Teleost Fundulus Heteroclitus Research Articles

Structural and functional differences in the promoter and 5' flanking region of Ldh-B within and between populations of the teleost Fundulus heteroclitus.

We have investigated the mechanisms underlying differences in the transcriptional regulation of lactate dehydrogenase-B (Ldh-B) between northern and southern populations of a teleost fish, Fundulus heteroclitus. A 1-kb region immediately 5' of the gene was sequenced from populations throughout the species range. There were two major allele classes in the sample, one containing alleles from Maine and another containing those from Florida. Populations from intermediate localities contained both allele classes. Some individuals from Georgia had sequences intermediate between the two classes, representing either ancestral alleles or recombinants. Tests of neutrality were applied to determine whether observed variation was consistent with neutral expectations. Significant deviations from neutral expectations were detected for the 5' flanking region, but not for other loci. The functional consequences of flanking sequence variation were assessed by transfection of reporter gene constructs into cultured cells and injection into living fish. Consistent with observed variation in Ldh-B transcription rate between populations, significant differences in reporter gene activity were driven by flanking regions from northern and southern populations both in cell culture and in vivo. This functional differentiation, coupled with departures from neutral expectations, suggests that selection may have acted on the regulation of Ldh-B in F. heteroclitus.

Cloning and Expression of a 5'-Iodothyronine Deiodinase from the Liver of Fundulus heteroclitus

Recent molecular cloning studies in mammals and amphibians have demonstrated that the types I, II, and III deiodinases constitute a family of selenoproteins of critical importance in metabolizing T4 to active (i.e. T3) and inactive (i.e. rT3) metabolites. In several tissues of teleost fish, various deiodinase processes have been described, but the structural and functional characteristics of these enzymes and their relationship to the deiodinases present in higher vertebrates remains uncertain. Using a complementary DNA library derived from the liver of the teleost Fundulus heteroclitus, we have identified a complementary DNA that codes for a deiodinase with functional characteristics virtually identical to those of the mammalian and amphibian type II deiodinase. Sequence analysis demonstrates a high degree of homology at both the nucleotide and predicted amino acid levels between the Fundulus clone and these previously characterized type II enzymes, including the presence of an in-frame TGA codon that codes for selenocysteine. These findings demonstrate that the deiodinase family of selenoproteins has been highly conserved during vertebrate evolution and underscores their importance in the regulation of thyroid hormone action.

Cloning and expression of a 5'-iodothyronine deiodinase from the liver of Fundulus heteroclitus.

Recent molecular cloning studies in mammals and amphibians have demonstrated that the types I, II, and III deiodinases constitute a family of selenoproteins of critical importance in metabolizing T4 to active (i.e. T3) and inactive (i.e. rT3) metabolites. In several tissues of teleost fish, various deiodinase processes have been described, but the structural and functional characteristics of these enzymes and their relationship to the deiodinases present in higher vertebrates remains uncertain. Using a complementary DNA library derived from the liver of the teleost Fundulus heteroclitus, we have identified a complementary DNA that codes for a deiodinase with functional characteristics virtually identical to those of the mammalian and amphibian type II deiodinase. Sequence analysis demonstrates a high degree of homology at both the nucleotide and predicted amino acid levels between the Fundulus clone and these previously characterized type II enzymes, including the presence of an in-frame TGA codon that codes for selenocysteine. These findings demonstrate that the deiodinase family of selenoproteins has been highly conserved during vertebrate evolution and underscores their importance in the regulation of thyroid hormone action.

A reverse transcription-polymerase chain reaction (RT-PCR) approach for cloning Ah receptors from diverse vertebrate species: Partial sequence of an Ah receptor from the teleost Fundulus heteroclitus

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related halogenated aromatic hydrocarbons. AhR proteins are expressed in most vertebrate animals, including teleost and elasmobranch fish, but the relationship between piscine and mammalian Ah receptors is uncertain. In the present studies, cloning of an Ah receptor cDNA from the teleost Fundulus heteroclitus was pursued using a reverse transcription-polymerase chain reaction (RT-PCR) approach. Degenerate oligodeoxynucleotides were designed based on conserved regions of mammalian Ah receptors and the related PAS proteins Per, ARNT and Sim. The oligonucleotides were used as primers in the RT-PCR to amplify a portion of an AhR expressed in Fundulus liver. A fragment of approximately 700 bp was amplified and directly sequenced. The deduced amino acid sequence of this fragment (212 residues) shares significant sequence identity with the PAS domains of the mouse (64%), rat (64%) and human (62%) Ah receptors. These data reveal conservation of Ah receptor structure between mammals and fish, vertebrate groups separated by over 400 million years of evolution. The successful use of RT-PCR to amplify a Fundulus AhR establishes the utility of this approach in the phylogenetic analysis of Ah receptor structure and function.

Evidence for Four Hox Clusters in the KillifishFundulus heteroclitus(Teleostei)

We analyzed the HOM/Hox cluster composition of the teleostFundulus heteroclitus(Killifish) using a PCR survey. We found a total number of 30 unique homeobox sequences which could be assigned to specific cognate groups of the Hox clusters by sequence comparisons. One sequence was of themshgroup. Ten homeobox fragments could be identified as orthologs with specific mammalian Hox genes of cluster A-D. The number of representatives in cognate groups 1, 4, 5, 6, 7, 8 and 9 differed from those of human and mouse. For groups 1 and 9 we found four representatives, which provides the first evidence for possible four Hox clusters in a diploid teleost, a lower vertebrate. Furthermore, it demonstrates significant differences of theFundulusclusters compared to those of mouse and humans. The implications of our data for the interpretation of the evolution of Hox clusters are discussed.

Functional Heterologous Gap Junctions in Fundulus Ovarian Follicles Maintain Meiotic Arrest and Permit Hydration during Oocyte Maturation

The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types. Octanol alone also initiated resumption of meiosis in follicle-enclosed oocytes, indicating that granulosa cells normally maintain meiotic arrest, as apparently occurs in mammalian and amphibian follicles. Both PMA and octanol also consistently inhibited the hydration process that normally accompanies meiotic maturation. These results support a previously suggested hypothesis that K+, which is the primary osmotic effector for oocyte hydration, is translocated via gap junctions from granulosa cells to the maturing oocyte.

The evolutionary significance of genetic variation at enzyme synthesizing loci in the teleost Fundulus heteroclitus

Using the teleost Fundulus heteroclitus as an experimental model, we demonstrate how a multi‐disciplinary approach to problems of intraspecies genetic variation can provide a better understanding of complex biodiversity problems than can be addressed by a more monolithic approach. We emphasize the importance of starting with simple molecular systems and making predictions that can be tested by experimentation at a higher level of biological complexity‐leading from molecules to cells to organ systems to organisms and eventually laboratory and field selection experiments. Using this interdisciplinary approach, we address the classical ‘selectionisti neutralist’ controversy.

The temporal relationships between P450E protein content, catalytic activity, and mRNA levels in the teleost Fundulus heteroclitus following treatment with β-naphthoflavone

P450 induction occurs in some marine organisms following chemical exposure. The mode of 3-methylcholanthrene (MC)-type induction was evaluated by examining hepatic isozyme P450E content, catalytic activity, and mRNA levels in the marine teleost Fundulus heteroclitus after exposure to a single dose of β-naphthoflavone (BNF). P450E is the major teleost P450 induced by MC-type compounds and is the catalyst for aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities. In a 20-day experiment, EROD activity was elevated in BNF-treated animals from Day 4 through Day 20. Increases in immunodetectable P450E showed the same trend as EROD activity, with consistently low control values and at least a 19-fold increase in the BNF-treated fish. Precipitation of liver RNA in vitro translation products with anti-P450E antibody gave no detectable signal from control fish, while the BNF-treated animals showed incorporation of [ 3H]leucine in a single 6,000 M r band. In a shorter term experiment, EROD activity and P450E levels were again coordinately increased in response to BNF treatment, and immunoprecipitation of translation products from these fish showed a clear trend of increased P450E mRNA levels for all time points 6 h or more post-treatment. Hybridization of RNA from BNF-treated Fundulus with a trout P450IA1 cDNA also showed increases in a single band with time. The increases in P450E mRNA preceded increases in P450E protein and enzyme activity by about 25 h. However, P450E mRNA declined rapidly, reaching control levels by 5 days, while protein levels remained elevated for at least 13 days. The results support a hypothesis that transcriptional enhancement is involved in induction of MC-inducible P450s in fish, but indicate that P450E induction is also under other forms of regulatory control.

A novel dehydrogenase reaction mechanism for hexose-6-phosphate dehydrogenase isolated from the teleost Fundulus heteroclitus.

Hexose-6-phosphate dehydrogenase (refers to hexose-6-phosphate dehydrogenase from any species in general) has been purified to apparent homogeneity from the teleost fish Fundulus heteroclitus. The enzyme was characterized for native (210 kDa) and subunit molecular mass (54 kDa), isoelectric point (6.65), amino acid composition, substrate specificity, and metal dependence. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucose 6-sulfate, glucosamine 6-phosphate, and glucose were found to be substrates in the reaction with NADP+, but only glucose was a substrate when NAD+ was used as coenzyme. A unique reaction mechanism for the forward direction was found for this enzyme when glucose 6-phosphate and NADP+ were used as substrates; ordered with glucose 6-phosphate binding first. NAD+ was found to be a competitive inhibitor toward NADP+ and an uncompetitive inhibitor with regard to glucose 6-phosphate in this reaction; Vmax = 7.56 mumol/min/mg, Km(NADP+) = 1.62 microM, Km(glucose 6-phosphate) = 7.29 microM, Kia(glucose 6-phosphate) = 8.66 microM, and Ki(NAD+) = 0.49 microM. The use of alternative substrates confirmed this result. This type of reaction mechanism has not been previously reported for a dehydrogenase.

Removal of enveloping follicle cells can trigger resumption of meiotic maturation in Fundulus heteroclitus oocytes

AbstractThe various somatic cell layers surrounding large (1.3–1.4 mm in diameter) meiosis‐arrested oocytes of the saltmarsh teleost Fundulus heteroclitus were successively removed by a combination of manual dissection and exposure to calcium/magnesium‐free medium. The efficacy of these methods was verified by light and scanning electron microscopy. Oocytes within intact follicles, and oocytes in follicles with the outermost surface epithelium and theca layers removed, typically resumed meiosis only when treated with either a steroid, 17α‐hydroxy‐20β‐dihydroprogesterone, or an aqueous extract of Fundulus pituitaries. In contrast, removal of the follicle cell layer immediately surrounding the oocyte frequently triggered the resumption of meiosis in the absence of exogenous hormonal stimulation. These results suggest that the follicle cells may somehow be responsible for the maintenance of meiotic arrest in non‐hormone‐treated oocytes.

Programmed Endocytosis During Epiboly ofFundulus heteroclitus

A major question in the analysis of teleost epiboly is the fate of the yolk cytoplasmic layer. It diminishes during epiboly and eventually disappears at the completion of epiboly. This paper is concerned with the fate of the surface of the yolk cytoplasmic layer during epiboly. When gastrulae during epiboly are bathed in lucifer yellow (CH) and then observed with fluorescent microscopy or bathed in ferritin and then fixed and observed with TEM, a thin circumferential ring of endocytic vesicles is observed, confined to the external yolk syncytial layer just peripheral to the advancing margin of the blastoderm. Even though the entire egg is immersed in the marker, endocytosis is confined to this limited region. More precisely, this endocytosis occurs only within the region of the external yolk syncytial layer, where the surface is most folded. The endocytic vesicles thus formed move downward and settle on the surface of the membrane separating the yolk from the cytoplasm in the yolk syncytial layer. They do not join the surface of the internal yolk syncytial layer; hence they do not contribute to its expansion. Prior to the onset of epiboly there is no such endocytosis at the surface of the egg. Since this endocytosis occurs only during epiboly and only at the surface of the external yolk syncytial layer just peripheral to the advancing margin of the blastoderm, and in the absence of large molecules in the medium, we conclude that it is programmed. We, therefore, present this as a case of programmed internalization of cell surface serving as the morphogenetic mechanism responsible for the disappearance of the surface of the yolk cytoplasmic layer during gastrulation of the teleost Fundulus heteroclitus

The role of K+ and Cl- conductances in chloride secretion by the opercular epithelium.

The opercular epithelium of the teleost Fundulus heteroclitus, when mounted in a lucite chamber under short-circuited conditions, secretes Cl- at a rate equivalent to the short-circuit current (Isc). The transepithelial Na+ movements are passive and proceed by a paracellular pathway. The addition of 2 X 10(-3) M Ba2+ to the serosal bathing solution inhibited the Isc 76.8% with no effect on the transepithelial conductance (Gt). The addition of 5 X 10(-4) M Cu2+ to the mucosal bathing solution inhibited the Isc 79.6% and reduced the Gt 35.6%. These inhibitory effects of Ba2+ and Cu2+ on the Isc were initiated within 1 minute after exposure with maximum effects occurring within 20 and 30 minutes, respectively. Simultaneous 36Cl- and 22Na+ unidirectional fluxes were performed on paired epithelia from the same fish. Serosal Ba2+ and mucosal Cu2+ inhibited the Cl- secretory flux 30.2 and 58.9%, respectively. The resulting net Cl- flux after inhibition was not significantly different from the mean measured Isc. Neither ion had significant effects on the Cl- influx (mucosa to serosa) or the unidirectional Na fluxes. These results indicated that the effects of both Ba2+ and Cu2+ were most likely exclusive to the transcellular Cl- pathway. Ba2+ is proposed to inhibit Cl- secretion by blocking the basolateral K+ channels, depolarizing the cell, and reducing the electrochemical driving force for Cl- across the apical membrane. Cu2+ is proposed to inhibit Cl- secretion by blocking the apical membrane Cl- channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.

Biochemical genetics of Fundulus heteroclitus (L.). IV. Spatial variation in gene frequencies of Idh-A, Idh-B, 6-Pgdh-A, and Est-S.

The spatial variation in gene frequencies of four unlinked polymorphic loci was studied in the teleost Fundulus heteroclitus. Three loci (Idh-A, Idh-B, and Est-S) exhibit significant north-south clinal variation in allelic frequencies along the Atlantic Coast of North America, while a fourth locus (6-Pgdh-A) shows a modest clinal variation. These data, together with our previous data for Ldh-B, Mdh-A, Gpi-B, and Pgm-A, reveal a pattern of low gene diversity in the colder northern extremes of the species range and high gene diversity in warmer southern latitudes.

Glucosephosphate isomerase (GPI) of the teleostFundulus heteroclitus (linnaeus): Isozymes, allozymes and their physiological roles

In order to evaluate the role of glucose-phosphate isomerase (GPI) inFundulus heteroclitus, the isozymes and allozymes were purified and some of their physical and kinetic properties determined.

Metabolic effects of pancreatectomy and hypophysectomy in the yellow American eel, Anguilla rostrata LeSueur

Metabolic effects of pancreatectomy and hypophysectomy were compared in the yellow American eel, Anguilla rostrata. It is reconfirmed that in the yellow eel pancreatectomy does not cause the classical symptoms of diabetes mellitus. In the present experiment, the operation was followed by a strong, stress-related hyperglycemic tendency. Liver glycogen was restored after an initial drop. Neither breakdown of adipose tissue nor an increase in serum free fatty acids or cholesterol was observed. Serum amino acid nitrogen was temporarily increased. Hypophysectomized eels showed a tendency to lower levels of serum glucose. Liver and muscle glycogen were statistically significantly lowered. Contrary to observations in the teleost Fundulus heteroclitus and the rat, the hypophysectomized eels showed a hypocholesteremia. No hyperuremia was seen, which again is in contrast to observations in F. heteroclitus. Both pancreatectomy and hypophysectomy were followed by a drop of the hematocrit and an increase in tissue hydration which persisted in the hypophysectomized eels 20 days after surgery. When both operations were combined, the eels survived only in 1:3 seawater, but not in fresh water or 2:3 seawater. The question of an osmoregulatory role of the islet organ is briefly discussed. Surgical stress affected probably all parameters with the possible exception of the abdominal fat stores. When the data of this study are integrated in an overall phylogenetic scale, it appears that in the intermediary metabolism the role of the pituitary is more conservative, while the importance of islet hormones increases in the tetrapodes.

Observations on the Structure of the Skin of the Teleost <i>Fundulus heteroclitus</i> (L)

Morphological and histochemical observations were realized on the body and fin skin of the teleost Fundulus heteroclitus. Attention was called to the following aspects:1. The presence and origin of a continuous mucous layer covering the superficial epithelium.2. The presence and characteristics of peculiar ridge-like structures on the superficial epithelium.3.The occurence of the so called “chloride cells” in this animal's epidermis.4. The presence of a cushion-like mucous connective tissue layer between the epidermis and the scales.5. An analysis of the scale's structure and its position in the dermis was performed.The functional significance of the above-mentioned structures is discussed.

Role of cell division in gastrulation of the viviparous toothcarp Xiphophorus helleri.

RECENTLY it was stated that morphogenetic movements in gastrulation of the viviparous teleost Fundulus heteroclitus occur in a normal fashion in cases in which cytokinesis is prevented by treatment with the antimitotic alkaloid colchicine1. To establish whether a more general significance should possibly be attached to this phenomenon a corresponding investigation was performed with a fish bearing live young, namely the viviparous cyprinodont Xiphophorus helleri, the swordtail. For this purpose pregnant females in the first phase of gestation were injected intraperitoneally with 0.5 ml. of 0.001 M colchicine solution. During gastrulation in stage 12 (beginning gastrula), 13 (advanced gastrula), 14 (further advanced gastrula) and 15 (closure of blastopore) both treated and control females were killed by decapitation and the ovaries were fixed in Bouin's fluid. The blastoderms were sectioned at 5µ and stained with Heidenhain's iron haematoxylin. The mitotic index, representing the ratio of cells in mitosis to the total number of cells, and expressed as per cent, was determined in each blastoderm on the basis of cell counts of the sections.