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>

<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>

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 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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JoVE Journal

Developmental Biology

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

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...