ hybridization showed that NXT2 transcripts were clearly detectable in embryonic heart as well as other tissues. Further analysis revealed that expression level of one form of alternative splicing NXT2 mRNA transcripts was significantly reduced, resulting in deficient myocardial cell differentiation and the malformation of cardiac valve at the atrioventricular boundary. The defects could be reproduced by morpholino anti-sense oligo knockdown of NXT2. CONCLUSION: NXT2 has a critical role in maintaining morphogenetic integrity of embryonic heart in vertebrate species.

Active Transport, Cell Nucleus↗
PubMed2004

Ectopic expression and knockdown of a zebrafish sox21 reveal its role as a transcriptional repressor in early development.

Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key roles in animal embryonic development. Zebrafish Sox21a is part of group B Sox proteins and its chicken and mouse orthologs have been described as transcriptional repressor and activator, respectively, in two different target gene contexts. Zebrafish sox21a is present as a maternal transcript in the oocyte and is mainly expressed at the developing midbrain-hindbrain boundary from the onset of neurulation. In order to understand its role in vivo, we ectopically expressed sox21a by microinjection. Ectopic expression of full length sox21a leads to dorsalization of the embryos. A subset of the dorsalized embryos shows a partial axis splitting, and hence an ectopic neural tube, as an additional phenotype. At gastrulation, injected embryos show expansion of the expression domains of organizer-specific genes, such as chordin and goosecoid. Molecular markers used in somitogenesis highlight that sox21a-injected embryos have shortened AP axis, undulating axial structures, enlarged or even radialized paraxial territory. The developmental abnormalities caused by ectopic expression of sox21a are suggestive of defects in convergence-extension morphogenetic movements. Antisense morpholino oligonucleotides, designed to functionally knockdown sox21a, cause ventralization of the embryos. Moreover, gain-of-function experiments with chimeric constructs, where Sox21a DNA-binding domain is fused to a transcriptional activator (VP16) or repressor (EnR) domain, suggests that zebrafish Sox21a acts as a repressor in dorso-ventral patterning.

Animals↗
PubMed1990

Identification of spinal neurons in the embryonic and larval zebrafish.

Previous studies indicated that the developing fish spinal cord was a simple system containing a small number of distinguishable neuronal cell types (Eisen et al., Nature 320:269-271, '86; Kuwada, Science, 233:740-746, '86). To verify this we have characterized the cellular anatomy of the spinal cord of developing zebrafish in order to determine the number, identities, and organization of the spinal neurons. Spinal neurons were labeled by intracellular dye injections, application of an axonal tracer dye to all or subsets of the axonal tracts, and application of antibodies which recognize embryonic neurons. We found that nine classes of neurons could be identified based on soma size and position, pattern of dendrites, axonal trajectory, and time of axonogenesis. These are two classes of axial motor neurons, which have been previously characterized (Myers, J. Comp. Neurol. 236:555-561, '85), one class of sensory neurons, and six classes of interneurons. One of the interneuron classes could be subclassified as primary and secondary based on criteria similar to those used to classify the axial motor neurons into primary and secondary classes. The early cord (18-20 hours) is an extremely simple system and contains approximately 18 lateral cell bodies per hemisegment, which presumably are post-mitotic cells. By this stage, five of the neuronal classes have begun axonogenesis including the primary motor neurons, sensory neurons, and three classes of interneurons. By concentrating on these early stages when the cord is at its simplest, pathfinding by growth cones of known identities can be described in detail. Then it should be possible to test many different mechanisms which may guide growth cones in the vertebrate central nervous system (CNS).

Animals↗
PubMed2006

Supraspinal input is dispensable to generate glycine-mediated locomotive behaviors in the zebrafish embryo.

The anatomy of the developing zebrafish spinal cord is relatively simple but, despite this simplicity, it generates a sequence of three patterns of locomotive behaviors. The first behavior exhibited is spontaneous movement, then touch-evoked coiling, and finally swimming. Previous studies in zebrafish have suggested that spontaneous movements occur independent of supraspinal input and do not require chemical neurotransmission, while touch-evoked coiling and swimming depend on glycinergic neurotransmission as well as supraspinal input. In contrast, studies in other vertebrate preparations have shown that spontaneous movement requires glycine and other neurotransmitters and that later behaviors do not require supraspinal input. Here, we use lesion analysis combined with high-speed kinematic analysis to re-examine the role of glycine and supraspinal input in each of the three behaviors. We find that, similar to other vertebrate preparations, supraspinal input is not essential for spontaneous movement, touch-evoked coiling, or swimming behavior. Moreover, we find that blockade of glycinergic neurotransmission decreases the rate of spontaneous movement and impairs touch-evoked coiling and swimming, suggesting that glycinergic neurotransmission plays critical yet distinct roles for individual patterns of locomotive behaviors.

Animals↗
PubMed2001

Zebrafish myelopoiesis and blood cell development.

The zebrafish (Danio rerio) animal model offers a unique opportunity to discover novel genes required for the control of normal vertebrate myeloid cell development. It is well suited for both developmental and genetic analyses: eg, genome-wide chemical mutagenesis screens have led to the identification of specific new genes affecting vertebrate erythropoiesis. Mutants defective in one or more hematopoietic functions will be useful as models of human disease and will assist in the elucidation of lineage-specific developmental programs. By using a combination of forward genetic mutagenesis screens and emerging strategies based on transgenic and antisense knockdown approaches, it should be possible to dissect the genetic programs that lead to myeloproliferative/myelodysplastic syndromes and to acute myeloid leukemia.

Animals↗
PubMed2004

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

Adenoviridae↗
PubMed1997

Effects of hirudin (thrombin specific inhibitor) in zebrafish embryos: a developmental role for thrombin.

To address the role of thrombin in early embryogenesis, hirudin, a thrombin specific inhibitor was microinjected into developing zebrafish embryos to inhibit the temporal activity of thrombin during early embryonic development. We found that the fibrin forming activity is inhibited by the presence of hirudin. We also found that hirudin affects development in zebrafish embryos suggesting thrombin's role in early embryogenesis. This ability to inhibit thrombin activity in developing embryos should facilitate studies on identifying signal transduction pathways affected by thrombin during embryogenesis.

Animals↗
PubMed2006

[Research progress on cell apoptosis during embryonic development of the zebrafish (Danio rerio)].

Cells have a built-in death program through the regulation of genes. Apoptosis (programmed cell death) plays a very important role in the development of the vertebrate embryo. The zebrafish (Danio rerio) has become a useful and ideal model in molecular developmental biology for in vivo study on the function of apoptosis in morphogenesis, sex differentiation and so on, and has already been garnering more and more attention. At present, much progress has been made regarding apoptotic pathways in zebrafish development. Caspases and other regulatory apoptotic genes have been cloned successfully. Moreover, transgenic zebrafish embryos have been induced to undergo apoptosis in vivo to study apoptotic signaling pathways and the abnormal morphological changes during embryogenesis. Transgenic zebrafish has provided a powerful tool for illuminating the interaction between regulatory apoptotic genes and vertebrate development.

Animals↗