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

Wij danken Prof. Christoph Englert en Prof. Didier Stainier voor gifting de Tg (wt1b: GFP) LI1 en Tg (kdrl: EGFP) s843 stammen, respectievelijk, en de Taiwan zebravis Core Faciliteit voor het verstrekken van Tg (kdrl: mCherry) CI5. Deze studie werd ondersteund door subsidies van het ministerie van Wetenschap en Technologie (96-2628-B-029-002-my3, 101-2313-B-029-001, 102-2628-B-029-002-my3 Taiwan, 102- 2321-B-400-018).

Ethiek Verklaring: Alle experimentele procedures op zebravis werden door de Institutional Animal Care en gebruik Comite van Tunghai University goedgekeurd (IRB Goedkeuring NO 101-12.) En in overeenstemming met de goedgekeurde richtlijnen uitgevoerd. 1. Voorraadoplossingen voor 3β-Hsd Enzymatische Activiteit Kleuring <ol><li> Bereid trans-dehydroandrosterone [10 mg / ml in dimethylsulfoxide (DMSO)]. </li><li> Bereid β-nicotinamide adenine dinucleotide hydraat (1,2 mg / ml in 0,1 M fosfaat...

<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. W., Chiu, C. H., 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=Fibronectin+mediates+correct+positioning+of+the+interrenal+organ+in+zebrafish.">Fibronectin mediates correct positioning of the interrenal organ in zebrafish.</a> Dev Dyn. 242, 432-443 (2013).</li><li>Chiu, C. H., Chou, C. W., Takada, S., 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=Development+and+fibronectin+signaling+requirements+of+the+zebrafish+interrenal+vessel.">Development and fibronectin signaling requirements of the zebrafish interrenal vessel.</a> PLoS One. 7, e43040 (2012).</li></ol>

De signaalsterkte van de 3β-Hsd activiteit verhoogd in de loop van de reactie. Duidelijke signalen van de 3β-Hsd activiteit werden gedetecteerd na 4 uur reactie voor de fasen van 28 HPF verder. De reactieduur vereist empirisch bepalen, afhankelijk van het doel van de test. Indien de kleuring vereist nachts verwerking, een licht blauwachtige achtergrond ontwikkelt zich meestal op de monsters. Dit probleem kan worden ondervangen door de tijdsduur fixatie (bijvoorbeeld 4 uur in 4% PFAT bij KT) vóór de verhogin...

De bijnier, een cruciaal onderdeel van de hypothalamus-hypofyse-bijnier as, scheidt steroïden en coördineert steroïde homeostase en de lichamelijke reactie op stress. De bijnier omvat de buitenste cortex, die steroïden scheidt in een zone-specifieke wijze en binnenste medulla, die catecholamines synthetiseert. De interrenal klier in teleosten is de tegenhanger van de bijnier bij zoogdieren en is samengesteld uit steroidogenic interrenal en chromaffiene cellen, die functionele equivalenten van de bijnierschors en merg, respectievelijk 1-3 zijn. Studies uitgevoerd met het zebravismodel hebben gemeld dat zowel steroidogenic en chromaffine cellijnen gevormd door moleculaire en cellulaire mechanismen die sterk lijken op die bij zoogdieren 1,2. Daarom is de zebravis is een potentieel krachtig model voor het bestuderen van genetische aandoeningen, neuro-endocriene controle en systeembiologie van de hypothalamus-hypofyse-bijnier (interrenal) as. <p class = &quot;jove_content&quot;&gt; In de bijnier, 3β-Hsd katalyseert de omzetting van progesteron uit pregnenolon, 17α-hydroxyprogesteron van 17α-hydroxypregnelolone en androstenedione uit dehydroepiandrosterone 4,5. 3β-Hsd is essentieel voor de biosynthese van alle klassen van hormonale steroïden, namelijk progesteron, glucocorticoïden, mineralocorticoïden, androgenen en oestrogenen. De twee menselijke 3β-Hsd isozymen HSD3B1 en HSD3B2 zijn differentieel tot expressie 6. HSD3B1 wordt uitgedrukt in de placenta en perifere weefsels, terwijl HSD3B2 uitgedrukt in de bijnierschors en de geslachtsklieren. Human HSD3B1 en HSD3B2 zijn co-orthologen van zebravis hsd3b1, die wordt uitgedrukt in het interrenal weefsel en volwassen geslachtsklieren; zebravis hsd3b2 is een voor het moederdier uitgedrukt gen waarvan de transcripten verdwijnen voordat organogenese 7. Het protocol van de hele-mount 3β-Hsd enzymatische activiteit test voor zebravis ontwikkeld door aanpassing Levy methode, eens beschreven door Milano et al. Op vriescoupes acht teleost species 8. Door het weefsel permeabiliteit en optische transparantie van het zebravisembryo, whole-mount 3β-Hsd histochemie succes kan worden gebruikt voor de vaste zebravis embryo&#39;s en larven en specifiek toespitsen op het gedifferentieerde interrenal weefsels. Deze gevoelige en snelle test is toegepast op verschillende mutanten en morphants tonen verschillende interrenal dysmorfogenese. De interrenal 3β-Hsd activiteit afwezig is in het embryo, waar specificatie van de interrenal weefsel wordt verstoord door een specifieke knockdown van de Ff1b transcriptiefactor en daalt als de interrenal differentiatie wordt beïnvloed door een knockdown van de Ff1b coregulator Prox-1 9,10. Met name kan de 3β-Hsd-activiteit worden gedetecteerd in mutanten met ernstige vroeg gebreken zoals eenogige pinhead en turen, waarbij de 3β-Hsd histochemie schetst hoe de interrenal celmigratie wordt aangetast 11. De differentiatie van de interrenal weefsel wordt niet aangetast, zelfs in de volledige afwezigheid van bloed en vaatstelsel. Daarom, hoe-endotheel afgeleide signalen geven vorm aan de ontwikkeling van interrenal orgaan kan worden bepaald 12,13. Kortom, deze histochemische assay is met succes gebruikt voor het bestuderen van de specificatie, differentiatie en migratie van steroidogenic cellen in de zebravis model. Daarom moet een efficiënt en betrouwbaar middel voor genetische of chemische screens gericht bijnieren interrenal orgaan stoornissen.

<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.

Om te bepalen hoe de steroidogenic interrenal weefsel codevelops met de pronephric nier glomerulus en de ontluikende vaatstelsel, de 3β-Hsd enzymatische activiteit test werd uitgevoerd op de dubbele transgene Tg (wt1b: GFP) LI1; Tg (kdrl: mCherry) CI5 embryo op 34 HPF (Figuur 1). In dit stadium is het 3β-Hsd activiteit-positieve steroidogenic weefsel ligt direct aan de middellijn en meteen caudaal van de pronephric nier glomerulus, terwij...

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

De interrenal klier in de zebravis is de teleostean tegenhanger van de zoogdieren bijnier. Dit protocol wordt beschreven hoe de 3-β-hydroxysteroïdedehydrogenase (Δ 5-4 isomerase, 3β-Hsd) verricht enzymatische activiteit test, die gedifferentieerde steroïdogene cellen in zebravisembryo detecteert.

Het visualiseren van de Interrenal steroïdogene Tissue en haar Vasculaire Microenvironment in zebravis

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

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