semi-automated-imaging-tissue-specific-fluorescence-zebrafish

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

University of Alabama at Birmingham

&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;&#x662F;&#x4E00;&#x5BB6;&#x4EBA;&#xFF0C;&#x79EF;&#x5206;&#x819C;&#x86CB;&#x767D;&#x7EC4;&#x6210;&#x4E24;&#x5B50;&#x65CF;&#xFF1A; &#x6B63;&#x7EDF;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;&#xFF0C;&#x8FD0;&#x8F93;&#x53EA;&#x6709;&#x6C34;&#x548C; aquaglyceroporins&#xFF0C;&#x5176;&#x4E2D;&#x8FD0;&#x8F93;&#x7518;&#x6CB9;&#x3001; &#x5C3F;&#x7D20;&#x3001; &#x6216;&#x5176;&#x4ED6;&#x5C0F;&#x7684;&#x6EB6;&#x8D28;&#x3002;&#x4E24;&#x4E2A;&#x6700;&#x8FD1;&#x63CF;&#x8FF0;&#x7684;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;&#xFF0C;&#x6570;&#x5B57; 11 &#x548C; 12&#xFF0C;&#x4F3C;&#x4E4E;&#x4E0E;&#x5176;&#x4ED6;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;&#x548C; aquaglyceroporins &#x66F4;&#x65E0;&#x8BBA;&#x8DDD;&#x79BB;&#x7684;&#x8FDC;&#x8FD1;&#x6709;&#x5173;&#x3002;

&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;-11&#xFF1A; &#x7F3A;&#x4E4F;&#x660E;&#x663E;&#x7684;&#x8FD0;&#x8F93;&#x529F;&#x80FD;&#x8868;&#x793A;&#x5728;&#x5927;&#x8111;&#x4E2D;&#x901A;&#x9053;&#x86CB;&#x767D;&#x3002;

The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins.

Aquaporin-11: a channel protein lacking apparent transport function expressed in brain.

Les aquaporines sont une famille de prot&#xE9;ines membranaires int&#xE9;grales compos&#xE9;es de deux sous-familles: les aquaporines orthodoxes, de l&#x26;#39;eau de transport qui ne, et les aquaglyc&#xE9;roporines, qui solut&#xE9;s glyc&#xE9;rol transports, de l&#x26;#39;ur&#xE9;e, ou d&#x26;#39;autres petits. Deux aquaporines r&#xE9;cemment d&#xE9;crits, les num&#xE9;ros de 11 et 12, semblent &#xEA;tre plus lointainement li&#xE9;e &#xE0; des aquaporines autres mammif&#xE8;res et aquaglyc&#xE9;roporines.

Aquaporin-11: une prot&#xE9;ine canal Faute Fonction Transports apparente exprim&#xE9;e dans le cerveau

Die Varianten sind eine Familie von integralen Membranproteinen, bestehend aus zwei Unterfamilien: die orthodoxen Varianten, die nur Wasser zu transportieren, und die Aquaglyceroporins, die Glycerin, Harnstoff oder anderen kleinen Analyten zu transportieren. Zwei k&uuml;rzlich beschriebene Varianten, Nummern 11 und 12, scheinen mehr entfernt verwandt sein mit den anderen S&auml;ugetieren Varianten und Aquaglyceroporins.

Aquaporin-11: ein Kanal-Protein fehlt offensichtlich Transportfunktion, ausgedr&uuml;ckt im Gehirn.

Le acquaporine sono una famiglia di proteine integrali di membrana composta da due sottofamiglie: le acquaporine ortodosso, che trasportano solo acqua, e il aquaglyceroporins, che trasportano il glicerolo, urea o altri piccoli soluti. Due recentemente descritti acquaporine, numeri 11 e 12, sembrano essere pi&ugrave; tiepidi agli altri mammiferi acquaporine e aquaglyceroporins.

Aquaporina-11: una proteina canale privo di funzione apparente trasporto espresso nel cervello.

&#x6B63;&#x7D71;&#x6D3E;&#x306E;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;&#x3001;&#x8F38;&#x9001;&#x306E;&#x307F;&#x6C34;&#x3001;aquaglyceroporins&#x3001;&#x8F38;&#x9001;&#x30B0;&#x30EA;&#x30BB;&#x30ED;&#x30FC;&#x30EB;&#x3001;&#x5C3F;&#x7D20;&#x3001;&#x307E;&#x305F;&#x306F;&#x4ED6;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x6EB6;&#x8CEA;&#xFF1A;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;&#x306F;&#x3001;2&#x3064;&#x306E;&#x4E9C;&#x79D1;&#x304B;&#x3089;&#x69CB;&#x6210;&#x3055;&#x308C;&#x308B;&#x5185;&#x5728;&#x6027;&#x819C;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x3067;&#x3042;&#x308B;&#x3002;&#x4E8C;&#x6700;&#x8FD1;&#x8A18;&#x8F09;&#x3055;&#x308C;&#x305F;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;&#x3001;&#x6570;&#x5B57;11&#x3068;12&#x306F;&#x3001;&#x3088;&#x308A;&#x9060;&#x304F;&#x3001;&#x4ED6;&#x306E;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;&#x3068;aquaglyceroporins&#x306B;&#x95A2;&#x9023;&#x3059;&#x308B;&#x3068;&#x601D;&#x308F;&#x308C;&#x308B;&#x3002;

&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;11&#xFF1A;&#x8133;&#x306B;&#x767A;&#x73FE;&#x898B;&#x304B;&#x3051;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30C8;&#x6A5F;&#x80FD;&#x3092;&#x6B20;&#x3044;&#x3066;&#x3044;&#x308B;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x30BF;&#x30F3;&#x30D1;&#x30AF;

Aquaporins&#xB294; &#xD544;&#xC218;&#xC801;&#xC778; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xB2E8;&#xBC31;&#xC9C8; 2 &#xC11C;&#xBE0C;&#xD328;&#xBC00;&#xB9AC;&#xAC00;&#xC758; &#xAD6C;&#xC131;&#xC758; &#xAC00;&#xC871;:&#xB9CC; &#xBB3C;&#xC744; &#xC218;&#xC1A1;, &#xC815;&#xD1B5; aquaporins &#xBC0F; &#xC218;&#xC1A1; &#xAE00;&#xB9AC;&#xC138;&#xB864;, &#xC6B0; &#xB808; &#xC544;, &#xB610;&#xB294; &#xB2E4;&#xB978; &#xC791;&#xC740; solutes aquaglyceroporins. &#xB450; &#xCD5C;&#xADFC; &#xC124;&#xBA85;&#xB41C; aquaporins, &#xC22B;&#xC790; 11&#xACFC; 12, &#xB354; &#xBA3C; &#xB2E4;&#xB978; &#xD3EC;&#xC720;&#xB958; aquaporins &#xBC0F; aquaglyceroporins&#xC5D0; &#xAD00;&#xB828; &#xB420; &#xB4EF;&#xD558;&#xB2E4;.

Aquaporin 11: &#xCC44;&#xB110; &#xB2E8;&#xBC31;&#xC9C8; &#xBD80;&#xC871; &#xD55C; &#xB450;&#xB1CC;&#xC5D0; &#xD45C;&#xD604; &#xB41C; &#xBA85;&#xBC31;&#xD55C; &#xC804;&#xC1A1; &#xAE30;&#xB2A5;.

Las acuaporinas son una familia de prote&#xED;nas integrales de membrana compuesta por dos subfamilias: las aquaporinas ortodoxos, que s&#xF3;lo el transporte de agua, y acuagliceroporinas los solutos, que el transporte de glicerol, urea, u otros peque&#xF1;os. Dos acuaporinas recientemente descrita, n&#xFA;meros 11 y 12, parecen estar m&#xE1;s alejadas a las acuaporinas otros mam&#xED;feros y acuagliceroporinas.

Aquaporin-11: A falta de una prote&#xED;na del canal de la funci&#xF3;n de transporte aparente expresa en el cerebro

Androgens play a central role in the regulation of male sexual differentiation and behavior in many vertebrates, including zebrafish. Their signaling is mediated by activation of the androgen receptor. A single androgen receptor (ar) gene was recently identified in zebrafish, which encodes a protein that binds androgens in vitro. However, the tissue-specific expression pattern of this receptor in vivo has not been described. Using whole-mount RNA in situ hybridization, we characterized expression of the ar gene in developing zebrafish and in the adult brain. In embryos, transcripts were found in the presumptive pronephros and in olfactory placodes. By 3-5 days postfertilization, ar transcripts were also detected in the pineal organ anlage and the retina. In the adult brain, ar was expressed in discrete regions of the telencephalon, in the preoptic area, and throughout the periventricular hypothalamus, regions previously implicated in the regulation of sexually dimorphic behaviors in mammals.

Androgen receptor gene expression in the developing and adult zebrafish brain.

Estrogens regulate a diverse range of physiological processes and affect multiple tissues. Estrogen receptors (ERs) regulate transcription by binding to DNA at conserved estrogen response elements, and such elements have been used to report ER activity in cultured cells and in transgenic mice. We generated stable, transgenic zebrafish containing five consecutive elements upstream of a c-fos minimal promoter and green fluorescent protein (GFP) to visualize and quantify transcriptional activation in live larvae. Transgenic larvae show robust, dose-dependent estrogen-dependent fluorescent labeling in the liver, consistent with er gene expression, whereas ER antagonists inhibit GFP expression. The nonestrogenic steroids dexamethasone and progesterone fail to activate GFP, confirming ER selectivity. Natural and synthetic estrogens activated the transgene with varying potency, and two chemicals, genistein and bisphenol A, preferentially induce GFP expression in the heart. In adult fish, fluorescence was observed in estrogenic tissues such as the liver, ovary, pituitary gland, and brain. Individual estrogen-responsive neurons and their projections were visualized in the adult brain, and GFP-positive neurons increased in number after 17β-estradiol exposure. The transgenic estrogen-responsive zebrafish allow ER signaling to be monitored visually and serve as in vivo sentinels for detection of estrogenic compounds.

Visualization of estrogen receptor transcriptional activation in zebrafish.

Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart.

Article	Year
Aquaporin-11: a channel protein lacking apparent transport function expressed in brain. BMC biochemistry\| PubMed ID: 16650285	2006
Androgen receptor gene expression in the developing and adult zebrafish brain. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 18816841	2008
Visualization of estrogen receptor transcriptional activation in zebrafish. Endocrinology\| PubMed ID: 21540282	2011
Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis. PloS one\| PubMed ID: 24223173	2013
Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples. Environmental health perspectives\| PubMed ID: 24425189	2014

Daniel A. Gorelick

Biography

JoVE Journal Publications

Other Publications