Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P elementmediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.targeted gene expression ͉ Gal4-UAS ͉ tetanus toxin ͉ touch response ͉ interneuron

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We identified visual opsin genes for three flounder species, including the spotted halibut (Verasper variegatus), slime flounder (Microstomus achne), and Japanese flounder (Paralichthys olivaceus). Structure and function of opsins for the three species were characterized together with those of the barfin flounder (V. moseri) that we previously reported. All four flounder species possessed five basic opsin genes, including lws, sws1, sws2, rh1, and rh2. Specific features were observed in rh2 and sws2. The rh2‐a, one of the three subtypes of rh2, was absent in the genome of V. variegatus and pseudogenized in V. moseri. Moreover, rh2‐a mRNA was not detected in M. achne and P. olivaceus, despite the presence of a functional reading frame. Analyses of the maximum absorption spectra (λmax) estimated by in vitro reconstitution indicated that SWS2A of M. achne (451.9 nm) and P. olivaceus (465.6 nm) were blue‐sensitive, whereas in V. variegatus (485.4 nm), it was green‐sensitive and comparable to V. moseri (482.3 nm). Our results indicate that although the four flounder species possess a similar opsin gene repertoire, the SWS2A opsin of the genus Verasper is functionally green‐sensitive, while its overall structure remains conserved as a blue‐sensitive opsin. Further, the rh2‐a function seems to have been reduced during the evolution of flounders. λmax values of predicted ancestral SWS2A of Pleuronectiformes and Pleuronectidae was 465.4 and 462.4 nm, respectively, indicating that these were blue‐sensitive. Thus, the green‐sensitive SWS2A is estimated to be arisen in ancestral Verasper genus. It is suggested that the sensitivity shift of SWS2A from blue to green may have compensated functional reduction in RH2‐A.

Melanocortin (MC) systems are composed of MC peptides such as adrenocorticotropic hormone (ACTH), several molecular forms of melanocyte-stimulating hormones (MSHs) and MC receptors (MCRs). Here we demonstrated that the cartilaginous fish, Dasyatis akajei (stingray) expresses five subtypes of MCR genes-mc1r to mc5r-as in the case of teleost and tetrapod species. This is the first evidence showing the presence of the full repertoire of melanocortin receptors in a single of cartilaginous fish. Expression of respective stingray mcr cDNAs in Chinese hamster ovary cells revealed that Des-acetyl-α-MSH exhibited cAMP-producing activity indistinguishable to ACTH(1-24) on MC1R and MC2R, while the activity of Des-acetyl-α-MSH on MC3R, MC4R, and MC5R were similar to or slightly greater than that of ACTH(1-24). Notably, in contrast to the other vertebrates, MC2R did not require coexpression with a melanocortin receptor-2 accessory protein 1 (mrap1) cDNA for functional expression. One of the roles of MC system resides in regulation of the pituitary-interrenal (PI) axis-a homologue of tetrapod pituitary-adrenal axis. In stingray, interrenal tissues were shown to express mc2r and mc5r as major MCR genes. These results established the presence of functional PI axis in stingray at the level of receptor molecule. While MC2R participates in adrenal functions together with MRAP1 in tetrapod species, the fact that sensitivity of MC5R to Des-acetyl-α-MSH and ACTH(1-24) were two order of magnitude higher than MC2R without coexpression with MRAP1 suggested that MC5R could play a more important role than MC2R to transmit signals conveyed by ACTH and MSHs if MRAP1 is really absent in the stingray.

The representative role of melanocyte-stimulating hormone (MSH) and melaninconcentrating hormone (MCH) in fish is regulation of pigment migration. However, our studies using barfin flounder Verasper moseri, a flatfish as a major experimental fish, have revealed that MSH and MCH are multifunctional because their receptors are widely distributed not only in the melanophores but also in the brain and systemic body. Their biological roles other than control of pigment migration would be regulation of feeding behavior, energy metabolism, cortisol release, etc. Among them, an interesting biological process on molecular level has been observed in the role of α-MSH. A fine difference in the structure-presence or absence of one acetyl group-modified the activities. Namely, desacetyl-α-MSH having no acetyl group at N-terminal stimulates pigment dispersion in melanophore and cortisol release from the interrenal gland, while α-MSH having one acetyl group has negligible effects. On the whole body level, MCH probably transfers information about photic conditions from the external environment to the body. MCH production is changeable, depending on the difference in the intensity of the light. A white background enhances production of MCH, and MCH turns body color pale by aggregating pigments in scales. It is suggested that this peptide stimulates feeding behavior. This monograph reveals molecular characteristic and biological significance of MSH and MCH systems in fish.

Proopiomelanocortin (POMC) is the precursor of several peptide hormones generated in the pituitary gland. After biosynthesis, POMC undergoes several posttranslational modifications, including proteolytic cleavage, acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation, which generate mature POMC-derived peptides. Therefore, POMC is a useful model for the investigation of posttranslational modifications. These processes have been extensively investigated in mammals, primarily in rodents. In addition, over the last decade, much information has been obtained about the posttranslational processing of POMC in non-mammalian animals such as fish, amphibians, reptiles, and birds through sequencing and peptide identification by mass spectrometry. One POMC modification, acetylation, is known to modulate the biological activities of POMC-derived α-melanocyte-stimulating hormone (α-MSH) having an acetyl group at N-terminal through potentiation or inhibition. This bidirectional regulation depends on its intrinsic roles in the tissue or cell; for example, α-MSH, as well as desacetyl (Des-Ac)-α-MSH, stimulates pigment dispersion in the xanthophores of a flounder. In contrast, α-MSH does not stimulate pigment dispersion in the melanophores of the same species, whereas Des-Ac-α-MSH does. Regulation of pigment-dispersing activities may be associated with the subtle balance in the expression of receptor genes. In this review, we consider the posttranslational modifications of POMC in vertebrates from an evolutionary aspect, with a focus on the relationship between acetylation and the biological activities of α-MSH as an important consequence of posttranslational modification.

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