Transient Ectopic Overexpression of Agouti-Signalling

While flatfish in the wild exhibit a pronounced countershading of the dorso-ventral pigment pattern, malpigmentation is commonly observed in reared animals. In fish, the dorso-ventral pigment polarity is achieved because a melanization inhibition factor (MIF) inhibits melanoblast differentiation and encourages iridophore proliferation in the ventrum. A previous work of our group suggested that asip1 is the uncharacterized MIF concerned. In order to further support this hypothesis, we have characterized asip1 mRNAs in both turbot and sole and used deduced peptide alignments to analyze the evolutionary history of the agouti-family of peptides. The putative asip precursors have the characteristics of a secreted protein, displaying a putative hydrophobic signal. Processing of the potential signal peptide produces mature proteins that include an N-terminal region, a basic central domain with a high proportion of lysine residues as well as a proline-rich region that immediately precedes the C-terminal poly-cysteine domain. The expression of asip1 mRNA in the ventral area was significantly higher than in the dorsal region. Similarly, the expression of asip1 within the unpigmented patches in the dorsal skin of pseudoalbino fish was higher than in the pigmented dorsal regions but similar to those levels observed in the ventral skin. In addition, the injection/electroporation of asip1 capped mRNA in both species induced long term dorsal skin paling, suggesting the inhibition of the melanogenic pathways. The data suggest that fish asip1 is involved in the dorsalventral pigment patterning in adult fish, where it induces the regulatory asymmetry involved in precursor differentiation into mature chromatophore. Adult dorsal pseudoalbinism seems to be the consequence of the expression of normal developmental pathways in an inaccurate position that results in unbalanced asip1 production levels. This, in turn, generates a ventral-like differentiation environment in dorsal regions.Publicado

Bases moleculares de las malformaciones pigmentarias en peces: implicación en el cultivo del rodaballo (Scophthalmus maximus L.)

In mammals the colour of skin is due to production of eumelanins (brown-black pigments) and pheomelanins (yellow-brownish pigments) in a specific type cell, the melanocytes. In contrast, in fish, pigmentation patternare determine by three different types of cells, melanophores, xantophores and iridophores. However, in both cases the melanogenesis is controlled by a similar mechanism. The α-MSH which stimulate the synthesis of melanin, and the ASP which antogonize its effects. Flounder (Scophthalmus maximus L.) agouti gen was cloned from skin tissue by using RACE-PCR with degenerated oligos. High expression of agouti gene was found in white epidermis areas compared with the black ones. Furthermore, dermal-injection of capped agouti mRNA produced a striking skin lightening. Thus, suggesting a central role of agouti gene in pigmentation pattern control in fish

Evaluating feed discrimination in seabream Sparus aurata using a dualchoice self-feeding system

Trabajo presentado en Aquaculture Europe 19, celebrado en Berlín (Alemania) del 7 al 10 de octubre de 2019.[Introduction]: Feed intake is a critical variable in aquaculture that limits growth and survival of reared animals. The inclusion of new raw materials in fish diets to meet cost-efficient production and sustainability goals may compromise the organoleptic quality of diets and, by extension, fish growth (Yaghoubi et al., 2016). Therefore, it seems reasonable to evaluate the fish s discriminatory capacity towards feed in order to discern the organoleptic preferences of aquacultured species. The goal of this study was to develop an experimental model to test feed discrimination in the gilthead seabream (Sparus aurata), an important species for Mediterranean seawater aquaculture.[Materials and methods]: We set up a dual-choice feeding system using self-feeders activated by a string sensor placed 3cm below the water surface. Feeders were connected to a computer system that recorded the date, time and tank from which each feed demand originated (Leal et al., 2009). The feed reward per sensor activation was set at approximately 1g/demand. Initially, 500 juvenile gilthead sea bream of approximately 10g were maintained in two 2500 l tanks provided with self-feeding systems during eight months for accommodation and learning. Subsequently, animals (body weight around 100g) were transferred to eleven 500 l experimental tanks (n=10/tank) provided with a dual choice feeding system consisting of two string sensors, each activating a different self-feeder. During 28 days, all feeders were provided with a control diet (44% CP and 18% CF, containing 12.5% fishmeal) for accommodation to the experimental tanks, but especially to the dual choice feeding system (phase I). Subsequently (phase II), in four tanks, one feeder was filled up with control diet supplemented with quinine (1.5%, negative diet) whereas the second feeder contained the control diet. In other three tanks, the tester-feeder distributed a positive diet (isoproteic and isolipidic but containing 46% fishmeal, 6% squid meal and 6% krill meal) and the second feeder delivered control diet. Finally, both positive and negative diets were confronted in the remaining four tanks. After eight consecutive days, the position of feeders was switched in the tanks and animals were allowed to feed for further 21 days (phase III). At the end of the experiment, the total amount of feed distributed was calculated by weighing the feed remaining in the feed hoppers. This quantity was used to calculate the delivery rate for each electronic feeder. The amount of feed delivered daily was calculated using the feeder delivery rate and number of daily demands. The experimental tanks were inspected daily to ensure the absence of feed on the bottom. Consecutively, and utilizing the same animals from the previous experiment, an additional trial was set up to corroborate the feeding deterrent effect of quinine using a single feeder per tank for 14 days. Three tanks were fed with quinine-supplemented diet and four tanks with control diet. Feed intake levels were calculated as before.[Results and discussion]: During phase I of the dual-choice experiment, using a univariate general linear model that analyzes all sensors jointly, independently of the tank, no significant differences in feeder activation were found, thus demonstrating that fish demanded feed in a similar way independently of feeder. Sensor activation differed according to time (hour of the day), day and tank. It was demonstrated that fish exhibited a similar daily pattern of demands (figure 1a) in all sensors/feeders. Number of demands was different between tanks but also between the different experimental days, thus indicating the instability of feed intake (data not shown). During phase II, when experimental diets were introduced, we found significant differences in sensor activation, with the lowest values being measured in feeders provided with quinine-supplemented diet and highest demand levels being found in those containing positive diet (figure 1b). This demonstrated that fish exhibited preferences towards sensors coupled to feeders delivering positive diets but avoided feeders supplying a deterrent feed (provided by quinine inclusion), when compared to the control diet. Similarly to the accommodation phase (I), feed intake levels differed according to day and tank, but this time we also recorded significant differences in the 24h-feeding pattern. In this respect, the activation period of sensors delivering quinine-rich diet was narrower (figure 1b). Finally, in phase III when the position of feeders was inverted, fish continued to demand less from feeders delivering the quinine diet, and had higher demands from feeders supplying the positive diet. Results suggest that fish are able to discriminate the position of the feeder according to the type of diet delivered or, what is the same, they can discriminate the diet independently of the feeder position. Similar to phase II, feeder sensor activation differed according to day, tank and time (figure 1c). Finally, using a different experimental set up in which animals were fed exclusively with one type of diet, either control or quinine-supplemented, we confirmed that, when presented with a diet containing quinine, gilthead seabream reduce their voluntary feed intake (data not shown), thus corroborating that the lower preference towards this feed, when an alternative feed is presented, is associated with a feeding deterrent effect.[Conclusions and future directions]: Seabream exhibited variable feed intake levels along different days and fish groups/tanks but were able to discriminate the diet’s organoleptic properties using a dual-choice self-feeding system. This opens the possibility to use the system to evaluate new raw materials in terms of feed acceptance/preference, as well as the potential of flavorings to overcome the negative effects of antinutritional compounds or medication on feed intake

Morphological and Molecular Characterization of Dietary-Induced Pseudo-Albinism during Post-Embryonic Development of Solea senegalensis (Kaup, 1858)

16 páginas, 5 tablas, 8 figurasThe appearance of the pseudo-albino phenotype was investigated in developing Senegalese sole (Solea senegalensis, Kaup 1858) larvae at morphological and molecular levels. In order to induce the development of pseudo-albinos, Senegalese sole larvae were fed Artemia enriched with high levels of arachidonic acid (ARA). The development of their skin pigmentation was compared to that of a control group fed Artemia enriched with a reference commercial product. The relative amount of skin melanophores, xanthophores and iridophores revealed that larval pigmentation developed similarly in both groups. However, results from different relative proportions, allocation patterns, shapes and sizes of skin chromatophores revealed changes in the pigmentation pattern between ARA and control groups from 33 days post hatching onwards. The new populations of chromatophores that should appear at post-metamorphosis were not formed in the ARA group. Further, spatial patterns of distribution between the already present larval xanthophores and melanophores were suggestive of short-range interaction that seemed to be implicated in the degradation of these chromatophores, leading to the appearance of the pseudo-albino phenotype. The expression profile of several key pigmentation-related genes revealed that melanophore development was promoted in pseudo-albinos without a sufficient degree of terminal differentiation, thus preventing melanogenesis. Present results suggest the potential roles of asip1 and slc24a5 genes on the down-regulation of trp1 expression, leading to defects in melanin production. Moreover, gene expression data supports the involvement of pax3, mitf and asip1 genes in the developmental disruption of the new post-metamorphic populations of melanophores, xanthophores and iridophores.This work was funded by the Ministry of Science and Innovation (MICIIN) of the Spanish Government (project AGL2008-03897-C04-01/ACU). MD was supported by a Juan de la Cierva post-doctoral contract (MICIIN, Spain)Peer reviewe

The C-terminal domains of melanocortin-2 receptor (MC2R) accessory proteins (MRAP1) influence their localization and ACTH-induced cAMP production

ACTH binding to the human melanocortin-2 receptor (MC2R) requires the presence of the MC2R accessory protein1 isoforms, MRAPα or MRAPβ. This study evaluated the role of the isoform-specific C-terminal domains of MRAP with regard to their cellular localization, topology, interaction with MRAP2 and cAMP production. When stably expressed in HEK293/FRT cells or in B16-G4F mouse melanoma cells (an MSH receptor-deficient cell clone), MRAPα and MRAPdCT (truncated MRAP1, N-terminal only) localized mainly around the nuclear envelope and within dense intracellular endosomes, while MRAPβ exhibited a strong localization at the plasma membrane, and partially with rapid recycling endosomes. MRAPβ and MRAPdCT both exhibited dual-topology (N cyto/C exo and N exo/C cyto) at the plasma membrane whereas MRAPα exhibited only N cyto/C exo topology at the plasma membrane while adopting dual-topology in intracellular compartments. Both MRAPα and MRAP2 colocalized in intracellular compartments, as opposed to weak colocalization between MRAPβ and MRAP2. MRAP2 and MC2R enhanced the expression of MRAP1 isoforms and vice versa. Moreover, in both HEK293/FRT and B16-G4F cells, ACTH failed to activate MC2R unless MRAP1 was present. MRAP1 expression enhanced MC2R cell-surface expression as well as concentration-dependent cAMP accumulation. In the presence of human or zebrafish MC2R, MRAPβ induced the highest cAMP accumulation while MRAPdCT induced the lowest. Together, the present findings indicate that the C-terminal domains of MRAP dictate their intracellular localization in addition to regulating ACTH-induced cAMP production. These preferential localizations suggest that MRAPα is involved in MC2R targeting to the plasma membrane, while MRAPβ may enhance ACTH-MC2R coupling to cAMP production. © 2012 Elsevier Inc.This work was supported by Grants from the Canadian Institutes of Health Research to N.G.-P. (MOP-82819) and to J.L.P. (MOP-69085) and by the Canada Research Chairs Program. N.G.-P. is a recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland; J.L.P. is a recipient of a Chercheur-boursier senior scholarship from the Fonds de la Recherche en Santé du Québec. N.G.-P. and J.-L.P. are members of the FRSQ-funded Centre de Recherche Clinique Étienne-le Bel. J.M.C-R is a recipient of research funds from the Spanish Science and Innovation Ministry (AGL2010-22247-C03-01, CSD 2007-00002). S.R. is a recipient of a studentship from the Fonds de la Recherche en Santé du Québec. MJA is a recipient of a ‘‘Juan de la Cierva’’ research contract (2009) from the Spanish Science and Innovation Ministry.Peer Reviewe

Melanocortin 4 receptor becomes an ACTH receptor by coexpression of melanocortin receptor accessory protein 2

Melanocortin 2 receptor (MC2R) is the only canonical ACTH receptor. Its functional expression requires the presence of an accessory protein, known as melanocortin receptor 2 accessory protein 1 (MRAP1). The vertebrate genome exhibits a paralogue gene called MRAP2, which is duplicated in zebrafish (MRAP2a and MRAP2b), although its function remains unknown. In this paper, we demonstrate that MRAP2a enables MC4R, a canonical MSH receptor, to be activated by ACTH with a similar sensitivity to that exhibited by MC2R. Both proteins physically interact and are coexpressed in the neurons of the preoptic area, a key region in the control of the energy balance and hypophyseal secretion in fish. ACTH injections inhibit food intake in wild-type zebrafish but not in fish lacking functional MC4R. Both MRAP1 and MRAP2a are hormonally regulated, suggesting that these proteins are substrates for feed-back regulatory pathways of melanocortin signaling. Fasting has no effect on the central expression of MRAP2a but stimulates MRAP2b expression. This protein interacts and is colocalized with MC4R in the tuberal hypothalamic neurons but has no effect on the pharmacologic profile of MC4R. However, MRPA2b is able to decrease basal reporter activity in cell lines expressing MC4R. It is plausible that MRAP2b decreases the constitutive activity of the MC4R during fasting periods, driving the animal toward a positive energy balance. Our data indicate that MRAP2s control the activity of MC4R, opening up new pathways for the regulation of melanocortin signaling and, by extension, for the regulation of the energy balance and obesity. © 2013 by The Endocrine Society.Peer Reviewe

Possible role of central the melanocortin system in the control of circadian locomotor activity rhythms

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017The melanocortin system integrates peptides derived from proopiomelanocortin that show melanocortinic and/or corticotropic activity. Endogenous melanocortin antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) compete with melanocortin peptide for binding to five different melanocortin receptors (MC1RMC5R). In zebrafish, AGRP and MC5R are duplicated. AGRP1 is specifically expressed in the hypothalamus but AGRP2 is expressed in the pineal. AGPR2 modulates the expression of the melanin-concentrating hormone (MCH) that is involved in the background adaptation. The expression of AGRP2 in the zebrafish pineal gland suggests the involvement of the melanocortin system in the regulation of circadian rhythms. We use a zebrafish transgenic strain overexpressing ASIP (Ag zebrafish) to study the role of melanocortin antagonist in the circadian structure of fish. We demonstrate that both diurnal and nocturnal melatonin levels in Ag zebrafish are statistically similar. Accordingly, comparison of brain transcriptome between Ag and wild type (WT) zebrafish revealed a significant enrichment of tryptophan and circadian rhythms go terms. Finally, the analysis of daily locomotor activity exhibited significant differences between Ag and WT fish further supporting a role for melanocortin system in the activity rhythms of zebrafish.Peer reviewe

Molecular cloning and characterization of the matricellular protein Sparc/osteonectin in flatfish, Scophthalmus maximus, and its developmental stage-dependent transcriptional regulation during metamorphosis

SPARC/osteonectin is amultifunctionalmatricellular glycoprotein,which is expressed in embryonic and adult tissues that undergo active proliferation and dynamic morphogenesis. Recent studies indicate that Sparc expression appears early in development, although its function and regulation during development are largely unknown. In this report, we describe the isolation, characterization, post-embryonic developmental expression and environmental thermal regulation of sparc in turbot. The full-length turbot sparc cDNA contains 930 bp and encodes a protein of 310 amino acids, which shares 77, 75 and 80% identity with human, frog and zebrafish, respectively. Results of whole-mount in situ hybridization reveal a dynamic expression profile during post-embryonic turbot development. Sparc is expressed differentially in the cranioencephalic region; mainly in jaws, branchial arches, fin folds and rays of caudal, dorsal and anal fins. Furthermore, ontogenetic studies demonstrated that Sparc gene expression is dynamically regulated during post-embryonic turbot development, with high expression during stage-specific post-embryonic remodeling. Additionally, the effect of thermal environmental conditions on turbot development and on ontogenetic sparc expression was evaluated.En prens

Papel de la proteína de señalización agouti (ASP) en la determinación del patrón de pigmentación en peces. Aproximación molecular, celular y bioquímica mediante la utilización de sistemas de expresión diferencial

Una de las adaptaciones cromáticas de mayor éxito en vertebrados es la presencia de un patrón dorso/ventral de coloración en el cual la piel dorsal es Oscura mientras que la piel ventral es clara. En los peces, al igual que en otros vertebrados, la determinación del patrón de pigmentación se inicia a partir de la diferenciación de células pluripotentes de la cresta neural en estadios tempranos del desarrollo larvario. La señalización de las melanocortinas via receptor de tipo 1 (MClR) es clave en la determinación genética de la pigmentación en peces. La activación de receptor por la hormona estimulante de los melanocitos (a-MSH) promueve la diferenciación de las células precursoras hacia melanocitos, su profileración y la slntesis de melanina. Atlpicarnente, el MClR es también regulado por un antagonista endógeno, la proteína de sellalización agouti (ASP). En peces, ASP se expresa diferencialmente en la piel de la región ventral, que habitualmente muestra tonalidades claras en contraste con la región dorsal. Varias hipótesis defienden que este antagonismo sobre MCIR es responsable del patrón de pigmentación dorso-ventral en peces. Oicha polaridad en el patrón de pigmentación es extrema en los peces planos que habitualmente presentan problemas de hiper- o hipopigmentación en cultivo. En este estudio, presentamos la identificación y caracterización molecular de la proteína de sel\alización agouti (ASP), en el pez cebra y en el rodaballo (Scophthalmus maximus L.), su importancia en la determinación del patrón de pigmentación y su posible implicación en la inducción de malformaciones pigmentarias mediante el uso de técnicas de ADN recombinante y de transferencia génica.Este trabajo ha sido financiado gracias a la concesión de los contratos JAEDoc (IIM-CSIC) y Ramón y Cajal (MEC-CSIC) a RMC y JR respectivamente, y al proyecto MICIN AGL2008-00392/ACU.Peer Reviewe