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PubMed2006

Gene duplication and separation of functions in alphaB-crystallin from zebrafish (Danio rerio).

We previously reported that zebrafish alphaB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish alphaB-crystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel alpha-crystallin transcript designated cryab2 and the resulting protein alphaB2-crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human alphaB-crystallin and zebrafish alphaB1-crystallin, respectively. RT-PCR showed that alphaB2-crystallin is expressed predominantly in lens but, reminiscent of mammalian alphaB-crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone-like activity of purified recombinant alphaB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of alpha-lactalbumin and lysozyme. At 25 degrees C and 30 degrees C, zebrafish alphaB2 showed greater chaperone-like activity than human alphaB-crystallin, and at 35 degrees C and 40 degrees C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that alphaB2-crystallin makes up approximately 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different alphaB-crystallins. Differences in primary structure, expression and chaperone-like activity suggest that the two zebrafish alphaB-crystallins perform divergent physiological roles. After gene duplication, zebrafish alphaB2 maintained the widespread protective role also found in mammalian alphaB-crystallin, while zebrafish alphaB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure-function relationships and the regulation of tissue-specific expression patterns.

Alternative Splicing↗
PubMed2001

Sparking new frontiers: using in vivo electroporation for genetic manipulations.

In vivo electroporation is a fascinating new approach by which gene expression, regulation, and function can be studied in developmental systems. This technique offers new opportunities for manipulations in animal models that lack genetic approaches, including avians. Furthermore, this approach is applicable to other embryo populations including mice, ascidians, zebrafish, Xenopus, and Drosophila. In this review, we discuss technical aspects of in vivo electroporation, review recent studies where this approach has been utilized successfully, and identify future directions.

Animals↗
PubMed2000

The need for a simple animal model to understand sleep.

Simple animal models have allowed biologists to apply the tools of modern molecular genetics to such complex behaviors as circadian rhythms and long-term memory consolidation. The mechanisms and molecules discovered in these simple animals are evolutionarily conserved in other species, including mammals. Sleep research lacks a simple animal model because criteria based on the electroencephalogram have been met only in birds and mammals. We argue that straightforward behavioral criteria could allow the identification of a sleep-like rest state that might be useful for molecular investigations to understand the regulation and function of sleep. Candidate model systems are discussed, leading to the conclusion that several species have complementary strengths. Specifically, techniques developed for larval zebrafish can be used to visualize neural firing patterns in the living animal, and the fruit fly Drosophila melanogaster has been used successfully for molecular and genetic dissection of complex behaviors. We conclude with a hypothesis that one putative function of sleep, the optimization of neural plasticity, would also have adaptive value in simple organisms and might therefore be evolutionarily conserved.

Animals↗
PubMed2003

Hey genes in cardiovascular development.

Hey genes encode a small family of basic helix-loop-helix (bHLH) transcription factors that are related to the Drosophila hairy and Enhancer-of-split genes. They belong to the still-limited number of direct targets of the Notch signaling pathway and are thus candidate molecules to effect critical developmental decisions like lateral inhibition, boundary formation, and inductive processes in numerous tissues. Human inherited mutations such as cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukencephalopathy and Alagille syndrome, as well as several mouse models, have highlighted the role of Notch signals in cardiovascular development and maintenance. Functional analyses in both mouse and zebrafish now have shown that Hey genes appear to be the most prominent transmitters for such signals to shape the cardiovascular system during development and perhaps also in later life.

Animals↗
PubMed2004

Early receptor current of wild-type and transducin knockout mice: photosensitivity and light-induced Ca2+ release.

We have used suction-electrode recording to measure the early receptor current (ERC) from single, isolated mammalian photoreceptors. When a wild-type mouse rod was illuminated with light sufficient to close all the cGMP-gated channels, a succeeding bright laser flash bleaching a large proportion of the visual pigment produced an ERC, which at 37 degrees C consisted primarily of a single component of transient positive current. The amplitude of total charge movement of this component declined exponentially with successive flashes, consistent with the direct proportionality of the ERC to the quantity of pigment bleached. From the constant of exponential decline, it was possible to estimate the in vivo photosensitivity of mouse rhodopsin to be about 6 x 10(-9)microm(2) per molecule. We have also measured the ERC from rods of transducin-knockout mice, for which previous illumination to close the cGMP-gated channels was not required. The ERC of these rods was similar to that of wild-type rods but was followed by a slow component of outward current whose maximum amplitude in some cells approached that of the normal light response. This slow current was blocked by l-cis diltiazem, indicating that it was produced by ion flux through the cyclic nucleotide-gated channels of the outer segment; however, it could not have been produced by the normal transduction cascade, since it was recorded from rods lacking transducin. Since it was depressed by prior incorporation of the Ca(2+) buffer BAPTA, it was probably generated by light-activated Ca(2+) release earlier demonstrated in salamander and zebrafish. Recordings of the ERC from normal and mutant mice may provide a useful tool for the analysis of models of retinal disease, as well as exploration of the molecular origin of light-activated Ca(2+) release.

Animals↗
PubMed1999

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue.

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.

Acetylgalactosamine↗
PubMed2002

Early induction of neural crest cells: lessons learned from frog, fish and chick.

The identification of genes in Xenopus, chick and zebrafish expressed early in prospective neural crest (NC) cells has challenged the previous view that the NC is induced during the closure of the neural tube. We compare here the early inductive molecular mechanisms in different organisms and, despite observed differences, propose a general common model for NC induction.

Animals↗
PubMed2002

Prey capture by larval zebrafish: evidence for fine axial motor control.

Swimming and turning behaviors of larval zebrafish have been described kinematically, but prey capture behaviors are less well characterized. High-speed digital imaging was used to record the axial kinematics of larval zebrafish as they preyed upon paramecia and also during other types of swimming. In all types of swim bouts, a series of traveling waves of bending is observed and these bends propagate along the trunk in the rostral to caudal direction. The prey capture swim bouts appeared to be more complex than other swim patterns examined. In the capture swim bouts, the initial bends were of low amplitude and were most prominent at far-caudal locations during each individual traveling wave of bending. Later bends in the bout (occurring just prior to prey capture) appeared to originate more rostrally and were of larger amplitude. These changes in bending pattern during capture swims were accompanied by a marked increase in tail-beat frequency. Associated with these axial kinematics were changes in heading and an abrupt increase in velocity close to the moment of prey capture. These changing patterns of bending suggest precise, bend-to-bend, neural control over both the timing and the rostral-caudal locus of bending. This degree of 'fine axial motor control' has not previously been described in the teleost behavioral literature and is notable because it occurs in larval zebrafish, where descending control signals are funneled through the roughly three-hundred neurons that project from brain into spinal cord. These findings will necessitate a significant increase in the complexity of current models of descending motor control in fishes.

Animals↗
PubMed2003

The role of epithelial remodelling in tooth eruption in larval zebrafish.

Based on light and transmission electron-microscopic observations on erupting first-generation teeth in the zebrafish, Danio rerio, we propose a biphasic mechanism for tooth eruption: (1). formation of an epithelial crypt prior to eruption of the tooth, possibly as a result of constraints in the epithelium resulting from the growth of adjacent tooth germs, and (2). detachment of cellular interdigitations both within the pharyngeal epithelium, at the pharyngeal epithelium/enamel organ boundary, and between the outer and inner dental epithelium, resulting in the exposure of the tooth tip in the crypt, immediately after tooth ankylosis. Later, further detachment of interdigitations between the inner and the outer enamel epithelium unfolds the epithelium even more and leads to a more pronounced exposure of the tooth tip. The presence of small patches of non-collagenous matrix on the outer surface of the tooth close to where it merges with the attachment bone is interpreted as a device to prevent complete detachment of the enamel organ. The biphasic nature of the mechanism for tooth eruption is supported by observations on in vitro cultured heads. First-generation teeth develop normally and crypts are formed, as under in vivo conditions, but the teeth fail to erupt. Taken together, our observations suggest that epithelial remodelling plays a crucial role in eruption of the teeth in this model organism.

Animals↗
PubMed2004

Early myocardial function affects endocardial cushion development in zebrafish.

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih(-/-)) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk(-/-)) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk(-/-) embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk(s11) variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.

Actins↗
PubMed2004

A crucial interaction between embryonic red blood cell progenitors and paraxial mesoderm revealed in spadetail embryos.

Zebrafish embryonic red blood cells (RBCs) develop in trunk intermediate mesoderm (IM), and early macrophages develop in the head, suggesting that local microenvironmental cues regulate differentiation of these two blood lineages. spadetail (spt) mutant embryos, which lack trunk paraxial mesoderm (PM) due to a cell-autonomous defect in tbx16, fail to produce embryonic RBCs but retain head macrophage development. In spt mutants, initial hematopoietic gene expression is absent in trunk IM, although endothelial and pronephric expression is retained, suggesting that early blood progenitor development is specifically disrupted. Using cell transplantation, we reveal that spt is required cell autonomously for early hematopoietic gene expression in trunk IM. Further, we uncover an interaction between embryonic trunk PM and blood progenitors that is essential for RBC development. Importantly, our data identify a hematopoietic microenvironment that allows embryonic RBC production in the zebrafish.

Animals↗
PubMed1999

Early postembryonic neural development in the zebrafish: a 3-D reconstruction of forebrain proliferation zones shows their relation to prosomeres.

Based on a section-by-section analysis of the morphology (combined silver/Nissl stain) and of the distribution of proliferation zones (immunohistochemical detection of the proliferating cell nuclear antigen) in the zebrafish (Danio rerio) forebrain at 5 days postfertilization, we created a three-dimensional reconstruction of proliferation zones of that developmental stage. The resulting model visualizes the size, number, location and morphology of forebrain proliferation zones. The latter foreshadow closely adult neuroanatomical forebrain entities. Furthermore, the detailed distribution of proliferation zones in the posterior forebrain - but not in the more anterior secondary prosencephalon - supports a segmental prosomeric organization.

Animals↗
PubMed2001

Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.

The connexin family of genes codes for proteins that oligomerize into a connexon of six subunits to form one half of the gap junction channel. Gap junctions are plasma membrane structures that mediate intercellular communication by joining the cytoplasm of two cells, allowing the passage of small molecules and metabolites, and contributing significantly to the maintenance of tissue homeostasis. The signaling mediated by these junctions appears to be necessary for the correct timing of key developmental events. This communication is especially important in the avascular lens where the intercellular passage of metabolites, second messengers, and ions is necessary to maintain the correct ionic balance in the lens fibre cells, and prevent cataract formation. To characterize the role that the connexin genes play in development, a novel connexin was cloned from zebrafish. A genomic clone was isolated that contained a 1,173 base open reading frame. The nucleotide sequence in this open reading frame shows extensive sequence similarity to mouse connexin50 (Cx50), chicken Cx45.6, sheep Cx49, and human Cx50. The protein encoded by this open reading frame contains 391 amino acids, with a predicted molecular weight of 44.1 kDa and a typical connexin transmembrane topology. By using the LN54 radiation hybrid panel, the Cx44.1 gene was mapped to linkage group 1. Whole-mount in situ hybridization and Northern blot analyses were performed on zebrafish embryos at various developmental stages to characterize the developmental expression of the Cx44.1 message. The ocular lens was the only tissue in which Cx44.1 transcripts were detected. The transcripts were first detected in the lens around 24 hr post fertilization and remained detectable until 120 hr post fertilization. Electrophysiological analysis of Cx44.1 channels revealed gating properties that were virtually identical to the mouse and chicken orthologues of Cx44.1.

Animals↗
PubMed2006

Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive ciliopathy characterized by progressive retinal degeneration, obesity, cognitive impairment, polydactyly, and kidney anomalies. The disorder is genetically heterogeneous, with 11 BBS genes identified to date, which account for ~70% of affected families. We have combined single-nucleotide-polymorphism array homozygosity mapping with in silico analysis to identify a new BBS gene, BBS12. Patients from two Gypsy families were homozygous and haploidentical in a 6-Mb region of chromosome 4q27. FLJ35630 was selected as a candidate gene, because it was predicted to encode a protein with similarity to members of the type II chaperonin superfamily, which includes BBS6 and BBS10. We found pathogenic mutations in both Gypsy families, as well as in 14 other families of various ethnic backgrounds, indicating that BBS12 accounts for approximately 5% of all BBS cases. BBS12 is vertebrate specific and, together with BBS6 and BBS10, defines a novel branch of the type II chaperonin superfamily. These three genes are characterized by unusually rapid evolution and are likely to perform ciliary functions specific to vertebrates that are important in the pathophysiology of the syndrome, and together they account for about one-third of the total BBS mutational load. Consistent with this notion, suppression of each family member in zebrafish yielded gastrulation-movement defects characteristic of other BBS morphants, whereas simultaneous suppression of all three members resulted in severely affected embryos, possibly hinting at partial functional redundancy within this protein family.

Animals↗
PubMedDate not supplied

The ghost of selection past: rates of evolution and functional divergence of anciently duplicated genes.

The duplication of genes and even complete genomes may be a prerequisite for major evolutionary transitions and the origin of evolutionary novelties. However, the evolutionary mechanisms of gene evolution and the origin of novel gene functions after gene duplication have been a subject of many debates. Recently, we compiled 26 groups of orthologous genes, which included one gene from human, mouse, and chicken, one or two genes from the tetraploid Xenopus and two genes from zebrafish. Comparative analysis and mapping data showed that these pairs of zebrafish genes were probably produced during a fish-specific genome duplication that occurred between 300 and 450 Mya, before the teleost radiation (Taylor et al. 2001). As discussed here, many of these retained duplicated genes code for DNA binding proteins. Different models have been developed to explain the retention of duplicated genes and in particular the subfunctionalization model of Force et al. (1999) could explain why so many developmental control genes have been retained. Other models are harder to reconcile with this particular set of duplicated genes. Most genes seem to have been subjected to strong purifying selection, keeping properties such as charge and polarity the same in both duplicates, although some evidence was found for positive Darwinian selection, in particular for Hox genes. However, since only the cumulative pattern of nucleotide substitutions can be studied, clear indications of positive Darwinian selection or neutrality may be hard to find for such anciently duplicated genes. Nevertheless, an increase in evolutionary rate in about half of the duplicated genes seems to suggest that either positive Darwinian selection has occurred or that functional constraints have been relaxed at one point in time during functional divergence.

Amino Acid Sequence↗
PubMed1998

Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio.

BACKGROUND: Transposable elements of the Tc1/mariner family are found in many species of the animal kingdom. It has been suggested that the widespread distribution of this transposon family resulted from horizontal transmission among different species. RESULTS: To test the ability of Tc1/mariner to cross species barriers, as well as to develop molecular genetic tools for studying zebrafish development, we determined the ability of the Tc3 transposon, a member of the Tc1/mariner family, to function in zebrafish. Tc3 transposons carrying sequences encoding the green fluorescent protein (GFP) were able to integrate in the fish genome by transposition. Integrated transposons expressed the GFP marker after germline transmission, and were capable of being mobilized upon introduction of transposase protein in trans. CONCLUSIONS: Our findings support models of horizontal transmission of Tc1/mariner elements between species. The work also establishes the basis for a novel method of transposon-mediated genetic transformation and for transposon-mediated genetic screens in zebrafish and other organisms.

Animals↗
PubMed2002

Non-canonical Wnt signalling and regulation of gastrulation movements.

Members of the Wnt family have been implicated in a variety of developmental processes including axis formation, patterning of the central nervous system and tissue morphogenesis. Recent studies have shown that a Wnt signalling pathway similar to that involved in the establishment of planar cell polarity in Drosophila regulates convergent extension movements during zebrafish and Xenopus gastrulation. This finding provides a good starting point to dissect the complex cell biology and genetic regulation of vertebrate gastrulation movements.

Animals↗
PubMed2007

Adaptability of the Vitamin D nuclear receptor to the synthetic ligand Gemini: remodelling the LBP with one side chain rotation.

The crystal structure of the ligand binding domain (LBD) of the wild-type Vitamin D receptor (VDR) of zebrafish bound to Gemini, a synthetic agonist ligand with two identical side chains branching at carbon 20 reveals a ligand-dependent structural rearrangement of the ligand binding pocket (LBP). The rotation of a Leu side chain opens the access to a channel that can accommodate the second side chain of the ligand. The 25% increase of the LBP's volume does not alter the essential agonist features of VDR. The possibility to adapt the LBP to novel ligands with different chemistry and/or structure opens new perspectives in the design of more specifically targeted ligands.

Adaptation, Biological↗