Jon Vidar Helvik

The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg hardening

The highly hydrophobic protein aggregate which constitutes the fish eggshell has for the first time been quantitatively solubilized. This study shows that the nonactivated eggshell from cod is composed primarily of only three protein monomers, designated alpha (74 kDa) beta (54 kDa) and gamma (47 kDa). Protein extraction studies of the eggshells before and after egg activation demonstrate that egg hardening is accompanied by a 10-fold decline in total protein solubility, which is due to nonextraction of the alpha, beta, and gamma chains. When present during the egg activation process monodansylcadaverine (MDC-a fluorescent lysine analog) inhibits eggshell hardening and at the same time becomes covalently incorporated into the eggshell. This MDC incorporation is calcium-dependent and suggests the induction of a perivitelline transglutaminase activity after egg activation. (Transglutaminases catalyze the formation of an amide bond (isopeptide bond) between the gamma-carbonyl group of glutamine and the epsilon-amino group of lysine with release of ammonia. Crosslinks between proteins are generated when the two amino acid residues are located on different proteins.) Protein solubilization studies and NaDodSO4 gel analysis of the eggshell proteins from eggs subjected to 5 mM MDC during egg activation, reveal that when eggshell hardening is blocked by MDC, the three main eggshell proteins remain extractable even after egg activation. Simultaneously we observed a covalent incorporation of MDC into the gamma protein.

Isolation and characterization of two teleost melanopsin genes and their differential expression within the inner retina and brain

Melanopsin is a newly discovered photopigment that is believed to be involved in the regulation of circadian rhythms in tetrapods. Here we describe the characterization of the first two teleost melanopsins (opn4a and opn4b) isolated from Atlantic cod (Gadus morhua). These two teleost genes belong to a subgroup of melanopsins that also include members from Xenopus, chicken, and Takifugu. In situ hybridization revealed that opn4a and opn4b are differentially expressed within the retina and brain. In the larval and adult retina, both melanopsins are expressed in a subset of cells in the inner retina, resembling amacrine and ganglion cells. In addition, opn4a is expressed in the horizontal cells, indicating a separate task for this gene. In the brain, the two melanopsins are separately expressed in two major retinal and extraretinal photosensitive integration centers, namely, the suprachiasmatic nucleus (opn4a) and the habenula (opn4b). The expression of opn4a in the suprachiasmatic nucleus in cod is similar to the melanopsin expression found in Xenopus. This suggests a conserved role for this opsin and an involvement in mediation of nonvisual photoreceptive tasks, such as entraining circadian rhythms and/or hypophysiotrophic systems. The differential expression of opn4b in the habenula suggests that this gene plays a role similar to that of opn4a, in that it is also situated in an area that integrates photic inputs from the pineal as well as other brain regions. Thus, the habenula may be an additional region that mediates photic cues in teleosts. J. Comp. Neurol. 456:84–93, 2003.

Ontogeny of ultraviolet-sensitive cones in the retina of rainbow trout (Oncorhynchus mykiss)

In order to facilitate emerging models of retinal development, we developed electroretinogram and in situ hybridization protocols to examine the ontogeny of photoreceptors in the retina of a land‐locked salmonid, the rainbow trout (Oncorhynchus mykiss). We cloned cDNA fragments corresponding to the rod opsin and each of the four cone opsin gene families, which we utilized to produce riboprobes. We established the specificity of the in situ hybridization protocol by examining subcellular signal localization and through double‐labeling experiments. We confirm the assumption that the accessory corner cones in the square mosaic are the ultraviolet wavelength‐sensitive (UVS) cone photoreceptor (i.e., they express an SWS1 opsin) and observed UVS cones throughout the retina of small trout. Larger fish have a decrease in sensitivity to short wavelength light stimuli and the distribution of UVS cones in the mature retina is limited to the dorsal‐temporal quadrant. These larger fish also possess differentiated UVS cones in the peripheral germinal zone (PGZ), including within areas peripheral to mature retina lacking UVS cones. These data are consistent with the loss of putative UVS cones from the PGZ of a migratory salmonid of another genus, and thus the disappearance of UVS cones appears to be general to the Family Salmonidae, regardless of life history strategy. The generation, differentiation, and subsequent loss of UVS cones in the smolt PGZ is a dramatic example of the supposition that the mechanisms of PGZ development recapitulate the retinal embryogenesis of that species. J. Comp. Neurol. 461:294–306, 2003.

Identification and localization of eight distinct hormone-producing cell types in the pituitary of male Atlantic halibut (Hippoglossus hippoglossus L.)

The eight distinct hormone-producing cell types in the adenohypophysis of male Atlantic halibut (Hippoglossus hippoglossus L.) were identified and localized using immunohistochemistry and in situ hybridization. Lactotropes either occupied most of the rostral pars distalis (RPD) or they were arranged in follicular structures located along the periphery of the RPD. Corticotropes were confined to a thin layer of RPD cells bordering the pars nervosa (PN). The somatotropes were arranged in multicellular layers bordering the highly convoluted PN penetrating the proximal pars distalis (PPD), while thyrotropes, scattered in small islets in between the somatotropes, were located in the centro-dorsal part of the PPD. Gonadotropes were found throughout the PPD. Immunoreactivity to glycoprotein-alpha and luteinizing hormone beta-subunit was also observed along the periphery of the pars intermedia (PI), indicating that a thin extension of the PPD surrounded the PI. In situ hybridization showed that follicle-stimulating hormone and luteinizing hormone were produced in distinct cells of the PPD. PI contained somatolactotropes bordering the highly convoluted PN, and melanotropes that showed positive immunostaining against both anti-alpha-melanocyte-stimulating hormone and anti-beta-endorphin. The general cellular organization was similar to that of other teleost fish. These results lay the basis for future investigations on Atlantic halibut pituitary physiology.

Molecular cloning and characterization of five opsin genes from the marine flatfish Atlantic halibut ( Hippoglossus hippoglossus )

Most molecular studies on the visual system in fish have been performed on freshwater teleosts such as goldfish and zebrafish where cones and rods appear simultaneously. Many marine fishes have long larval phase in the upper pelagic zone before transformation into a juvenile and a benthic life style. The retina at the larval stages consists of only single cone cells; later during metamorphosis double cones and rods develop. The flatfish Atlantic halibut (Hippoglossus hippoglossus) is a typical example of a marine species with such a two-step retina development. In this study, we have cloned five different opsins from Atlantic halibut larvae and juvenile retinas. Sequence comparisons with other opsins and phylogenetic analysis show that the five genes belong to the opsins of long-wavelength sensitive (L); middle-wavelength sensitive, M(Cone) and M(Rod); and short-wavelength sensitive, S(Blue) and S(Ultraviolet), respectively. In situ hybridization analysis reveals expression in double cone (L and M(Cone)), single cone (S(Blue) and S(Ultraviolet)), and rod (M(Rod)) types of photoreceptor cells in juvenile halibut retina. The visual system in Atlantic halibut seems therefore to have all four types of cone photoreceptors in addition to rod photoreceptors. This work shows for the first time molecular isolation of a complete set of retinal visual pigment genes from a marine teleost and describes the first cloning of an ultraviolet-sensitive opsin type from a marine teleost.

Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos

MicroRNAs (miRNAs) are generated from primary transcripts through sequential processing by two RNase III enzymes, Drosha and Dicer, in association with other proteins. This maturation is essential for their function as post‐transcriptional regulators. Notably, Dicer is also a component of RNA‐induced silencing complexes, which incorporate either miRNA or small interfering RNA (siRNA) as guides to target specific mRNAs. In zebrafish, processed miRNAs belonging to the miR‐430 family have previously been shown to promote deadenylation and degradation of maternal mRNAs during early embryogenesis. We show that injection of one‐cell‐stage zebrafish embryos with siRNA causes a significant reduction in the endogenous levels of processed miR‐430 and other miRNAs, leading to unspecific developmental defects. Coinjection of siRNA with preprocessed miR‐430 efficiently rescued development. This indicates that the abnormalities generally observed in siRNA‐treated zebrafish embryos could be due to inhibition of miR‐430 processing and/or activity. Our results also suggest that the miRNA pathway in mammals, under some experimental or therapeutic conditions, may be affected by siRNA.

Cholecystokinin mRNA in Atlantic herring, Clupea harengus--molecular cloning, characterization, and distribution in the digestive tract during the early life stages.

The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.

Corticotropin-Releasing Factor Neurogenesis during Midlife Development in Salmon: Genetic, Environmental and Thyroid Hormone Regulation

Salmon parr–smolt transformation (smoltification) is a mid‐life transitional stage between life in freshwater and seawater that entails a wide range of neural, endocrine and physiological modifications. In salmon, the neuroendocrine corticotropin‐releasing factor (CRF) system regulates pituitary adrenocorticotrophic hormone and thyrotrophin release. Four experimental groups of Atlantic salmon, Salmo salar, were used to investigated CRF neurogenesis and its regulation during smoltification. We compared: (i) developmental stages (parr and early‐smolt) in anadromous controls; (ii) a developmentally arrested model: anadromous reared under continuous light (LL) with anadromous controls; (iii) a natural hypoendocrine/incomplete smolt development salmon model (landlocked) with anadromous controls; and (iv) landlocked treated with thyroxine to anadromous control smolt levels. CRF neurogenesis between groups was studied with bromodeoxyuradine (BrdU) incorporation followed by double‐labelling CRF and BrdU immunhistochemistry. The rate of CRF neurogenesis in the preoptic area (POA) increased from parr to early‐smolts in anadromous salmon. By contrast, neurogenesis was inhibited in the LL group and reduced in the landlocked salmon. The administration of thyroxine in landlocked salmon to match anadromous levels increased the rate of CRF neurogenesis to anadromous levels. In conclusion, newly‐formed CRF cells in the POA during smoltification are associated with increased retinal innervation to the POA and endocrine responsiveness to increased photoperiod. Both genetic and environmental factors influence the degree of salmon brain development. Thyroid hormones increase CRF neurogenesis during this critical period of development in salmon. We hypothesise that a positive‐feedback of thyroid hormones on CRF neurogenesis may be an important event in reaching the developmental climax during critical periods.

Expression of the eight AMPA receptor subunit genes in the developing central nervous system and sensory organs of zebrafish

The AMPA type glutamate receptors mediate the majority of fast synaptic transmission in the vertebrate nervous system. Whereas mammals have four subunit genes, Gria1‐4, zebrafish has retained a duplicated set of eight genes named gria1‐4a and b. We give here a detailed overview of the expression patterns of all eight zebrafish subunits within the developing central nervous system and sensory organs at 24, 48, and 72 hr after fertilization. Expression domains include distinct neuronal subsets in the developing forebrain, midbrain, hindbrain, and spinal cord, as well as in the ganglion‐ and inner nuclear layers of the retina. As a general rule, each pair of duplicated gria genes is differentially expressed, indicating subfunctionalization of AMPA receptor subunit expression in the teleost lineage. Our findings suggest that zebrafish can serve as a useful model system to investigate the role of AMPA receptors and their differential expression in the vertebrate nervous system. Developmental Dynamics 237:788–799, 2008.

Emergence of Axonal Tracts in the Developing Brain of the Turbot (Psetta maxima)

In this study we have investigated the pattern of morphogenesis and axogenesis in the turbot brain during embryonic and early larval stages with immunohistochemistry using an antibody against acetylated tubulin. The first immunoreactive elements were detected at 74 h post-fertilization in fibers running in the medial and lateral longitudinal fascicles. Newly positive axonal bundles are progressively added during development forming rostrocaudally directed tracts. The tract of the postoptic commissure appears at 86 h post-fertilization located rostrally to the medial longitudinal fascicle. Together, the medial longitudinal fascicle and the tract of the postoptic commissure constitute a major longitudinal axonal pathway, which is extended rostrally in embryos of 98 h post-fertilization by the supraoptic tract. In the forebrain, two vertical tracts, the tract of the posterior commissure (appearing around 98 h post-fertilization) and the tract of the anterior commissure (detected at 110 h post-fertilization) project descending axons to the pre-existing axonal longitudinal pathway. These early tracts are connected by four associated commissures (ventral tegmental, postoptic, posterior and anterior commissure). Some groups of labeled cell bodies are identified either as the origin of the embryonic tracts or contributing axons to the axonal pathways. Additionally, a conspicuous cluster of large cells, not clearly associated with any axonal bundle, was observed from 98 h post-fertilization lining the caudal floor of the presumptive hypothalamus. Several hypotheses are proposed to determine the nature of these cells. A comparison of the emergence of the axonal circuitry in turbot and that of other teleosts reveals significant analogies, suggesting that a common pattern underlies the establishment of the embryonic tracts in this vertebrate group. The minor differences observed between different teleost species, associated with the absence of some axonal fascicles, is also considered.