This work was supported by grants from the National Key Research and Development Program of China-stem cell and translational research [grant number 2016YFA0102300 (to Jun Yang)];the Nature Science Foundation of China [grant number 81870051, 81670054 (to Jun Yang)]; the CAMS Innovation Fund for Medical Sciences (CIFMS) [grant number 2016-I2M-4-003 (to Jun Yang)]; the PUMC Youth Found and the Fundamental Research Funds for the Central Universities [grant number 2017310013 (to Fang Zhou)].

All animal experiments described were approved by the Research Ethics Committee of Zhejiang University school of medicine.
1. Whole-mount in situ hybridization
<ol>
	<li>Zebrafish line husbandry and reproduction
	<ol>
		<li>Feed and raise all adult zebrafish at 26-28 &#176;C in a recirculating aquaculture system with 14 h light/10 h dark cycle for each day. Use AB (wild-type) zebrafish lines for the following procedures.</li>
		<li>Put a layer of pebbles in the bottom of the breeding box as a shelter for eggs. Group parental fish in the ratio of 2:1 (i.e., two females and one male) into each box. Let the female fish spawn for 1 day. At the end of spawning, remove the parent fish immediately to prevent them from eating the eggs.</li>
		<li>Inject the 2 ng of morpholinos and 500 pg of mRNA into one-cell stage embryos using the Femto Jet injection system under a microscope (endoglin-MOs sequence: 5&#39;-GATGAACTCAACACTCGTGTCTGAT-3&#39;; 5-mispair control MOs sequence: 5&#39;-AAACAGACCACATCCTCTTCATCTC-3&#39;).</li>
		<li>Incubate the zygotes in acidic sea water at 27 &#176;C for approximately 48 h.</li>
	</ol>
	</li>
	<li>Collection and fixation of zebrafish embryos
	<ol>
		<li>Use a plastic transfer pipette to collect 20-40 zebrafish embryos from the circulating system water (pH = 5.0) in a 1.5 mL tube when they are dechorionated and reach a specific period of development. Add 1 mL of freshly prepared 4% PFA solution at room temperature (RT). 
		NOTE: The period at which the embryo chosen is based on the purpose of the experiment. Table 1 shows the fixation time required for different embryonic periods.</li>
		<li>Remove the fixation solution, wash embryos with phosphate-buffered solution with Tween-20 (PBST, dilute 1 mL of Tween into 1000 mL of PBS solution) 3x for 5 min each at RT.</li>
		<li>Dehydrate the embryos through incubation in a series of 25%, 50% and 75% methanol (diluted in PBST) for 5 min each. Transfer embryos to 100% methanol for 5 min and replace with fresh methanol. Store the embryos at -20 &#176;C for overnight or longer.</li>
	</ol>
	</li>
	<li>Hydration and digestion
	<ol>
		<li>Hydrate embryos that were preserved in 100% methanol using a sieve with nylon mesh at the bottom to sequentially wash embryos in the wells of 12 well plates with a series of 75%, 50%, and 25% methanol (diluted in PBST) for 5 min each. Wash the embryos with PBST 3x for 5 min each at RT.</li>
		<li>Add 50 &#181;L of proteinase K (10 mg/mL) into the tube with embryos and follow the digestion time in the Table 2 at RT.</li>
		<li>Remove the proteinase K and wash embryos with PBST 3x for 5 min each at RT.</li>
	</ol>
	</li>
	<li>Probe hybridization and post-fixation
	<ol>
		<li>Put the probes designed according to each gene&#39;s mRNA sequences in a 40 &#176;C hybridizatin system until the precipitate is dissolved, which should take ~10 min. Mix the target probes of eg (XM_007116.7) and an additional two important genes involve in the early mesoderm endotheial progenitor formation(here, aplnr [NM_001075105.1] and nrp1a [NM_001040326.1]) in a 1.5 mL tube at a 50:1:1 ratio.</li>
		<li>Add 50-100 &#181;L of mixed target probes into each tube with embryos, then incubate overnight at 40 &#176;C. 
		NOTE: Pre-mixed probes must be pre-warmed to 40 &#176;C and cooled to RT before use.</li>
		<li>Prepare a 0.2x saline sodium citrate solution with Tween-20 (SSCT): dissolve 175.3 g of NaCl and 88 g of sodium citrate into 1 L of dH2O to obtain a 20x SSC solution. Then, dilute the solution 1:100 in dH2O to obtain a 0.2 x SSCT solution. Dilute Tween-20 1:1000 in the 0.2x SSCT solution.</li>
		<li>Transfer the recycled probes to a new tube. Wash the embryos in 0.2x SSCT solution 3x for 15 min each at RT. The recycled probes can be reused 5x-10x.</li>
		<li>Use 4% paraformaldehyde (PFA) to fix the embryos for 30 min at RT. Wash the embryos in 0.2x SSCT solution 3x for 15 min each at RT. 
		NOTE: The work of Gross-Thebing et al. suggests a post-fixation period of 10 min14, but the actual amount must be adjusted accordingly. Here, we tested 10 min post-fixation 3x, and the sensitivity of DAB dying is significantly influenced. After investigating, ensure that 0.5-1.0 h of fixation is the optimal condition (a shorter fixation period cannot produce a clear signal from the target RNA, and the appropriate extension of fixation time helps strengthen the signal and provide less background noise).</li>
	</ol>
	</li>
	<li>Sequential amplifier and label probe hybridization
	<ol>
		<li>Remove the SSCT solution and replace with 50 &#181;L of Amp 1. Allow the embryos to settle in Amp 1 at 40 &#176;C for 30 min. Then, wash the embryos in 0.2x SSCT solution 3x for 15 min each at RT.</li>
		<li>Remove the SSCT solution and add 50 &#181;L of Amp 2. Allow the embryos to settle at 40 &#176;C for 15 min. Then, wash the embryos in 0.2x SSCT solution 3x for 15 min each at RT.</li>
		<li>Remove the SSCT solution, add 50 &#181;L of Amp 3 and tap the tube mildly. Then, incubate the embryos at 40 &#176;C for 30 min. Wash the embryos in 0.2x SSCT solution 3x for 15 min each at RT.</li>
		<li>Remove the SSCT solution, add 50 &#181;L of Amp 4 dropwise, and carefully tap the tube. Then, incubate the embryos at 40 &#176;C for 15 min. Wash the embryos in 0.2x SSCT 3x for 15 min each at RT.</li>
	</ol>
	</li>
	<li>Counterstaining and microscopy
	<ol>
		<li>Remove the SSCT solution and add 50 &#181;L of DAPI to each tube. Incubate the embryos overnight at 4 &#176;C.</li>
		<li>Wash the embryos with PBST and prepare them for imaging using a 1% low melting point agarose (LMP) solution (1 g of LMP dissolved in 100 mL of dH2O) in a Petri dish filled with PBST.</li>
		<li>Use a DAB peroxidase substrate kit to stain the specimen. Add color reagents A, B, and C (50 &#181;L each) to 1 mL of distilled water and mix well to obtain a complete DAB working fluid. Add the fluid to the specimen and cover for 10 min.</li>
		<li>Wash the specimen thoroughly with PBST after staining.</li>
		<li>Use an optical microscope with a photographic function for imaging the samples. 
		NOTE: If taking images later, tubes should be wrapped in aluminum foil and stored at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Tube formation assay
NOTE: The eng mutant and wildtype ECs (control) were differentiated from iPSCs derived from an HHT patient (here, a 62 year-old female patient with recurrent epistaxis since 22 years old, gastrointestinal bleeding, pulmonary arteriovenous malformations, carrying a missense eng mutation in position c.88T&#62;C of exon 2) and a healthy donor (without eng mutation), which were provided by Peking Union Medical College Hospital with approval from the college research ethics committee.
<ol>
	<li>Control and HHT iPSC cell cultures
	<ol>
		<li>Add a certain volume of basement membrane matrix (e.g., Matrigel) to a 6 well cell culture plate to cover the bottom of the well and incubate the plates for 1 h at 37 &#176;C. 
		NOTE: When first using the basement membrane matrix stored at -20 &#176;C, incubate on ice or in a frost-free 4 &#176;C refrigerator until thawed. Then, dilute at 1:100 in DMEM/F12, then distribute the diluent into 200 &#181;L tubes containing 100 &#181;L each. Store the tubes at -20 &#176;C and avoid repeated freezing and thawing. When adding the diluted basement membrane matrix, do not create bubbles. After adding the diluted basement membrane matrix, shake the 6 well plate gently by hand so that it evenly covers the bottom.</li>
		<li>After coating the plate, remove the used basement membrane matrix solution from the wells. Then, add 2 mL of mTeSR1 medium into each well, and plate the iPSCs harvested from the last passage at approximately 1 x 106 per well.</li>
	</ol>
	</li>
	<li>Generation and expansion of ECs from iPSCs
	<ol>
		<li>After approximately 4 days of iPSC culture, exchange the culture medium with BEL medium supplemented with activin A (25 ng/mL), BMP4 (30 ng/mL), VEGF165 (50 ng/mL), and CHIR99021 (1.5 &#181;M). Grow the cells for 3 days to generate mesoderm cells.</li>
		<li>Replace the medium described in step 2.2.1 with BEL medium supplemented with VEGF (50 ng/mL) and SB431542 (10 &#181;M) for 4 days to expand vascular ECs. Treat the cells with the same medium and culture for another 3-4 days.</li>
		<li>Use CD31-dynabeads to purify the mature vascular ECs: refer to the instructions in the CD31-dynabeads kit, which links human CD31 antibody with magnetic beads to combine CD31 molecules expressed on ECs, then collect the CD31-positive cells by elution buffer. Maintain and expand the purified ECs in EC-SFM medium containing VEGF165 (30 ng/mL), bFGF (30 ng/mL), and FBS (1%).</li>
	</ol>
	</li>
	<li>Plating endothelial cells on Matrigel-coated plates and microscopy
	<ol>
		<li>Add 10 &#181;L of basement membrane matrix per well to coat the angiogenesis plates (see Table of Materials) and incubate at 30 min at 37 &#176;C.</li>
		<li>Harvest endothelial cells after checking endothelial markers CD31 and VE-cadherin with immunofluorescence staining and resuspend them in endothelial growth medium 2 by pipetting repeatedly. Add 50 &#181;L of cell suspension (2 x 105 cells/mL) per well onto the solidified matrix and incubate the cells for 3-5 h at 37 &#176;C. 
		NOTE: Before the tube formation experiment, endothelial markers should be checked to make sure the proper phenotype of functional endothelial cells. 1 x 104 cell per well is an ideal number for tube formation. Too many or too few cells are not conducive to tube formation. Add cells from the side of the dish and do not touch the basement membrane matrix. Use light microscope in high magnification field to check the cells and take photos.</li>
	</ol>
	</li>
	<li>Quantitative results of tube formation
	<ol>
		<li>Observe the results with a microscope with high resolution and take pictures of at least 10 areas for each group to obtain reliable data statistics.</li>
		<li>Examine endothelial tube formation: estimate the extent of tube formation by inspecting the overall tube length, tube number, and branch points. Assess and count the number and length of branches using ImageJ software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol applied an improved whole-mount in situ RNA analysis platform for zebrafish and tube formation assays on iPSC-ECs derived from an HHT patient. The traditional ISH method requires a longer experimental cycle with extra experimental steps. The protocol has some important improvements, the use of independent double Z probes and iPSCs derived from a patient with HHT that were applied in WISH assays and tube formation, respectively. These refinements are crucial for enhancing the detection sensitivity compared with what is observed with traditional ISH. The uniquely designed probes and target signal amplification allow the detection of rarely expressed transcripts (aplnra is a poorly expressed gene in 24 hpf embryos). One modification is the extra fixation after probe hybridization. The extra fixation can enhance the combination of probes and transcripts. DAB staining is also applied rather than fluorescence staining, because it was found that it was difficult to perform fluorescence staining in certain low-abundance genes. Then, iPSCs were used for angiogenesis assays, which increases the significance and clinic relevance of this work. Generation of iPSCs requires strict culture conditions and represents a limitation of the protocol.
Some critical steps must be highlighted. In WISH analysis, the digestion time of zebrafish embryos should be followed in strict accordance with the text. A shorter digestion time cannot guarantee that the probe combines with transcripts successfully. In contrast, extended digestion time will destroy the integrity of embryos. In the tube formation experiments, the imaging timing should be well-controlled. Generally, the endothelial cells will become tubes within 12 h but the tubular structure tends to disintegrate after 24 h. The cell number is also important for tube formation, as a cell density that is too high or too low can lead to failed tube formation. If it is difficult to induce endothelial cells to form tubes, it is helpful to include adding growth factors such as VEGF to the culture medium as a positive control to test the ability of ECs to form tubular structures.
In summary, the improved WISH method has been considered an advantageous technique for laboratory workflows. Recently, our research group has started to test this assay in clinical samples. Considering higher sensitivity and more efficient detection than observed in traditional methods, this improved WISH method holds significant promise for developing and implementing RNA-based molecular diagnostics22.
Performance of tube formation assay using patient iPSC-ECs allows confirmation of results from animal studies as well as identification of disease-causing mutations from single nucleotide polymorphism in the eng gene. The combination of these methods clarifies the role of eng in vascular formation and hold potential in gene correction of patient iPSC-ECs for cardiovascular cell therapy23.

A single mutation on eng (CD105) has been reported in patients with HHT. The mutation leads to increased EC proliferation and reduced flow-mediated EC elongation1,2. It has also been previously reported that ENG deficiency reduces endothelial markers expression (i.e., kdrl, cdh5, hey2) in zebrafish3. Endoglin, mainly expressed in endothelial cells, is a transmembrane glycoprotein and functions as a co-receptor for transforming growth factor &#946; (TGF-&#946;) family members. It directs BMP9 binding on the cell surface to regulate downstream gene, including Id1 expression, to induce stem cell differentiation toward ECs4. Thus, the eng gene plays important roles in vasculogenesis and human vascular disease5,6. We have previously examined the effects of endoglin knockdown on vessel formation in zebrafish embryos, followed by analysis of iPSCs-derived ECs acquired from an HHT patient bearing an eng mutation7. This protocol demonstrates the effects of ENG deficiency on endothelial progenitor marker expression and tube formation, which is a quantifiable method for measuring in vitro angiogenesis.
To study eng spatial and temporal expression, WISH is employed to detect gene expression in vivo8. In situ hybridization (ISH) is a method of using labeled probes with complement sequences of target nucleic acids (DNA or mRNA) to detect and visualize target nucleic acid hybrids in a fixed specimen. The process amplifies gene expression signals in vivo and is used to detect the expression of genes by microscopy. WISH has been widely used in various model animals, especially in zebrafish9. It is also used to acquire the following data: 1) gene spatial/temporal expression patterns, which provide information about gene function and classification; and 2) specifically expressed gene markers that are used in high-throughput drug or mutant screening10.
Chromogenic probes are easily degraded&#160;with traditional chromosome in situ hybridization (CISH), which results in high background noise and nonspecific signals11,12. The WISH method uses two independent double Z probes, which are designed to hybridize to target RNA sequences. Each probe contains an 18-25 sequence complementary to the target RNA and a 14 base tail sequence (conceptualized as Z). The target probes are used in a pair (double Z). The two tail sequences together form a 28 base hybridization site for the preamplifier, which contains 20 binding sites for the amplifier. The amplifier, in turn, contains 20 binding sites for the label probe and can theoretically yield up to 8,000 labels for each target RNA sequence.
This advanced technology facilitates simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology13. Further modification of the WISH methods is based on previous research14, including extra fixation and DAB staining. Provided here is an improved WISH protocol that can work even if the target RNA is partially decreased or degraded. Advantages include that it can be completed in 24 h without RNase-free conditions. Signals can also be simultaneously detected through multiple channels from multiple targets, and the results are consistent and compatible with results from different high-throughput automation platforms.
Results from animal models do not necessary reflect the same phenomenon that occurs in humans. ENG contains two pairs of conserved cysteines, C30-C207 and C53-C182, which form disulfide bridges in orphan regions. To further study the role of eng in HHT patients, tube formation assays with iPSCs derived from HHT patients have been carried out in cells without/with eng mutations (Cys30Arg, C30R)15. After Kubota et al. first reported the tube formation experiment16, the assay has been developed in several ways. It has been used to identify angiogenic or antiangiogenic factors, define the signaling pathways in angiogenesis, and identify genes regulating angiogenesis17.
Prior to the availability of patient-derived iPSC-ECs, researchers used primarily cultured ECs to study angiogensis16. However, for endothelial cells, it is a technical challenge to transduce exogenous genes with a virus, because of the limited passage number that ECs can undergo. This is because there is hardly enough cellular material to be collected from human vessels either from surgery or matched approved donors. With the invention of the iPSC generation technique by Shinya Yamanaka, human ECs derived from iPSCs can be used reliably in in vitro experiments, as reported previously18.
Using virally transduced ECs with limited numbers and passages may be sufficient for signaling studies, but for functional studies, it is better to generate mutant pluripotent stem cell lines, (either iPSCs or CRISPER/Cas9-targeted hESCs), then differentiate them into ECs for angiogenesis studies that use tube formation assays19. Tube formation can be used to evaluate the function of endothelial cells bearing mutations. This protocol also describes tube formation on an &#181;-slide angiogenesis plate, which is an easy, cost-effective, and reproducible method.
The protocol below provides a reliable and systemic method for studying the role of specific genes in vascular formation, along with details for in vivo expression pattern and in vitro functional quantification for modeling human disease.

<table><tbody><tr><td>&amp;micro;-Slide Angiogenesis</td><td>ibidi</td><td>81506</td><td>Cell culture</td></tr><tr><td>Amp 1-FL</td><td>ACD</td><td>SDS 320852</td><td>Signal Amplification</td></tr><tr><td>Amp 2-FL</td><td>ACD</td><td>SDS 320853</td><td>Signal Amplification</td></tr><tr><td>Amp 3-FL</td><td>ACD</td><td>SDS 320854</td><td>Signal Amplification</td></tr><tr><td>Amp 4 Alt B-FL</td><td>ACD</td><td>SDS 320856</td><td>Signal Amplification</td></tr><tr><td>Corning Matrigel Matrix</td><td>Corning</td><td>354234</td><td>Growth factor-reduced Matrigel</td></tr><tr><td>DEPC</td><td>Sigma</td><td>D5758</td><td>RNAase-free Water</td></tr><tr><td>Human Endothelial-SFM</td><td>Thermofisher</td><td>11111044</td><td>Cell culture</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>30525-89-4</td><td>Fixed embryos</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>30525-89-4</td><td>Fixed Cells</td></tr><tr><td>Protease K</td><td>ACD</td><td>SDS 322337</td><td>Digest tissue</td></tr><tr><td>Sodium Citrate</td><td>Sigma</td><td>6132-04-3</td><td>SSCT solution: Wash Buffer</td></tr><tr><td>VEGF-165</td><td>STEMCELL Technologies</td><td>78073</td><td>Growth factor</td></tr></tbody></table>

whole-mount in situ hybridization in zebrafish embryos and tube formation assay in ipsc-ecs to study the role of endoglin in vascular development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Whole mount in situ hybridization is based on a principle similar to fluorescence resonance energy transfer. It is designed to improve both sensitivity and specificity in zebrafish ISH as well as amplify target-specific signals without affecting the background signal.
In 24 hpf zebrafish embryos, endoglin is highly expressed in the posterior cardinal vein (PCV), intersegmental vessels (ISVs), and blood islands3. It is hard to control the staining time in traditional chromogenic in situ hybridization (CISH), with non-positive signals occurring in some regions, such as yolk sac (Figure 1A). To investigate the role of eng in early vascular development, a hemogenic endothelial marker was used (aplnra) as well as another endothelial progenitor marker (nrp1a) to examine which type of endothelium was affected by eng silencing20. The expression of aplnra and nrp1a was weak in 24 hpf embryos. Compared to CISH, the weak signal from the two genes was clearly demonstrated in the tail region after eng knockdown in WISH (Figure 1B). The knockdown of eng RNA caused reduced expression of two types of endothelial cells marker genes expression in an HHT animal model.
Before conducting experiments on differentiated iPSCs, the cells were identified and confirmed as ECs. Morphologically, they appeared as cobblestone shape. Expression of endothelial cell surface molecular markers CD31, CD146, VE-cadherin, and vWF was confirmed with IF staining21. After magnetic-based isolation using CD31, the functional assay was conducted as described below.
Here, the tube formation assay was performed using the eng mutant and control iPSC-derived endothelial cells. Statistical analysis was performed based on the number, branches, and lengths of tubes. A novel parameter was also introduced based on previous work3, points of angiogenesis, in order to reflect tube formation of endothelial cells. Eventually, it was found that eng mutant endothelial cells formed fewer branches than control endothelial cells, and that branches in eng mutant endothelial cells significantly increased after stimulation with vascular endothelial growth factor (VEGF) (Figure 2A,B). The mutation in eng resulted in defective vessel tube formation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60498/60498fig1.jpg" /> 
Figure 1: Endoglin knockdown decreased the expression of endothelial markers. (A) CISH and WISH were performed to determine the expression of endoglin in 24 hpf embryos (n &#62; 30). The red box represents the enlarged region. Scale bars = 200 &#181;m. (B) aplnra and nrp1a expression by WISH in 24 hpf zebrafish embryos of the control-MO and eng-MO group. The red arrow indicates regions where the expression of these genes significantly decreased. <a href="https://www.jove.com/files/ftp_upload/60498/60498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60498/60498fig2ab.jpg" /> 
Figure 2: Tube formation of eng mutant and control iPSC-ECs. (A) The tube formation by the four groups, including the control group, the control + VEGF (control endothelial cells + 30 ng/mL VEGF), the eng mutant (eng mutant endothelial cells) group, and the eng mutant + VEGF (eng mutant endothelial cells + 30 ng/mL VEGF) group. Tube formation was assessed and photographed after 3 h. Scale bars = 100 &#181;m. (B) Quantitative results of tube formation are shown. At the covered area the number of branches and their length of tubes were calculated by Image J. Error bars represent the SD of the mean values from three independent experiments. A value of P was considered statistically significant when *p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/60498/60498fig2ablarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Embryonic period</td>
			<td>Fixation time</td>
		</tr>
		<tr>
			<td>4-cells to 8 hpf</td>
			<td>4 hours</td>
		</tr>
		<tr>
			<td>8 hpf to 24 hpf</td>
			<td>1 hours</td>
		</tr>
		<tr>
			<td>24 hpf to 4 dpf</td>
			<td>30 minutes</td>
		</tr>
	</tbody>
</table>
Table 1: Fixation time required for different embryonic stages.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Embryonic period</td>
			<td>Digestion time</td>
		</tr>
		<tr>
			<td>4-cells to 8 hpf</td>
			<td>2 minutes</td>
		</tr>
		<tr>
			<td>8 hpf to 24 hpf</td>
			<td>5 minutes</td>
		</tr>
		<tr>
			<td>24 hpf to 4 dpf</td>
			<td>10 minutes</td>
		</tr>
	</tbody>
</table>
Table 2: Digestion time required for different embryonic stages.

Watch this Scientific Journal Video about Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development at JoVE.com

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

whole-mount-situ-hybridization-zebrafish-embryos-tube-formation-assay

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.7K Views.  Zhejiang University School of Medicine. Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Video: Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

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

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 ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biology

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Neuroscience

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

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Chapters in this video

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

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function