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

We thank Prof. Christoph Englert and Prof. Didier Stainier for gifting the Tg(wt1b:GFP) li1 and Tg(kdrl:EGFP)s843 strains, respectively, and the Taiwan Zebrafish Core Facility for providing Tg(kdrl:mCherry)ci5. This study was supported by grants from Taiwan&#39;s 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).

Ethics Statement: All experimental procedures on zebrafish were approved by the Institutional Animal Care and Use Committee of Tunghai University (IRB Approval NO. 101-12) and carried out in accordance with the approved guidelines.
1. Stock Solutions for 3&#946;-Hsd Enzymatic Activity Staining
<ol>
	<li>Prepare trans-dehydroandrosterone [10 mg/ml in dimethyl sulfoxide (DMSO)].</li>
	<li>Prepare &#946;-nicotinamide adenine dinucleotide hydrate (1.2 mg/ml in 0.1 M phosphate buffer, pH 7.2).</l...

<ol>
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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>

The signal strength of the 3&#946;-Hsd activity increased over the course of reaction. Clear signals of the 3&#946;-Hsd activity were detected after 4 hours of reaction for stages from 28 hpf onward. However, the reaction duration requires empirical determination, depending on the purpose of the assay. In cases where the staining requires overnight processing, a slightly bluish background tends to develop on the samples. This problem can be overcome by increasing the fixation duration (e.g., 4 hr in 4% PFAT at R...

The adrenal gland, a crucial component of the hypothalamo-pituitary-adrenal axis, secretes steroids and coordinates steroid homeostasis and the bodily response to stress. The adrenal gland comprises the outer cortex, which secretes steroids in a zone-specific manner, and inner medulla, which synthesizes catecholamines. The interrenal gland in teleosts is the counterpart of the adrenal gland in mammals and is composed of steroidogenic interrenal and chromaffin cells, which are functional equivalents of the adrenal cortex and medulla, respectively1-3. Studies conducted using the zebrafish model have reported that both steroidogenic and chromaffin cell lineages are formed by molecular and cellular mechanisms highly resembling those in mammals1,2. Therefore, the zebrafish is a potentially powerful model for studying genetic disorders, neuroendocrine control, and systems biology of the hypothalamo-pituitary-adrenal (interrenal) axis.
In the adrenal gland, 3&#946;-Hsd catalyzes the conversion of progesterone from pregnenolone, 17&#945;-hydroxyprogesterone from 17&#945;-hydroxypregnelolone, and androstenedione from dehydroepiandrosterone4,5. 3&#946;-Hsd is essential for biosynthesizing all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. The two human 3&#946;-Hsd isozymes HSD3B1 and HSD3B2 are differentially expressed6. HSD3B1 is expressed in the placenta and peripheral tissues, whereas HSD3B2 is expressed in the adrenal cortex and gonads. Human HSD3B1 and HSD3B2 are co-orthologs of zebrafish hsd3b1, which is expressed at the interrenal tissue and adult gonads; zebrafish hsd3b2 is a maternally expressed gene whose transcripts disappear before organogenesis7. The protocol of the whole-mount 3&#946;-Hsd enzymatic activity assay for zebrafish was developed by modifying Levy&#39;s method, as described by Milano et al., on frozen sections of eight teleost species8. Because of the tissue permeability and optical transparency of the developing zebrafish, whole-mount 3&#946;-Hsd histochemistry can be successfully used for the fixed zebrafish embryo and larvae and specifically delineate the differentiated interrenal tissues.
This sensitive and rapid assay has been applied to various mutants and morphants demonstrating different types of interrenal dysmorphogenesis. The interrenal 3&#946;-Hsd activity is absent in the embryo where specification of the interrenal tissue is disrupted through a specific knockdown of the Ff1b transcription factor and is decreased as the interrenal differentiation is affected by a knockdown of the Ff1b coregulator Prox19,10. Notably, the 3&#946;-Hsd activity can be detected in mutants with severe early stage defects, such as one-eyed pinhead and squint, where the 3&#946;-Hsd histochemistry delineates how the interrenal cell migration is affected11. The differentiation of the interrenal tissue is not compromised even in the complete absence of blood and vasculature. Therefore, how endothelium-derived signals shape the developing interrenal organ can be determined12,13. Overall, this histochemical assay has been successfully used for studying specification, differentiation, and migration of steroidogenic cells in the zebrafish model. Therefore, it should be an efficient and a reliable tool for any genetic or chemical screens targeting adrenal and interrenal organ disorders.

<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.

To determine how the steroidogenic interrenal tissue codevelops with the pronephric kidney glomerulus and its nascent vasculature, the 3&#946;-Hsd enzymatic activity assay was performed on the double transgenic Tg(wt1b:GFP)li1;Tg(kdrl:mCherry)ci5 embryo at 34 hpf (Figure 1). At this stage, the 3&#946;-Hsd activity-positive steroidogenic tissue is located right to the midline and immediately caudal to the pronephric kidney glomerulus, whereas...

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

The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-&#946;-hydroxysteroid dehydrogenase (&#916;5-4 isomerase; 3&#946;-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing zebrafish.

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

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