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PubMed2004

A second insulin gene in fish genomes.

The recent characterization of diverse vertebrate genomes has revealed the importance of gene duplication in vertebrate evolution. Evidence suggests that a genome duplication event occurred on the lineage leading to teleost fish-species that are often used to understand human biology. The existence of a genome duplication event complicates the use of fish as a model for human diseases as there are often two fish homologues for a single copy human gene. Often the second homologue has not been recognized. Our searches of the near complete zebrafish and fugu fish genomes indicate that both species have two insulin genes. Phylogenetic analysis indicates that the two genes are likely the product of the fish-specific genome duplication. The maintenance of two insulin genes within the fish suggests that the two genes have different functions. Thus the well-characterized insulin genes in some fish species may not be complete homologues of the human insulin gene.

Amino Acid Sequence↗
PubMed2005

Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation.

Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRalpha, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRalpha in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRalpha cDNA from a goldfish ovarian cDNA library. Northern blot analysis indicates the presence of a major 2.6kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafish (93%), and human (55%) mPRalphas. Western blot analysis using a polyclonal goldfish mPRalpha antibody shows a major immunoreactive band of the predicted molecular weight (40kDa) in goldfish ovarian membranes. Computer modeling predicts that the deduced protein has seven transmembrane domains, typical of G protein-coupled receptors. Treatment of full grown, late vitellogenic stage follicle-enclosed oocytes in vitro with gonadotropin increased mPRalpha protein levels. A correlation between mPRalpha protein levels and the ability of oocytes to undergo GVBD in response to the MIH (maturational competence) was observed after treatment with gonadotropin. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRalpha blocked both the induction of oocyte maturational competence and mPRalpha protein upregulation by gonadotropin. These results with the goldfish mPRalpha protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRalpha acts as an intermediary in MIH induction of oocyte maturation in teleosts.

Amino Acid Sequence↗
PubMed2003

Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor.

Recently we discovered a previously uncharacterized gene with the characteristics of a membrane progestin receptor (mPR) in a fish model, spotted seatrout. Here, we report the identification, cloning, and characteristics of other members of this hitherto unknown family of putative mPRs from several vertebrate species, including human, mouse, pig, Xenopus, zebrafish, and Fugu, with highly conserved nucleotide and deduced amino acid sequences and similar structures to the spotted seatrout mPR. The 13 vertebrate genes identified seem to belong to an unknown gene family. Phylogenetic analysis indicates these cDNAs comprise three distinct groups (named alpha, beta, and gamma) within this gene family. Structural analyses of the translated cDNAs suggest they encode membrane proteins with seven transmembrane domains. The transcript sizes of the human alpha, beta, and gamma putative mPR mRNAs varied from 2.8 to 5.8 kb and showed distinct distributions in reproductive, neural, kidney and intestinal tissues, respectively. Recombinant human alpha, gamma, and mouse beta proteins produced in an Escherichia coli expression system demonstrated high affinity (K(d) = 20-30 nM) saturable binding for progesterone. Further analysis of binding to the gamma-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation (t(1/2) = 2-8 min). These results suggest this is a new family of steroid receptors unrelated to nuclear steroid receptors, but instead having characteristics of G protein-coupled receptors.

Amino Acids↗
PubMed2002

FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain.

The segmentation of the vertebrate hindbrain into rhombomeres is highly conserved, but how early hindbrain patterning is established is not well understood. We show that rhombomere 4 (r4) functions as an early-differentiating signaling center in the zebrafish hindbrain. Time-lapse analyses of zebrafish hindbrain development show that r4 forms first and hindbrain neuronal differentiation occurs first in r4. Two signaling molecules, FGF3 and FGF8, which are both expressed early in r4, are together required for the development of rhombomeres adjacent to r4, particularly r5 and r6. Transplantation of r4 cells can induce expression of r5/r6 markers, as can misexpression of either FGF3 or FGF8. Genetic mosaic analyses also support a role for FGF signaling acting from r4. Taken together, our findings demonstrate a crucial role for FGF-mediated inter-rhombomere signaling in promoting early hindbrain patterning and underscore the significance of organizing centers in patterning the vertebrate neural plate.

Animals↗
PubMed2003

Asian medaka fishes offer new models for studying mechanisms of seawater adaptation.

Japanese medaka (Oryzias latipes) is a freshwater (FW) teleost that is popular throughout the world for laboratory use. In this paper, we discuss the utility of Japanese medaka and related species for studying mechanisms of seawater (SW) adaptation. In addition to general advantages as an experimental animal such as their daily spawning activity, transparency of embryos, short generation time and established transgenic techniques, Japanese medaka have some adaptability to SW unlike the strictly stenohaline zebrafish (Danio rerio). Since other species in the genus Oryzias exhibit different degrees of adaptability to SW, comparative studies between Japanese medaka, where molecular-biological and genetic information is abundant, and other Oryzias species are expected to present varying approaches to solving the problems of SW adaptation. We introduce some examples of interspecies comparison for SW adaptabilities both in adult fish and in embryos. Oryzias species are good models for evolutionary, ecological and zoogeographical studies and a relationship between SW adaptability and geographic distribution has been suggested. Medaka fishes may thus deliver new insights into our understanding of how fish have expanded their distribution to a wide variety of osmotic environments.

Adaptation, Physiological↗
PubMedDate not supplied

Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition.

Summary The evolutionary origin of vertebrate hindbrain segmentation is unclear since the amphioxus, the closest living invertebrate relative to the vertebrates, possesses a hindbrain homolog that displays no gross morphological segmentation. Three of the estrogen-receptor related (ERR) receptors are segmentally expressed in the zebrafish hindbrain, suggesting that their common ancestor was expressed in a similar, reiterated manner. We have also cloned and determined the developmental expression of the single homolog of the vertebrate ERR genes in the amphioxus (AmphiERR). This gene is also expressed in a segmented manner in a region considered homologous to the vertebrate hindbrain. In contrast to the expression of amphioxus islet (a LIM-homeobox gene that also labels motoneurons), AmphiERR expression persists longer in the hindbrain homolog and does not later extend to additional posterior cells. In addition, AmphiERR and one of its vertebrate homologs (ERRalpha) are expressed in the developing somitic musculature of amphioxus and zebrafish, respectively. Altogether, our results are consistent with fine structural evidence suggesting that the amphioxus hindbrain is segmented, and indicate that chordate ERR gene expression is a marker for both hindbrain and muscle segmentation. Furthermore, our data support an evolution model of chordate brain segmentation: originally, the program for anterior segmentation in the protochordate ancestors of the vertebrates resided in the developing axial mesoderm which imposed reiterated patterning on the adjacent neural tube; during early vertebrate evolution, this segmentation program was transferred to and controlled by the neural tube.

Amino Acid Sequence↗
PubMed2003

The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm.

Nodal signals, a subclass of the TGFbeta superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt(-/-); cyc(+/+) and sqt(-/-); cyc(+/-) mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt(-/-); cyc(+/-) or sqt(-/-); cyc(+/+) embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.

Animals↗
PubMed2005

Highly conserved regulatory elements around the SHH gene may contribute to the maintenance of conserved synteny across human chromosome 7q36.3.

Comparative genomic analysis reveals an exceptionally large section of conserved shared synteny between the human 7q36 chromosomal region and the pufferfish (Fugu rubripes) genome. Remarkably, this conservation extends not only to gene order across 16 genes, but also to the position and orientation of a number of prominent conserved noncoding elements (CNEs). A functional assay using zebrafish has shown that most of the CNEs have reproducible and specific enhancer activity. This enhancer activity is often detected in a subset of tissues which reflect the endogenous expression pattern of a proximal gene, though some CNEs may act over a long range. We propose that the distribution of CNEs, and their probable association with a number of genes throughout the region, imposes a critical constraint on genome architecture, resulting in the maintenance of such a large section of conserved synteny across the vertebrate lineage.

Animals↗
PubMed1998

Determination of the zebrafish forebrain: induction and patterning.

We report an analysis of forebrain determination and patterning in the zebrafish Danio rerio. In order to study these events, we isolated zebrafish homologs of two neural markers, odd-paired-like (opl), which encodes a zinc finger protein, and fkh5, which encodes a forkhead domain protein. At mid-gastrula, expression of these genes defines a very early pattern in the presumptive neurectoderm, with opl later expressed in the telencephalon, and fkh5 in the diencephalon and more posterior neurectoderm. Using in vitro explant assays, we show that forebrain induction has occurred even earlier, by the onset of gastrulation (shield stage). Signaling from the early gastrula shield, previously shown to be an organizing center, is sufficient for activation of opl expression in vitro. In order to determine whether the organizer is required for opl regulation, we removed from late blastula stage embryos either the presumptive prechordal plate, marked by goosecoid (gsc) expression, or the entire organizer, marked by chordin (chd) expression. opl was correctly expressed after removal of the presumptive prechordal plate and consistently, opl was correctly expressed in one-eyed pinhead (oep) mutant embryos, where the prechordal plate fails to form. However, after removal of the entire organizer, no opl expression was observed, indicating that this region is crucial for forebrain induction. We further show that continued organizer function is required for forebrain induction, since beads of BMP4, which promotes ventral fates, also prevented opl expression when implanted during gastrulation. Our data show that forebrain specification begins early during gastrulation, and that a wide area of dorsal mesendoderm is required for its patterning.

Amino Acid Sequence↗
PubMed2006

Quantifying cardiovascular flow dynamics during early development.

The relationship between developing biologic tissues and their dynamic fluid environments is intimate and complex. Increasing evidence supports the notion that these embryonic flow-structure interactions influence whether development will proceed normally or become pathogenic. Genetic, pharmacological, or surgical manipulations that alter the flow environment can thus profoundly influence morphologic and functional cardiovascular phenotypes. Functionally deficient phenotypes are particularly poorly described as there are few imaging tools with sufficient spatial and temporal resolution to quantify most intra-vital flows. The ability to visualize biofluids flow in vivo would be of great utility in functionally phenotyping model animal systems and for the elucidation of the mechanisms that underlie flow-related mechano-sensation and transduction in living organisms. This review summarizes the major methodological advances that have evolved for the quantitative characterization of intra-vital fluid dynamics with an emphasis on assessing cardiovascular flows in vertebrate model organisms.

Animals↗
PubMed2004

Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling.

Graded Hedgehog (Hh) signaling patterns the spinal cord dorsoventral axis by inducing and positioning distinct precursor domains, each of which gives rise to a different type of neuron. These domains also generate glial cells, but the full range of cell types that any one precursor population produces and the mechanisms that diversify cell fate are unknown. By fate mapping and clonal analysis in zebrafish, we show that individual ventral precursor cells that express olig2 can form motoneurons, interneurons and oligodendrocytes. However, olig2+ precursors are not developmentally equivalent, but instead produce subsets of progeny cells in a spatially and temporally biased manner. Using genetic and pharmacological manipulations, we provide evidence that these biases emerge from Hh acting over time to set, maintain, subdivide and enlarge the olig2+ precursor domain and subsequently specify oligodendrocyte development. Our studies show that spatial and temporal differences in Hh signaling within a common population of neural precursors can contribute to cell fate diversification.

Animals↗
PubMed2003

Ash1a and Neurogenin1 function downstream of Floating head to regulate epiphysial neurogenesis.

The homeodomain transcription factor Floating head (Flh) is required for the generation of neurones in the zebrafish epiphysis. It regulates expression of two basic helix loop helix (bHLH) transcription factor encoding genes, ash1a (achaete/scute homologue 1a) and neurogenin1 (ngn1), in epiphysial neural progenitors. We show that ash1a and ngn1 function in parallel redundant pathways to regulate neurogenesis downstream of flh. Comparison of the epiphysial phenotypes of flh mutant and of ash1a/ngn1 double morphants reveals that reduced expression of ash1a and ngn1 can account for most of the neurogenesis defects in the flh-mutant epiphysis but also shows that Flh has additional activities. Furthermore, different cell populations show different requirements for ash1a and ngn1 within the epiphysis. These populations do not simply correspond to the two described epiphysial cell types: photoreceptors and projection neurones. These results suggest that the genetic pathways that involve ash1a and ngn1 are common to both neuronal types.

Animals↗
PubMed2006

An enzyme-linked immunosorbent assay for rare minnow (Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish (Danio rerio).

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow (Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp (Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17alpha-ethinylestradiol (EE(2)) of rare minnow and zebrafish (Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE(2) l(-1) for rare minnow and 4 ng EE(2) l(-1) for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC-MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment.

Animal Feed↗
PubMed2004

Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1-8) in pufferfish (Fugu rubripes).

The guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins of the calmodulin (CaM) gene superfamily that function in the regulation of photoreceptor guanylate cyclases (GCs). In the mammalian retina, two GCAPs (GCAP 1-2) and two transmembrane GCs have been identified as part of a complex regulatory system responsive to fluctuating levels of free Ca(2+). A third GCAP, GCAP3, is expressed in human and zebrafish (Danio rerio) retinas, and a guanylate cyclase-inhibitory protein (GCIP) has been shown to be present in frog cones. To explore the diversity of GCAPs in more detail, we searched the pufferfish (Fugu rubripes) and zebrafish (Danio rerio) genomes for GCAP-related gene sequences (fuGCAPs and zGCAPs, respectively) and found that at least five additional GCAPs (GCAP4-8) are predicted to be present in these species. We identified genomic contigs encoding fuGCAPl-8, fuGCIP, zGCAPl-5, zGCAP7 and zGCIP. We describe cloning, expression and localization of three novel GCAPs present in the zebrafish retina (zGCAP4, zGCAP5, and zGCAP7). The results show that recombinant zGCAP4 stimulated bovine rod outer segment GC in a Ca(2+)-dependent manner. RT-PCR with zGCAP specific primers showed specific expression of zGCAPs and zGCIP in the retina, while zGCAPl mRNA is also present in the brain. In situ hybridization with anti-sense zGCAP4, zGCAP5 and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. The presence of at least eight GCAP genes suggests an unexpected diversity within this subfamily of Ca(2+)-binding proteins in the teleost retina, and suggests additional functions for GCAPs apart from stimulation of GC. Based on genome searches and EST analyses, the mouse and human genomes do not harbor GCAP4-8 or GCIP genes.

Amino Acid Sequence↗
PubMedDate not supplied

The emergence of physiological genomics.

'Physiological genomics' represents a research paradigm shift emerging to define the functions of tens of thousands of newly discovered genes which are expected to emerge from the sequencing of the human genome and other model organisms. Genomic tools, which will allow a higher efficiency of identification of gene function, are being developed at remarkable speed. This article discusses some of the genomic and bioinformatic tools currently available or under development to provide the infrastructure for mapping and identification of gene function in simple organisms (bacteria, zebrafish, fly, worm) and complex mammalian organisms (mouse and rat). The problems facing the scientific community in the implementation of this functional approach are discussed as it is now evident that new technological and organizational infrastructures are emerging to link genes to overall function of whole organisms.

Animals↗
PubMed1999

WNT targets. Repression and activation.

Several puzzling observations made previously suggested that target genes that are activated by WNT signaling during development were actively repressed in the absence of the signal. Recent work sheds light on how this switch between repression and activation is regulated.

Animals↗
PubMed2001

Wnt receptors and Wnt inhibitors are expressed in gradients in the developing telencephalon.

The caudomedial margin of the medial pallium, known as the cortical hem, expresses several Wnt genes that have been shown to be crucial for cortical development. We examined the expression of members of the Frizzled (mFz) family of Wnt receptors and the Secreted Frizzled Related Protein (SFRP) family of Wnt inhibitors during telencephalic development. We found that mFz-5 and mFz-8 are specifically expressed in the neocortical neuroepithelium and excluded from the hippocampal neuroepithelium in early telencephalic development, whereas mFz-9 and mFz-10 have expression domains confined to the medial pallium. In addition, SFRP-1 and SFRP-3 are expressed in opposing anterolateral to caudomedial gradients within the telencephalic ventricular zone throughout corticogenesis.

Animals↗