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).</li>
	<li>Prepare nicotinamide (vitamin B3, 50 mg/ml in H2O).</li>
	<li>Prepare 4-nitro blue tetrazolium (50 mg/ml in 70% dimethylformamide).</li>
	<li>Aliquot all these components into microfuge tubes and store at -20 &#176;C. 
	NOTE: In our experience, repeated freezing and thawing of the aliquots of all the aforementioned solutions do not affect the intensity of 3&#946;-Hsd activity staining.</li>
</ol>
2. Whole-mount 3&#946;-Hsd Activity Staining
NOTE: The 3&#946;-Hsd enzymatic activity is detectable in zebrafish embryos as early as 28 hours post fertilization (hpf) when cultured at 28.5 &#176;C, which is consistent with the onset of mRNA expression of hsd3b12,7. The whole-mount 3&#946;-Hsd activity staining protocol in this study is based on a previously described method8 and was determined to be successful in the developing zebrafish up to 7 days post fertilization9. For analyzing the vascular microenvironment of the interrenal steroidogenic tissue, whole-mount 3&#946;-Hsd activity staining must be applied to transgenic lines expressing endothelium-specific fluorescence, such as Tg(kdrl:EGFP)s843 14 and Tg(kdrl:mCherry)ci5 15. To analyze the interrenal steroidogenic tissue considering zebrafish kidney development, 3&#946;-Hsd activity staining can be applied to the Tg(wt1b:GFP)li1 fish16, where the formation of the glomerulus and pronephric tubules is delineated.
<ol>
	<li>Treat the developing zebrafish by incubating them in 0.03% phenylthiourea in egg water (0.06 mg/L reef salt in deionized water), starting from a stage between 12 and 24 hpf, to inhibit pigment formation.</li>
	<li>Fix dechorionated embryos or larvae in (A) 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) supplemented with 0.1% Tween 20 (PFAT) for 1 hr at room temperature (RT) or (B) 2% PFAT overnight at 4 &#176;C or for 2 hr at 4 &#176;C, followed by 4 hr at RT. 
	Caution: PFA is toxic by inhalation and by skin contact. It is destructive to the skin, mucous membranes, eyes, and upper respiratory tract.</li>
	<li>Wash the embryos 4x for 10 min with PBS supplemented with 0.1% Tween 20 (PBST) at RT. 
	NOTE: It is critical to not dry the samples while changing the washing solutions.</li>
	<li>Freshly prepare the staining solution before the reactions (Table 1). Prepare, vortex, and centrifuge parts A and B in separate tubes. 
	NOTE: Adding all the components straight into one single tube would cause severe precipitation.</li>
	<li>Add the entire solution of part A to the entire solution of part B, mix well to prepare the staining solution, and immediately distribute 1 ml into each microfuge tube containing fixed and washed embryos.</li>
	<li>Wrap microfuge tubes with aluminum foil to protect them from light and place on either a rotator or a rocking platform for gentle shaking at RT.</li>
	<li>Empirically determine the reaction time by monitoring chromogenic signals through microscopy. Slight precipitations will develop over time; however, they will not interfere with the reaction process. 
	NOTE: For embryos fixed at 28 hpf, staining at 25 &#176;C for 4 hr generates a clear signal at the interrenal tissue.</li>
	<li>Stop the reaction by washing 4x for 10 min in PBST. If no further immunohistochemical (IHC) analysis is required, post-fix the stained embryos with 4% PFAT for 60 min at RT. Otherwise, store the washed embryos at 4 &#176;C until further use.</li>
	<li>For whole-mount microscopy, clear the stained, post-fixed, and washed embryos with 50% glycerol in PBS, orient them with the dorsal side facing up on a slide, and observe using an upright microscope under bright field illumination. 
	NOTE: To make a high-resolution image of the interrenal tissue morphology, flat-mount analysis of the 3&#946;-Hsd activity-stained and deyolked embryo is recommended. As the interrenal steroidogenic tissue is positioned right above the yolk sac, a ventral flat-mount analysis of the interrenal tissue allows a direct and clear observation of the interrenal morphology17.</li>
</ol>
3. IHC Analysis of 3&#946;-Hsd Activity-stained Embryos
NOTE: For analyzing the vascular microenvironment of the steroidogenic tissue, the 3&#946;-Hsd activity-stained embryos with a transgenic endothelium-specific fluorescent reporter are subjected to transverse vibratome sectioning and subsequent IHC analysis12,18.
<ol>
	<li>Post-fixation
	<ol>
		<li>Post-fix the 3&#946;-Hsd activity-stained and washed embryos with 2% PFA supplemented with 1%Triton X-100 for 1 hr at 4 &#176;C and wash 4x for 10 min in PBS containing 1% Triton X-100 (PBSTx) at RT.</li>
	</ol>
	</li>
	<li>Embedding
	<ol>
		<li>Prepare 4% low-melting agarose by dissolving the agarose in PBS at 95 &#176;C, aliquot into microfuge tubes, and store at 4-8 &#176;C.</li>
		<li>Melt an aliquot of 4% low-melting agarose at 70 &#176;C for 10 min, and then keep the aliquot at 47 &#176;C. Embed each embryo in molten 4% low-melting agarose in a detached cap of a 1.5 ml microfuge tube which serves as a mold, and allow to cool until solid.</li>
	</ol>
	</li>
	<li>Vibratome Sectioning
	<ol>
		<li>Stick a piece of paper tape to the dry platform of the vibratome.</li>
		<li>Remove the hardened agarose block from the mold by a razor blade. Apply superglue to the paper tape on the platform, and fix the agarose block onto the paper tape, with the anterior side of the sample facing up. Trim the agarose block to a trapezoidal prism shape, with approximately 4 mm and 8 mm on each side of the upper and lower facets, respectively.</li>
		<li>Rinse the sample-containing agarose block with cold PBS supplemented with 0.5% Triton X-100 (0.5% PBSTx) by using a dropper.</li>
		<li>Slice the agarose block into 100 &#181;m sections according to the instruction manual of the vibratome. Constantly rinse the agarose block to prevent drying. As each slice is cut, remove it from the vibratome using a 26 G syringe needle, and transfer to a spot plate containing cold 0.5% PBSTx (500 &#181;l /well).</li>
		<li>Collect the 3&#946;-Hsd activity-positive tissue sections by checking under a dissection microscope, and transfer the sections by a pipette tip with its end cut-off (about 1 mm inner diameter at the opening) into a 96-well plate containing cold 0.5% PBSTx (150 &#181;l/well). The tissue samples usually dissociate from the agarose block after this step.</li>
	</ol>
	</li>
	<li>Permeabilization and Washing
	<ol>
		<li>Wash the slices 6x for 15 min in 0.5% PBSTx at RT, 10 min in PBS, and 4x for 10 min in PBS supplemented with 1% bovine serum albumin/1% DMSO/0.1% Triton X-100 (PBDTx).</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Incubate the slices for 1 hr in 2% fetal calf serum (FCS) in PBDTx at RT.</li>
	</ol>
	</li>
	<li>Primary Antibody Reaction
	<ol>
		<li>Incubate the slices for 1.5 days in PBDTx containing a primary antibody (e.g., rabbit polyclonal anti-human fibronectin and mouse anti-human focal adhesion kinase) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Washing
	<ol>
		<li>Wash the slices 6x for 10 min in PBDTx at RT.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Incubate the slices for 1 hr in 2% FCS in PBDTx at RT.</li>
	</ol>
	</li>
	<li>Secondary Antibody Reaction
	<ol>
		<li>Incubate the slices for 1.5 days in PBDTx containing a secondary antibody (fluorophore-conjugated goat anti-rabbit or anti-mouse IgG) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Washing
	<ol>
		<li>Wash the slices 6x for 10 min in PBDTx at RT and 4x for 10 min in PBST at RT. 
		NOTE: For confocal microscopic analysis, clear the samples with 50% glycerol in PBS.</li>
	</ol>
	</li>
</ol>
4. Confocal Analysis
<ol>
	<li>Observe the whole mount embryo and the section by a confocal microscope equipped with 10X/0.5 and 20X/0.75 objective lens.</li>
	<li>For simultaneous detection of the fluorescence and the 3&#946;-Hsd staining, set the multitrack mode as follows: 488 nm argon laser and 505-530 nm bandpass filter for the detection of GFP; 543 nm Helium-Neon laser and 560-615 nm bandpass filter for the detection of mCherry; and transmitted light channel for the detection of 3&#946;-Hsd staining. The pinhole size is 1 Airey unit, and the resolution is 1,024 x 1,024 pixel.</li>
	<li>Assemble the z stacks (with 6 &#956;m interval) as a 3D projection, and merge the projection and the bright field, by using the built-in confocal software.</li>
</ol>

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	<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>

The authors have no competing financial interests to declare.

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><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></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 ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.9K Views.  Tunghai University. The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-β-hydroxysteroid dehydrogenase (Δ5-4 isomerase; 3β-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing zebrafish.

Video: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Chemistry

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Cancer Research

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Biology

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medicine

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

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Chapters in this video

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Dissection of Larval Zebrafish Gonadal Tissue

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

Dissection of the Adult Zebrafish Kidney

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization