Name: visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish
Uploaded: 2016-12-21T13:00:00
Description: Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Vi takker professor Christoph Englert og professor Didier Stainier for gifting Tg (wt1b: GFP) LI1 og Tg (kdrl: EGFP) s843 stammer, henholdsvis, og Taiwan Sebrafisk Kjerne Facility for å gi Tg (kdrl: mCherry) ci5. Denne studien ble støttet med tilskudd fra Taiwan Ministry of Science and Technology (96-2628-B-029-002-My3, 101-2313-B-029-001, 102-2628-B-029-002-My3, 102- 2321-B-400-018).

Etikk Uttalelse: Alle eksperimentelle prosedyrer på sebrafisk ble godkjent av Institutional Animal Care og bruk komité Tunghai-universitetet (IRB godkjenning 101-12.) Og utføres i samsvar med vedtatte retningslinjer. 1. Stock Løsninger for 3β-Hsd enzymatisk aktivitet Farging <ol><li> Fremstille trans-dehydroandrosterone [10 mg / ml i dimetylsulfoksid (DMSO)]. </li><li> Fremstille β-nikotinamid-adenin-dinukleotid-hydrat (1,2 mg / ml i 0,1 M fosfatbuffer, pH 7,2). </li><li> Forbered ni...

<ol>
	<li>Hsu, H. J., Lin, G., Chung, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+early+development+of+zebrafish+interrenal+glands+and+pronephros:+differential+control+by+wt1+and+ff1b.">Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b.</a> Development. 130, 2107-2116 (2003).</li><li>To, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary-interrenal+interaction+in+zebrafish+interrenal+organ+development.">Pituitary-interrenal interaction in zebrafish interrenal organ development.</a> Mol Endocrinol. 21, 472-485 (2007).</li><li>Liu, Y. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+zebrafish+hsd3b+genes+are+distinct+in+function,+expression,+and+evolution.">Two zebrafish hsd3b genes are distinct in function, expression, and evolution.</a> Endocrinology. 156, 2854-2862 (2015).</li><li>Grassi Milano, E., Basari, F., Chimenti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+and+adrenomedullary+homologs+in+eight+species+of+adult+and+developing+teleosts:+morphology,+histology,+and+immunohistochemistry.">Adrenocortical and adrenomedullary homologs in eight species of adult and developing teleosts: morphology, histology, and immunohistochemistry.</a> Gen Comp Endocrinol. 108, 483-496 (1997).</li><li>Chai, C., Liu, Y. W., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ff1b+is+required+for+the+development+of+steroidogenic+component+of+the+zebrafish+interrenal+organ.">Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ.</a> Dev Biol. 260, 226-244 (2003).</li><li>Liu, Y. W., Gao, W., Teh, H. L., Tan, J. H., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prox1+is+a+novel+coregulator+of+Ff1b+and+is+involved+in+the+embryonic+development+of+the+zebra+fish+interrenal+primordium.">Prox1 is a novel coregulator of Ff1b and is involved in the embryonic development of the zebra fish interrenal primordium.</a> Mol Cell Biol. 23, 7243-7255 (2003).</li><li>Chai, C., Liu, Y. W., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ff1b+is+required+for+the+development+of+steroidogenic+component+of+the+zebrafish+interrenal+organ.">Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ.</a> Dev. Biol. 260, 226-244 (2003).</li><li>Chou, C. W., Zhuo, Y. L., Jiang, Z. Y., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hemodynamically-regulated+vascular+microenvironment+promotes+migration+of+the+steroidogenic+tissue+during+its+interaction+with+chromaffin+cells+in+the+zebrafish+embryo.">The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.</a> PLoS One. 9, e107997 (2014).</li><li>Liu, Y. W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium+is+required+for+the+promotion+of+interrenal+morphogenetic+movement+during+early+zebrafish+development.">Endothelium is required for the promotion of interrenal morphogenetic movement during early zebrafish development.</a> Dev Biol. 297, 44-58 (2006).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Proulx, K., Lu, A., Sumanas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+vasculature+in+zebrafish+forms+by+angioblast+cluster-derived+angiogenesis.">Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis.</a> Dev Biol. 348, 34-46 (2010).</li><li>Perner, B., Englert, C., Bollig, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wilms+tumor+genes+wt1a+and+wt1b+control+different+steps+during+formation+of+the+zebrafish+pronephros.">The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros.</a> Dev Biol. 309, 87-96 (2007).</li><li>Chou, C. 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Signalstyrken til den 3β-Hsd aktivitet økes i løpet av reaksjonen. Klare signaler om den 3β-Hsd-aktivitet ble påvist etter 4 timers reaksjon for trinn fra 28 HPF framover. Imidlertid krever reaksjonen varighet empiriske bestemmelse, avhengig av formålet med analysen. I de tilfeller hvor fargingen krever behandling over natten, har en tendens til en lett blåaktig bakgrunn å utvikle seg på prøvene. Dette problemet kan overvinnes ved å øke fiksering varighet (for eksempel 4 timer i 4% PFAT ved romtempe...

Binyrene, en viktig del av hypothalamo-hypofyse-binyre-aksen, skiller steroider og koordinerer steroid homeostase og kroppslig reaksjon på stress. Binyrene omfatter den ytre cortex, som utskiller steroider i en sone-spesifikk måte, og indre marg, som syntetiserer katekolaminer. Den interrenal kjertel i teleoster er motstykket i binyrene hos pattedyr og er sammensatt av steroidogenic interrenal og chromaffin celler, som er funksjonelle ekvivalenter av binyrebarken og medulla, henholdsvis 1-3. Studier utført ved hjelp av sebrafisk-modellen har rapportert at både steroidogenic og chromaffin celle linjene er dannet av molekylære og cellulære mekanismer sterkt ligner de i pattedyr 1,2. Derfor sebrafisk er en potensielt kraftig modell for å studere genetiske lidelser, nevroendokrin kontroll og systembiologi av hypothalamo-hypofyse-binyre (interrenal) akse. <p class = &quot;jove_content&quot;&gt; I binyrene, katalyserer 3β-Hsd konvertering av progesteron fra pregnenolone, 17α-hydroxyprogesterone fra 17α-hydroxypregnelolone, og androstendion fra Dehydroepiandrosteron 4,5. 3β-Hsd er viktig for biosynthesizing alle klasser av hormonelle steroider, nemlig progesteron, glukokortikoider, mineralkortikoider, androgener og østrogener. De to menneskelige 3β-Hsd isozymene HSD3B1 og HSD3B2 er forskjellig uttrykt 6. HSD3B1 er uttrykt i morkaken og perifert vev, mens HSD3B2 er uttrykt i binyrebarken og gonadene. Menneskelig HSD3B1 og HSD3B2 er co-ortologer av sebrafisk hsd3b1, som uttrykkes ved interrenal vev og voksne gonader; sebrafisk hsd3b2 er en moderlig uttrykt gen hvis transkripsjoner forsvinne før organogeneseperioden 7. Protokollen av hele montering 3β-Hsd enzymatisk aktivitetsanalyse for sebrafisk ble utviklet ved å modifisere Levy metode, ens beskrevet av Milano et al. , På frosne deler av åtte teleost arter 8. På grunn av den vev permeabilitet og den optiske gjennomsiktigheten av det fremkallende sebrafisk, hel-mount 3β-Hsd histokjemi kan med hell brukes for det faste sebrafisk embryo og larver og spesielt avgrense de differensierte interrenal vev. Dette følsom og rask analyse har blitt brukt til ulike mutanter og morphants demonstrere ulike typer interrenal dysmorphogenesis. Den interrenal 3β-Hsd aktivitet er fraværende i embryo der spesifikasjonen av interrenal vevet er forstyrret gjennom en spesifikk knockdown av Ff1b transkripsjonsfaktor og reduseres ettersom interrenal differensiering er påvirket av en knockdown av Ff1b coregulator Prox1 9,10. Spesielt kan 3β-Hsd aktivitet påvises i mutanter med alvorlige tidlig stadium defekter, slik som enøyde knappenålshode og myse, hvor 3β-Hsd histokjemi skisserer hvordan interrenal celle migrasjon påvirkes 11. Differensieringen av interrenal vevet er ikke i fare selv i fullstendig fravær av blod og blodkar. Derfor, hvordan endotel-avledede signaler forme det fremkallende interrenal organ kan bestemmes 12,13. Alt i alt har denne histokjemisk analyse blitt brukt til å studere beskrivelsen, differensiering og migrering av steroidogenic celler i sebrafisk modell. Derfor bør det være en effektiv og pålitelig verktøy for eventuelle genetiske eller kjemiske skjermer rettet mot binyrene og interrenal organ lidelser.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Confocal microscope</td>
			<td style="width:187px;">Carl Zeiss&#160;</td>
			<td style="width:72px;">LSM510&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D8418&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>USB</td>
			<td>US16374</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Fetal Calf Serum&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30073</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Nicotinamide adenine dinucleotide hydrate</td>
			<td>Sigma</td>
			<td>N1636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Nitro blue tetrazolium</td>
			<td>Promega</td>
			<td>S380C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nusieve GTG</td>
			<td>Lonza</td>
			<td>50081</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylthiourea</td>
			<td>Sigma&#160;</td>
			<td>P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-100Tab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PYREX Spot Plate</td>
			<td>Corning</td>
			<td>7220-85</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reef Salt</td>
			<td>AZOO</td>
			<td>AZ28001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-Dehydroandrosterone</td>
			<td>Sigma</td>
			<td>D4000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000M</td>
		</tr>
	</tbody>
</table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

For å finne ut hvordan de steroidogenic interrenal vev codevelops med pronephric nyre glomerulus og sin begynnende blodkar, ble 3β-Hsd enzymatisk aktivitetsanalyse utført på dobbelttransgene Tg (wt1b: GFP) LI1, Tg (kdrl: mCherry) ci5 embryo ved 34 HPF (Figur 1). På dette trinn blir 3β-Hsd-aktivitet-positive steroidogenic vev lokalisert rett til midtlinjen og umiddelbart kaudalt for pronephric nyre glomerulus, mens noen steroidogenic c...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Den interrenal kjertel i sebrafisk er teleostean motstykket til pattedyr binyrene. Denne protokollen introduserer hvordan å utføre tre-β-hydroksysteroiddehydrogenase (Δ 5-4 isomerase, 3β-Hsd) enzymatisk aktivitetsanalyse, som oppdager differensierte steroidogenic celler i å utvikle sebrafisk.

Visualisere Interrenal Steroidogenic Tissue og dens Vaskulær mikromiljøet i Sebrafisk

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

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