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

Ringraziamo il Prof. Christoph Englert e il Prof. Didier Stainier per regalare il Tg (wt1b: GFP) LI1 e Tg (kdrl: EGFP) s843 ceppi, rispettivamente, e il meccanismo di Taiwan Zebrafish Nucleo per la fornitura di Tg (kdrl: mCherry) CI5. Questo studio è stato sostenuto da sovvenzioni dal Ministero della Scienza e della Tecnologia (96-2628-B-029-002-MY3, 101-2313-B-029-001, 102-2628-B-029-002-MY3 di Taiwan, 102- 2321-B-400-018).

Etica Dichiarazione: Tutte le procedure sperimentali su zebrafish sono stati approvati dal Comitato di Cura e uso istituzionale degli animali di Tunghai University (IRB Soddisfazione NO 101-12.) E realizzato in conformità con le linee guida approvate. 1. Soluzioni azioni 3β-Hsd enzimatico attività di colorazione <ol><li> Preparare trans-dehydroandrosterone [10 mg / ml in dimetilsolfossido (DMSO)]. </li><li> Preparare β-nicotinammide adenina dinucleotide idrato (1,2 mg / ml in tampone f...

<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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrenal+organogenesis+in+the+zebrafish+model.">Interrenal organogenesis in the zebrafish model.</a> Organogenesis. 3, 44-48 (2007).</li><li>Cravioto, M. D., 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=A+new+inherited+variant+of+the+3+beta-hydroxysteroid+dehydrogenase-isomerase+deficiency+syndrome:+evidence+for+the+existence+of+two+isoenzymes.">A new inherited variant of the 3 beta-hydroxysteroid dehydrogenase-isomerase deficiency syndrome: evidence for the existence of two isoenzymes.</a> J Clin Endocrinol Metab. 63, 360-367 (1986).</li><li>Lachance, Y., 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=Characterization+of+human+3+beta-hydroxysteroid+dehydrogenase/delta+5-delta+4-isomerase+gene+and+its+expression+in+mammalian+cells.">Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.</a> J Biol Chem. 267, 3551 (1992).</li><li>Simard, J., 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=Molecular+biology+of+the+3beta-hydroxysteroid+dehydrogenase/delta5-delta4+isomerase+gene+family.">Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.</a> Endocr Rev. 26, 525-582 (2005).</li><li>Lin, J. 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>

La forza dell&#39;attività 3β-Hsd segnale aumentato nel corso della reazione. chiari segnali di attività 3β-HSD sono stati rilevati dopo 4 ore di reazione per fasi, dalla 28 in poi HPF. Tuttavia, la durata della reazione richiede determinazione empirica, a seconda dello scopo del test. Nei casi in cui la colorazione richiede elaborazione notturna, uno sfondo leggermente bluastra tende a svilupparsi sui campioni. Questo problema può essere superato aumentando la durata di fissaggio (ad esempio, 4 ore a 4% P...

La ghiandola surrenale, una componente fondamentale dell&#39;asse ipotalamo-ipofisi-surrene, secerne gli steroidi e coordina l&#39;omeostasi di steroidi e la risposta del corpo allo stress. La ghiandola surrenale comprende la corteccia esterna, che secerne steroidi in maniera specifica per la zona, e midollo interni, che sintetizza catecolamine. La ghiandola interrenale in teleostei è la controparte della ghiandola surrenale nei mammiferi ed è composto da cellule interrenale e cromaffini steroidogenici, che sono equivalenti funzionali della corteccia surrenale e midollare, rispettivamente, 1-3. Studi condotti utilizzando il modello zebrafish hanno riferito che entrambe le linee cellulari steroidogenici e cromaffini sono formate da meccanismi molecolari e cellulari altamente simili a quelle dei mammiferi 1,2. Pertanto, il pesce zebra è un modello potenzialmente potente per lo studio di malattie genetiche, controllo neuroendocrino, e la biologia dei sistemi dell&#39;asse ipotalamo-ipofisi-surrene (interrenale). <p class = &quot;jove_content&quot;&gt; Nella ghiandola surrenale, 3β-Hsd catalizza la conversione del progesterone dal pregnenolone, 17α-idrossiprogesterone da 17α-hydroxypregnelolone, e androstenedione da deidroepiandrosterone 4,5. 3β-HSD è essenziale per biosynthesizing tutte le classi di steroidi ormonali, vale a dire il progesterone, glucocorticoidi, mineralcorticoidi, androgeni ed estrogeni,. I due umana 3β-Hsd isoenzimi HSD3B1 e HSD3B2 sono espressi in modo differenziale 6. HSD3B1 è espressa nella placenta e nei tessuti periferici, mentre HSD3B2 si esprime nella corteccia surrenale e gonadi. HSD3B1 umana e HSD3B2 sono co-ortologhi di hsd3b1 zebrafish, che si esprime al tessuto interrenale e gonadi adulti; hsd3b2 zebrafish è un gene maternamente espresso le cui trascrizioni scomparire prima organogenesi 7. Il protocollo di tutto il montaggio 3β-Hsd saggio di attività enzimatica per zebrafish è stato sviluppato modificando il metodo di Levy, uns descritto da Milano et al. , Su sezioni congelate di otto specie di teleostei 8. A causa della permeabilità dei tessuti e la trasparenza ottica del pesce zebra in via di sviluppo, tutto il montaggio 3β-Hsd istochimica può essere utilizzato con successo per l&#39;embrione zebrafish fisso e larve e specificamente delineare i tessuti interrenale differenziati. Questo test sensibile e rapida è stata applicata a vari mutanti e morphants dimostrano diversi tipi di dysmorphogenesis interrenale. L&#39;attività interrenale 3β-HSD è assente nell&#39;embrione se le specifiche del tessuto interrenale è interrotta attraverso una specifica atterramento del fattore di trascrizione Ff1b ed è diminuito come la differenziazione interrenale è influenzata da un atterramento del coregulator Ff1b Prox1 9,10. In particolare, l&#39;attività 3β-Hsd può essere rilevato in mutanti con gravi difetti nelle fasi iniziali, come ad esempio un occhio capocchia di spillo e strabismo, dove il 3β-Hsd istochimica delinea come la migrazione delle cellule interrenale è influenzata 11. La differenziazione del tessuto interrenale non è compromessa anche in completa assenza di sangue e vascolare. Pertanto, come forma di segnali endotelio-derivati in via di sviluppo dell&#39;organo interrenale può essere determinato 12,13. Nel complesso, questo saggio istochimico è stato utilizzato con successo per lo studio specificazione, differenziazione e migrazione delle cellule steroidogeniche nel modello zebrafish. Pertanto, dovrebbe essere un efficiente e uno strumento affidabile per eventuali schermi genetici o chimici di targeting disturbi degli organi surrenali e interrenale.

<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.

Per determinare come i codevelops steroidogenici tessuto interrenale con il glomerulo renale pronephric e la sua vascolarizzazione nascente, il saggio di attività enzimatica 3β-HSD è stato eseguito sul doppio transgenici Tg (wt1b: GFP) LI1; Tg (kdrl: mCherry) CI5 embrione a 34 HPF (Figura 1). In questa fase, il tessuto steroidogenico activity-positive 3β-Hsd si trova alla linea mediana e caudale immediatamente glomeruli renali pronephri...

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

La ghiandola interrenale in zebrafish è la controparte teleostei della ghiandola surrenale mammiferi. Questo protocollo introduce come eseguire la deidrogenasi 3-β-idrossisteroide (Δ 5-4 isomerasi; 3β-HSD) saggio di attività enzimatica, che rileva le cellule differenziate steroidogeniche nel zebrafish in via di sviluppo.

Visualizzare il Tissue interrenale Steroidogenic e la sua Vascolare Microenvironment in Zebrafish

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

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