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PubMed2002

First steps of otolith formation of the zebrafish: role of glycogen?

The first steps of otolith formation were studied by electron microscopy in zebrafish embryos at different postfertilization (PF) time intervals. Between 19 and 22 h PF, the otic cavity contains glycogen particles derived by an apocrine process from the apical portions of the epithelial cells of the inner ear. The particles are arranged in parallel arrays, then in pseudocrystalloid structures, and finally in concentric arrays to form dense clusters referred to as "spherules". At 23 h PF, a group of "globules", consisting of modified aggregated "spherules" surrounded by several free "spherules", forms the nascent otolith. At 30 h PF, fused globules form a roughly spherical otolith. Spherules undergoing their process of modification and aggregation, are located in its central part, and constitute the so-called "nucleus". At 50 h PF, the otolith is a flattened hemisphere. It is made up of fused globules surrounded by two concentric layers whose organization is similar to that observed in the otolith of the adult fish. At this stage, calcium may be detected in the otolith except in its nucleus. We suggest that glycogen molecules found in the nascent otolith might allow the insertion of molecules such as glycoproteins (collagens) which are known to fix calcium. As a result, glycogen might play a key role in initiating the formation of otoliths and possibly that of other calcified tissues.

Animals↗

PubMed2003

A 90-degree rotation of the mitotic spindle changes the orientation of mitoses of zebrafish neuroepithelial cells.

In the neural plate and neural tube in the trunk region of the zebrafish embryo, dividing cells are oriented parallel to the plane of the neuroepithelium, while in neural keel/rod, cells divide perpendicular to it. This change in the orientation of mitosis is brought about by a 90 degrees rotation of the mitotic spindle. As the two halves of the neural primordium in keel/rod stage are in apposition, the perpendicular orientation of mitoses in this stage determines that daughter cells become allocated to both sides of the neural tube. To assess the role played by cell junctions in controlling the orientation of dividing cells, we studied the expression of components of adherens and tight junctions in the neuroepithelial cells. We find that these proteins are distributed irregularly at the neural plate stage and become polarised apically in the cell membrane only during the keel/rod stage. The stereotypic orientation of mitoses is perturbed only weakly upon loss of function of the cell junction components ASIP and aPKClambda, suggesting that mitotic orientation depends in part on the integrity of cell junctions and the polarity of the epithelium as a whole. However, the 90-degree rotation of the spindle does not require perfectly polarised cell junctions between the neuroepithelial cells.

Animals↗

PubMed2004

The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish.

To examine a putative role for neuronal nitric oxide synthase (nNOS) in early vertebrate development we investigated nNOS mRNA expression and cGMP production during development of the zebrafish Danio rerio. The nNOS mRNA expression in the central nervous system (CNS) and periphery showed a distinct spatio-temporal pattern in developing zebrafish embryo and young larvae. nNOS mRNA expression was first detected at 19 h postfertilisation (h.p.f.), in a bilateral subpopulation of the embryonic ventrorostral cell cluster in the forebrain. The number of nNOS mRNA-expressing cells in the brain slowly increased, also appearing in the ventrocaudal cell cluster from about 26 h.p.f., and in the dorsorostral and hindbrain cell cluster and in the medulla at 30 h.p.f. A major increase in nNOS mRNA expression started at about 40 h.p.f., and by 55 h.p.f. the expression constituted cell populations in differentiated central nuclei and in association with the proliferation zones of the brain, and in the medulla and retina. In parts of the skin, nNOS mRNA expression started at 20 h.p.f. and ended at 55 h.p.f. Between 40 and 55 h.p.f., nNOS mRNA expression started in peripheral organs, forming distinct populations after hatching within or in the vicinity of the presumptive swim bladder, enteric ganglia, and along the alimentary tract and nephritic ducts. Expression of nNOS mRNA correlated with the neuronal differentiation pattern and with the timing and degree of cGMP production. These studies indicate spatio-temporal actions by NO during embryogenesis in the formation of the central and peripheral nervous system, with possible involvement in processes such as neurogenesis, organogenesis and early physiology.

Animals↗

PubMed2001

Estrogen and xenoestrogens upregulate the brain aromatase isoform (P450aromB) and perturb markers of early development in zebrafish (Danio rerio).

Estrogen synthesized in the brain itself by the action of cytochrome P450 aromatase (P450arom) is known to have permanent organizing effects on the developing CNS. In fish, estrogen upregulates the predominant brain isoform (P450aromB), implying that xenoestrogens (XE) could act as neurodevelopmental toxicants by altering P450aromB. To test this hypothesis, zebrafish embryos were exposed to 17beta-estradiol (E(2)), diethylstilbestrol (DES, a potent agonist), and bisphenol A (BPA, a weak agonist). RT-PCR/Southern transfer analysis showed that E(2) (0.01-10 microM) upregulated P450aromB in a dose-response manner. The effect of DES (0.01 microM) was similar to 1 microM E(2) (three- to four-fold higher than control), but BPA was less effective (<threefold increase at 10 microM). mRNA levels of the predominant ovarian isoform (P450aromA) were unchanged by estrogen. Treatment with E(2) (0.1-10 microM) between 2 and 72 hpf had dose-response effects on mortality and hatching and induced a 'curved tail down' phenotype characteristic of mutants with defects of early CNS development. The critical period of estrogen sensitivity for effects on mortality and curved tails was 2-24 hpf, whereas hatching effects were both stage- and duration-dependent. Developmental effects of DES and BPA were similar to E(2) but testosterone, and 5alpha-dihydrotestosterone were ineffective. 17alpha-Estradiol showed a small but significant effect on curved tails. We conclude that P450aromB mRNA is a sensitive marker of XE effect during embryogenesis, but further studies are required to determine whether changes in neural aromatase expression and estrogen biosynthesis have consequences for CNS development.

Animals↗

PubMed1998

Myocyte enhancer factor 2C and Nkx2-5 up-regulate each other's expression and initiate cardiomyogenesis in P19 cells.

The Nkx2-5 homeodomain protein plays a key role in cardiomyogenesis. Ectopic expression in frog and zebrafish embryos results in an enlarged myocardium; however, expression of Nkx2-5 in fibroblasts was not able to trigger the development of beating cardiac muscle. In order to examine the ability of Nkx2-5 to modulate endogenous cardiac specific gene expression in cells undergoing early stages of differentiation, P19 cell lines overexpressing Nkx2-5 were differentiated in the absence of Me2SO. Nkx2-5 expression induced cardiomyogenesis in these cultures aggregated without Me2SO. During differentiation into cardiac muscle, Nkx2-5 expression resulted in the activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In order to compare the abilities of Nkx2-5 and MEF2C to induce cellular differentiation, P19 cells overexpressing MEF2C were aggregated in the absence of Me2SO. Similar to Nkx2-5, MEF2C expression initiated cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2-5, GATA-4, cardiac alpha-actin, and myosin heavy chain expression. These findings indicate the presence of a positive regulatory network between Nkx2-5 and MEF2C and show that both factors can direct early stages of cell differentiation into a cardiomyogenic pathway.

Actins↗

PubMed2006

Specification of the primitive myeloid precursor pool requires signaling through Alk8 in zebrafish.

In the zebrafish embryo, primitive hematopoiesis initiates in two spatially distinct regions. Rostrally, the cells of the anterior lateral plate mesoderm (ALPM) give rise exclusively to cells of the myeloid lineage in a pu.1-dependent manner. Caudally, in the posterior lateral plate mesoderm (PLPM), the expression of gata1 defines a precursor pool that gives rise predominantly to the embryonic erythrocytes. The transcription factor scl acts upstream of both gata1 and pu.1 in these precursor pools, activating a series of conserved transcription factors that cell-autonomously specify either myeloid or erythroid fates. However, the mechanisms underlying the spatial separation of the hematopoietic precursor pools and the induction of differential gene expression within these pools are not well understood. We show here that the Bmp receptor lost-a-fin/alk8 is required for rostral pu.1 expression and myelopoiesis, identifying an early genetic event that distinguishes between the induction of anterior and posterior hematopoiesis. Introducing a constitutively active version of the Alk8 receptor led to increased pu.1 expression, but the role of alk8 was independent of the scl-dependent cell-fate pathway. Furthermore, the role of Alk8 in myelopoiesis was genetically separable from its earlier role in dorsal-ventral embryonic patterning.

Activin Receptors, Type I↗

PubMed1998

Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis.

We have taken advantage of the optical transparency of zebrafish embryos to investigate the patterns of cell division, movement and shape during early stages of development of the central nervous system. The surface-most epiblast cells of gastrula and neurula stage embryos were imaged and analysed using a computer-based, time-lapse acquisition system attached to a differential interference contrast (DIC) microscope. We find that the onset of gastrulation is accompanied by major changes in cell behaviour. Cells collect into a cohesive sheet, apparently losing independent motility and integrating their behaviour to move coherently over the yolk in a direction that is the result of two influences: towards the vegetal pole in the movements of epiboly and towards the dorsal midline in convergent movements that strengthen throughout gastrulation. Coincidentally, the plane of cell division becomes aligned to the surface plane of the embryo and oriented in the anterior-posterior (AP) direction. These behaviours begin at the blastoderm margin and propagate in a gradient towards the animal pole. Later in gastrulation, cells undergo increasingly mediolateral-directed elongation and autonomous convergence movements towards the dorsal midline leading to an enormous extension of the neural axis. Around the equator and along the dorsal midline of the gastrula, persistent AP orientation of divisions suggests that a common mechanism may be involved but that neither oriented cell movements nor shape can account for this alignment. When the neural plate begins to differentiate, there is a gradual transition in the direction of cell division from AP to the mediolateral circumference (ML). ML divisions occur in both the ventral epidermis and dorsal neural plate. In the neural plate, ML becomes the predominant orientation of division during neural keel and nerve rod stages and, from late neural keel stage, divisions are concentrated at the dorsal midline and generate bilateral progeny (C. Papan and J. A. Campos-Ortega (1994) Roux's Arch. Dev. Biol. 203, 178-186). Coincidentally, cells on the ventral surface also orient their divisions in the ML direction, cleaving perpendicular to the direction in which they are elongated. The ML alignment of epidermal divisions is well correlated with cell shape but ML divisions within the neuroepithelium appear to be better correlated with changes in tissue morphology associated with neurulation.

Animals↗

PubMed2004

Inhibition of Wnt/Axin/beta-catenin pathway activity promotes ventral CNS midline tissue to adopt hypothalamic rather than floorplate identity.

Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, we have explored the role of the Wnt/Axin/beta-catenin pathway in this process. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.

Animals↗

PubMed2003

Effects of 17alpha-ethynylestradiol in a partial life-cycle test with zebrafish (Danio rerio): effects on growth, gonads and female reproductive success.

In this study, the impact of an exposure of zebrafish embryos (Danio rerio) until 3 months post fertilization to 17alpha-ethynylestradiol (EE2: 0; 0,1; 1; 10; 25 ng/l) was evaluated for growth and development, gonad development and body vitellogenin (VTG) content. After a recovery period of 5 months, the female reproductive success was evaluated. The results demonstrated a significant reduction in total body length, body weight, whole body Ca and P content and an increase in morphological abnormalities for fish exposed to 25 ng/l EE2 as a function of exposure time. An increase in total body VTG content was observed for fish exposed for a period of 3 months down to levels of 1 ng/l EE2. At the age of 3 months, a dose-dependent increase of the number of fish with no macroscopic recognizable gonads was observed (up to 100% at 25 ng/l EE2). After a recovery period on clean tap water for 5 months, all fish had developed either ovaries or testis with a gonadosomatic index not different from control fish and the sex ratio was similar in EE2 treatment groups and controls. Nevertheless, a reduced number of spawning females and a reduced egg production were found for the female fish exposed to 10 or 25 ng/l EE2 for 3 months and which were allowed to recover for 5 months. Although the underlying mechanism could not be elucidated, these findings did indicate that the reproduction potential of fish populations might be disturbed by a long-term exposure to EE2 (> or =10 ng/l) from fertilization until sexual maturity.

Animals↗

PubMed2002

Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin.

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

Alternative Splicing↗

PubMed1996

Jaw and branchial arch mutants in zebrafish I: branchial arches.

Jaws and branchial arches together are a basic, segmented feature of the vertebrate head. Seven arches develop in the zebrafish embryo (Danio rerio), derived largely from neural crest cells that form the cartilaginous skeleton. In this and the following paper we describe the phenotypes of 109 arch mutants, focusing here on three classes that affect the posterior pharyngeal arches, including the hyoid and five gill-bearing arches. In lockjaw, the hyoid arch is strongly reduced and subsets of branchial arches do not develop. Mutants of a large second class, designated the flathead group, lack several adjacent branchial arches and their associated cartilages. Five alleles at the flathead locus all lead to larvae that lack arches 4-6. Among 34 other flathead group members complementation tests are incomplete, but at least six unique phenotypes can be distinguished. These all delete continuous stretches of adjacent branchial arches and unpaired cartilages in the ventral midline. Many show cell death in the midbrain, from which some neural crest precursors of the arches originate. lockjaw and a few mutants in the flathead group, including pistachio, affect both jaw cartilage and pigmentation, reflecting essential functions of these genes in at least two neural crest lineages. Mutants of a third class, including boxer, dackel and pincher, affect pectoral fins and axonal trajectories in the brain, as well as the arches. Their skeletal phenotypes suggest that they disrupt cartilage morphogenesis in all arches. Our results suggest that there are sets of genes that: (1) specify neural crest cells in groups of adjacent head segments, and (2) function in common genetic pathways in a variety of tissues including the brain, pectoral fins and pigment cells as well as pharyngeal arches.

Animals↗

PubMed2005

XTbx1 is a transcriptional activator involved in head and pharyngeal arch development in Xenopus laevis.

The development of pharyngeal arch derivatives in mouse and zebrafish embryos depends on the activity of the transcription factor Tbx1. We cloned the Xenopus laevis orthologue of Tbx1 (XTbx1) and show that the pattern of expression is similar to that in other vertebrate species. Zygotic transcripts are first detected shortly after the mid-blastula transition and are localized to the presumptive mesoderm at mid-gastrula stages. XTbx1 expression persists in the lateral plate mesoderm at neurula stages and is found in the pharyngeal arches and otic vesicles from early tail bud stages onward. We demonstrate that XTbx1 is a transcriptional activator and that this trans-activation requires the C-terminal region of the protein. A dominant interfering mutant of XTbx1 disrupts the development of Xenopus head structures and pharyngeal arch derivatives. Lineage labeling reveals a requirement for XTbx1 function in cells that contribute to the pharyngeal mesoderm and for fgf8 expression.

Animals↗

PubMed2004

Development of the nervus terminalis: origin and migration.

The origin of the nervus terminalis is one of the least well understood developmental events involved in generating the cranial ganglia of the forebrain in vertebrate animals. This cranial nerve forms at the formidable interface of the anteriormost limits of migrating cranial neural crest cells, the terminal end of the neural tube and the differentiating olfactory and adenohypophyseal placodes. The complex cellular interactions that give rise to the various structures associated with the sensory placode (olfactory) and endocrine placode (adenohypophysis) surround and engulf this enigmatic cranial nerve. The tortured history of nervus terminalis development (see von Bartheld, this issue, pages 13-24) reflects the lack of consensus on the origin (or origins), as well as the experimental difficulties in uncovering the origin, of the nervus terminalis. Recent technical advances have allowed us to make headway in understanding the origin(s) of this nerve. The emergence of the externally fertilized zebrafish embryo as a model system for developmental biology and genetics has shed new light on this century-old problem. Coupled with new developmental models are techniques that allow us to trace lineage, visualize gene expression, and genetically ablate cells, adding to our experimental tools with which to follow up on studies provided by our scientific predecessors. Through these techniques, a picture is emerging in which the origin of at least a subset of the nervus terminalis cells lies in the cranial neural crest. In this review, the data surrounding this finding will be discussed in light of recent findings on neural crest and placode origins.

Animals↗

PubMed2007

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein betagamma dimers: consequences on G protein activation and stability.

It is generally accepted that G protein coupled receptors (GPCR) activate heterotrimeric G proteins by inducing a GDP/GTP exchange at the G protein alpha subunit. In addition, the transfer of high energetic phosphate by nucleoside diphosphate kinase (NDPK) and/or the beta subunit of G proteins (Gbeta) can induce G protein activation. Recent evidence suggests that the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers. In this complex, NDPK B acts as a protein histidine kinase phosphorylating Gbeta at histidine residue 266 (His266). The high energetic phosphoamidate bond on His266 allows for a phosphate transfer specifically onto GDP and thus local formation of GTP, which binds to and thereby activates the respective G protein alpha subunit. Apparently, this process occurs independent of the classical GPCR-induced GDP/GTP exchange at least for members of the G(s) and G(i) subfamilies of heterotrimeric G proteins. By using a mutant of Gbeta(1) in which His266 was replaced by Leu, it was recently demonstrated that NDPK B/Gbetagamma-mediated G(s) activation contributes by about 50% to basal cAMP formation and contractility in rat cardiac myocytes. Besides its apparent role in G protein activation, the complex formation of NDPK B with Gbetagamma dimers might be essential for G protein stability. Depletion of either the NDPK B orthologue or Gbeta(1) isoforms in zebrafish embryos led to a similar phenotype displaying contractile dysfunction in the heart accompanied by a complete loss of heterotrimeric G protein expression. In conclusion, the interaction of NDKP B with Gbetagamma dimers might play an important role in signal transduction, and alterations in this novel pathway might be of pathophysiological importance.

Animals↗

PubMed2004

Analysis of EphA4 in the lesser spotted catshark identifies a primitive gnathostome expression pattern and reveals co-option during evolution of shark-specific morphology.

The Eph family is the largest known group of structurally related receptor tyrosine kinases (RTKs). Each Eph receptor has a specific Ephrin ligand, and these function to define spatial boundaries during development. Analyses of EphA4 in mouse, chick, frog and zebrafish embryos have implicated this gene in a number of developmental processes, including maintenance of segmental boundaries, axon guidance, limb development, neural crest migration and patterning of the ear. In order to determine which components of EphA4 function may be primitive for gnathostomes, we cloned EphA4 from the lesser spotted catshark ( Scyliorhinus canicula) and examined its expression pattern during shark embryonic development. Consistent with the patterns reported for bony fish and tetrapods, we observed segmental expression of EphA4 in the developing hindbrain and later in the pharyngeal arches of shark embryos. EphA4 was also detected during sensory organogenesis, in the developing ear, eye, nasal pits and lateral line. A dynamic pattern of EphA4expression occurs during shark fin development, suggesting an early role in outgrowth and patterning of the fin buds and a later role in tissue differentiation. We also observed several novel domains of EphA4expression that have not been reported in other vertebrates, including external gill buds, dermal denticles, median fins and claspers. While some of these domains may reflect co-option of EphA4expression to novel sites for development of shark-specific characters, others are more likely to be ancestral patterns of expression that were lost in other vertebrate lineages.

Amino Acid Sequence↗

PubMed2002

Generation of two-color transgenic zebrafish using the green and red fluorescent protein reporter genes gfp and rfp.

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F(1) transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.

Journal Article↗

PubMed2000

Overexpression of a Zebrafish ARNT2-like Factor Represses CYP1A Transcription in ZLE Cells.

Aryl hydrocarbon receptor nuclear translocator (ARNT) factors belong to a novel basic-helix-loop-helix-PAS (bHLH-PAS) transcription factor family that controls a variety of physiological and developmental processes. In a previous study, we obtained a partial complementary DNA fragment of an ARNT2-like factor from zebrafish embryo, liver, and other tissues by reverse transcription-polymerase chain reaction. In an effort to characterize the function of this factor, we screened an embryonic complementary DNA library and obtained a complete cDNA of this ARNT2-like factor, zARNT2A. The deduced protein sequence of zARNT2A encompasses the basic-helix-loop-helix and PAS-A/B motifs and shares highest sequence similarity with the amino-terminal half of mouse ARNT2 factor. However, it lacks a carboxy-terminal transactivation motif following the PAS-A/B motifs. Transient expression of zARNT2A in cultured cells resulted in repression of TCDD-dependent CYP1A transcription. Whole-mount in situ hybridization revealed that zARNT2A is expressed in brain and pronephros at prime-5 stages. In adult fish, zARNT2A messenger RNA is transcribed in a wide range of tissues, which indicates that zARNT2A and its corresponding signal transduction mechanisms have important roles in fish development and other physiological aspects.

Journal Article↗

PubMed2005

Isolation and characterization of two cytochrome P450 aromatase forms in killifish (Fundulus heteroclitus): differential expression in fish from polluted and unpolluted environments.

Populations of killifish (Fundulus heteroclitus) persist in many different highly polluted environment indicative of adaptation or tolerance. In this study, we sought to determine whether long term, multigenerational exposures to environmental contaminants has affected reproductively relevant genes and biological processes. A homology cloning strategy was used to isolate the killifish cytochrome P450 aromatase (P450arom, estrogen synthetase) cDNAs. Consistent with previous fish studies, killifish were found to have two P450arom forms, which segregated into A- and B-gene clades and were differentially expressed in brain (B >> A) and ovary (A >> B). Comparison of killifish from highly polluted (New Bedford Harbor, NBH) and unpolluted (Scorton Creek, SC) environments revealed no site-related differences in P450arom coding sequences or in overall tissue distribution patterns. As measured by real-time quantitative PCR (QPCR) analysis, however, P450arormB (a known marker of estrogen effect) was approximately two-fold higher in the brain of NBH than of SC fish, a difference seen in reproductively active and inactive males and females. Providing further evidence of exposure to estrogen-like pollutants or metabolites in NBH, vitellogenin (vtg) mRNA and protein were elevated in seasonally active and inactive males, and in reproductively inactive females, when compared to SC fish. By contrast, during the period of reproductive activity, NBH females had a lower gonadosomatic index, lower plasma estrogen, a decreased hepatosomatic index, and reduced vtg expression as compared to SC females, indicating that the female hypothalamic-pituitary-gonadal (HPG)-liver axis is impaired in the polluted environment. As measured by a decrease in plasma androgen (but not GSI), the male HPG axis was impaired in reproductively active NBH versus SC fish. In agreement with reports that NBH killifish are resistant to dioxin-like chemicals (DLC) that activate arylhydrocarbon receptor (AhR) signaling, ovarian P450aromA (a marker of dioxin-like effect in zebrafish embryos) did not differ in SC and NBH fish. In conclusion, the killifish population at the NBH Superfund site maintains a level of reproductive competence in the face of evidence of exposure to estrogen-like pollutants and endocrine disruption.

Amino Acid Sequence↗