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Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum.

ABSTRACT: Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR(RH2)-RH2-1 or LCR(RH2)-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR(RH2)-RH2-1:GFP)(pt112) and Tg(LCR(RH2)-RH2-2:GFP)(pt115) transgenic zebrafish lines express GFP in the pineal gland and certain types of photoreceptors. In addition, some of these lines also express GFP in the hatching gland, optic tectum, or olfactory bulb. Some of the expression patterns differ significantly from previously published similar transgenic fish lines, making them useful tools for studying the development of the corresponding tissues and organs. In addition, the variations of GFP expression among different lines corroborate the notion that transgenic expression is often subjected to position effect, thus emphasizing the need for careful verification of expression patterns when transgenic animal models are utilized for research.

SUBMITTER: Fang W

PROVIDER: S-EPMC3675662 | biostudies-literature | 2013 Jun-Jul

REPOSITORIES: biostudies-literature

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Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum.

Fang Wei W Bonaffini Sarah S Zou Jian J Wang Xiaolei X Zhang Cen C Tsujimura Taro T Kawamura Shoji S Wei Xiangyun X

Gene expression patterns : GEP 20130314 5-6

Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR(RH2)-RH2-1 or LCR(RH2)-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR(RH2)-RH2-1:GFP)(pt112) and Tg(LCR(RH2)-RH2-2:GFP)(pt115) transgenic zebrafish lines express GFP in the pineal gland and certain ...[more]

PMID: 23499733

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Project description:NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown. To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype. The Cre/loxP recombination strategy was used to target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout mice (NeuroD1floxed::Crx-Cre+/-). NeuroD1 floxed mice (NeuroD1floxed::Crx-Cre-/-) served as the controls. Pineal glands and retinas from two-month-old control and conditional knockout mice were collected at ZT6 and ZT20. 3 pools of 6 pineal glands per genotype and respective time of day were collected for each sample. Similarly, 3 pools of 6 retinas each were also collected. RNA from each pool was extracted and hybridized on the Affymetrix Mouse 430 2.0 array.

Dataset Information

Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum.

Publications

Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum.

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