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 tackar Prof. Christoph Englert och professor Didier Stainier för Gifting Tg (wt1b: GFP) LI1 och Tg (kdrl: EGFP) s843 stammar, respektive, och Taiwan Zebrafish Core Facility för att tillhandahålla Tg (kdrl: mCherry) CI5. Denna studie har finansierats med bidrag från Taiwans ministeriet för vetenskap och teknik (96-2628-B-029-002-My3, 101-2313-B-029-001, 102-2628-B-029-002-My3, 102- 2321-B-400 till 018).

Etik uttalande: Alla experimentella procedurer på zebrafisk har godkänts av Institutional Animal Care och användning kommittén av Tunghai University (IRB godkännande nr 101-12.) Och genomförs i enlighet med de godkända riktlinjerna. 1. Stamlösningar för 3β-Hsd Enzymatisk aktivitet Färgning <ol><li> Framställa trans-dehydroandrosterone [10 mg / ml i dimetylsulfoxid (DMSO)]. </li><li> Förbereda β-nikotinamid-adenin-dinukleotid-hydrat (1,2 mg / ml i 0,1 M fosfatbuffert, pH 7,2)....

<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>

Signalstyrkan för den 3β-HSD-aktivitet ökade under loppet av reaktionen. Tydliga signaler från 3β-HSD-aktivitet detekterades efter 4 timmars reaktion för stegen från 28 HPF framåt. Kräver emellertid reaktionstiden empiriska bestämningen, beroende på syftet med analysen. I de fall där färgningen kräver över natten bearbetning, tenderar en något blåaktig bakgrund att utveckla på proverna. Detta problem kan övervinnas genom att öka fixerings varaktighet (t.ex., 4 h i 4% PFAT vid RT) innan 3β-H...

Binjuren, en viktig komponent i hypotalamus-hypofys-binjure-axeln, utsöndrar steroider och samordnar steroid homeostas och kroppslig reaktion på stress. Binjuren innefattar det yttre cortex, som utsöndrar steroider i en zon specifikt sätt, och inre medulla, som syntetiserar katekolaminer. Den interrenal körtel i teleosts är motsvarigheten till binjuren hos däggdjur och består av steroidogena interrenal och kromaffinceller, som är funktionella ekvivalenter av binjurebarken och märg, respektive 1-3. Studier utförda med hjälp av zebrafisk modellen har rapporterat att både steroidogena och kromaffina cellinjer bildas genom molekylära och cellulära mekanismer mycket liknar de hos däggdjur 1,2. Därför är zebrafisk en potentiellt kraftfull modell för att studera genetiska sjukdomar, neuroendokrin kontroll och systembiologi i hypotalamus-hypofys-binjure (interrenal) axel. <p class = &quot;jove_content&quot;&gt; I binjuren, 3β-HSD katalyserar omvandlingen av progesteron från pregnenolon, 17α-hydroxiprogesteron från 17α-hydroxypregnelolone och androstendion från dehydroepiandrosteron 4,5. 3β-HSD är avgörande för biosyntes alla typer av hormonella steroider, nämligen progesteron, glukokortikoider, mineralkortikoider, androgener och östrogener. De två mänskliga 3β-HSD isozymer HSD3B1 och HSD3B2 är differentiellt uttryckta 6. HSD3B1 uttrycks i placentan och perifera vävnader, medan HSD3B2 uttrycks i binjurebarken och gonader. Human HSD3B1 och HSD3B2 är co-ortologer av zebrafisk hsd3b1, som uttrycks på interrenal vävnad och vuxna könskörtlar; zebrafisk hsd3b2 är för modern uttryckt gen vars transkript försvinna innan organ 7. Protokollet av hela montering 3β-HSD enzymatisk aktivitet analys för zebrafisk utvecklades genom att modifiera Levy metod, ens beskrivs av Milano et al. På frysta sektioner av åtta teleost arter 8. På grund av vävnads permeabilitet och optisk transparens av utvecklingszebrafisk, hel-mount 3β-HSD histokemi med framgång kan användas för fasta zebrafisk embryot och larver och specifikt beskriva de differentierade interrenal vävnader. Denna känsliga och snabb analys har tillämpats på olika mutanter och morphants visar olika typer av interrenal dysmorphogenesis. Den interrenal 3β-HSD-aktivitet är frånvarande i embryot där specificering av interrenal vävnaden störs genom en särskild knockdown av Ff1b transkriptionsfaktor och minskar som interrenal differentiering påverkas av en knockdown av Ff1b coregulator Prox1 9,10. Noterbart kan 3β-HSD-aktivitet detekteras i mutanter med allvarliga defekter tidigt stadium, såsom enögd knappnålshuvud och kisa, där 3β-Hsd histokemi skisserar hur interrenal cellmigration påverkas 11. Differentieringen av interrenal vävnaden inte äventyras även i total avsaknad av blod och kärl. Därför hur endotelhärledd signaler forma utveckla interrenal organ kan bestämmas 12,13. Sammantaget har detta histokemisk analys med framgång använts för att studera specifikation, differentiering och migration av steroidogena celler i zebrafisk modellen. Därför bör det vara ett effektivt och tillförlitligt verktyg för eventuella genetiska eller kemiska skärmar riktar binjurarna och interrenal störningar organ.

<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.

För att bestämma hur steroidogena interrenal vävnads codevelops med pronephric njuren glomeruli och dess begynnande kärl var 3β-HSD enzymatisk aktivitet analysen utförs på dubbeltransgena Tg (wt1b: GFP) LI1, Tg (kdrl: mCherry) CI5 embryo vid 34 HPF (Figur 1). I detta skede är den 3β-HSD-aktivitet-positiva steroidogena vävnad ligger rätt med mittlinjen och omedelbart kaudalt om pronephric njurglomerulus, medan vissa steroidogena 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 körtel i zebrafisk är teleostean motsvarigheten till däggdjur binjuren. Detta protokoll beskriver hur du utför tre-β-hydroxisteroiddehydrogenas (Δ 5-4 isomeras, 3β-HSD) enzymatisk aktivitet analys som detekterar differentierade steroidogena celler i utvecklingszebrafisk.

Visualisera Interrenal steroidogena Tissue och dess Vascular mikromiljö i Zebrafish

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

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