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 Prof. Didier Stainier for gifting Tg (wt1b: GFP) LI1 og Tg (kdrl: EGFP) s843 stammer henholdsvis og Taiwan zebrafisk Core Facility for at give Tg (kdrl: mCherry) CI5. Denne undersøgelse blev støttet af tilskud fra Taiwans Ministeriet for Videnskab og Teknologi (96-2628-B-029-002-MY3, 101-2313-B-029-001, 102-2628-B-029-002-MY3, 102- 2321-B-400-018).

Etik Statement: Alle eksperimentelle procedurer på zebrafisk blev godkendt af Institutional Animal Care og brug Udvalg Tunghai University (IRB Godkendelse NO 101-12.) Og udføres i overensstemmelse med de godkendte retningslinjer. 1. Stock Løsninger til 3β-HSD Enzymatisk aktivitet Farvning <ol><li> Forbered trans-dehydroandrosterone [10 mg / ml i dimethylsulfoxid (DMSO)]. </li><li> Forbered β-Nikotinamidadenindinucleotid hydrat (1,2 mg / ml i 0,1 M phosphatpuffer, pH 7,2). </li><li> Fo...

<ol>
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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 af ​​3β-HSD-aktivitet steg i løbet af reaktionen. blev påvist klare signaler af 3β-HSD-aktivitet efter 4 timers reaktion for stadier fra 28 HPF fremefter. Imidlertid kræver reaktionen varighed empiriske bestemmelse, afhængigt af formålet med analysen. I tilfælde, hvor farvningen kræver indlæggelse behandling, en lidt blålig baggrund tendens til at udvikle på prøverne. Dette problem kan overvindes ved at øge varigheden fiksering (fx 4 timer i 4% PFAT ved stuetemperatur), før 3β-...

Binyrerne, en afgørende komponent i hypothalamus-hypofyse-binyre-aksen, udskiller steroider og koordinerer steroid homeostase og den kropslige reaktion på stress. Binyrerne omfatter den ydre cortex, som udskiller steroider i en zone-specifik måde, og indre medulla, som syntetiserer catecholaminer. Den interrenal kirtel i benfisk er modstykket til binyrerne i pattedyr, og er sammensat af steroidogene interrenal og kromaffinceller, som er funktionelle ækvivalenter af binyrebarken og medulla henholdsvis 1-3. Undersøgelser udført ved hjælp af zebrafisk model har rapporteret, at både steroidogene og chromaffin cellelinier er dannet af molekylære og cellulære mekanismer meget ligner dem i pattedyr 1,2. Derfor er zebrafisk er et potentielt magtfuldt model til at studere genetiske sygdomme, neuroendokrine kontrol og systemer biologi hypothalamus-hypofyse-binyre (interrenal) akse. <p class = &quot;jove_content&quot;&gt; I binyrerne, 3β-HSD katalyserer omdannelsen af progesteron fra pregnenolon, 17α-hydroxyprogesteron fra 17α-hydroxypregnelolone, og androstendion fra dehydroepiandrosteron 4,5. 3β-HSD er afgørende for biosyntese af alle klasser af hormonale steroider, nemlig progesteron, glucocorticoider, mineralcorticoider, androgener og østrogener. De to menneskelige 3β-HSD isozymer HSD3B1 og HSD3B2 udtrykkes forskelligt 6. HSD3B1 udtrykkes i placenta og perifere væv, mens HSD3B2 udtrykkes i binyrebarken og gonader. Menneskelig HSD3B1 og HSD3B2 er co-ortologer af zebrafisk hsd3b1, som udtrykkes ved interrenal væv og voksne gonader; zebrafisk hsd3b2 er et maternelt udtrykt gen, hvis udskrifter forsvinder før organogenese 7. Protokollen for hel-mount 3β-HSD enzymatisk aktivitet assay for zebrafisk blev udviklet ved at modificere Levy metode, ens beskrevet af Milano et al. På frosne snit af otte teleost arter 8. På grund af vævet permeabilitet og optisk transparens af det udviklende zebrafisk, hel-mount 3β-HSD histokemi held kan anvendes til den faste zebrafisk embryoner og larver og specifikt afgrænse de differentierede interrenal væv. Dette følsomme og hurtige assay er blevet anvendt på forskellige mutanter og morphants udvise forskellige typer interrenal dysmorphogenesis. Den interrenal 3β-HSD-aktivitet er fraværende i embryo hvor specifikation af interrenal væv forstyrres gennem en særlig knockdown af Ff1b transskription faktor, og er faldet, idet interrenal differentiering er påvirket af en knockdown af Ff1b coregulator Prox1 9,10. Især kan detekteres 3β-HSD-aktivitet i mutanter med alvorlige fase defekter tidligt, såsom enøjet pinhead og skele, hvor 3β-Hsd histokemi skildrer hvordan interrenal cellevandring påvirkes 11. Differentieringen af ​​interrenal væv ikke kompromitteres, selv i den fuldstændige mangel på blod og kar. Derfor, hvordan endotelafledt signaler forme udvikle interrenal organ kan bestemmes 12,13. Alt i alt har denne histokemisk assay med succes blevet brugt til at studere specifikation, differentiering og migration af steroidogene celler i zebrafisk model. Derfor bør det være et effektivt og pålideligt værktøj til eventuelle genetiske eller kemiske skærme rettet mod adrenale og interrenal orgel 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 at bestemme, hvordan de steroidogene interrenal væv codevelops med pronephric nyre glomerulus og dens spirende vaskulatur blev 3β-HSD enzymatisk aktivitet assay udført på den dobbelte transgene Tg (wt1b: GFP) LI1; Tg (kdrl: mCherry) CI5 embryo på 34 HPF (Figur 1). På dette stadium er det 3β-HSD-aktivitet-positive steroidogene væv placeret lige med midterlinjen og umiddelbart kaudalt for pronephric nyre glomerulus, mens nogle ste...

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 kirtel i zebrafisk er teleostean modstykke til pattedyr binyrerne. Denne protokol introducerer hvordan du udfører den 3-β-hydroxysteroiddehydrogenase (Δ 5-4 isomerase; 3β-HSD) enzymatisk aktivitet assay, der detekterer differentierede steroidogene celler i udviklingslandene zebrafisk.

Visualisere Interrenal steroidogeniske Tissue og dens Vaskulær mikromiljø i Zebrafisk

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

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