Name: whole-mount in situ hybridization in zebrafish embryos and tube formation assay in ipsc-ecs to study the role of endoglin in vascular development
Uploaded: 2020-05-28T13:00:00
Description: 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

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 ...

<ol>
	<li>Jin, 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=Endoglin+prevents+vascular+malformation+by+regulating+flow-induced+cell+migration+and+specification+through+VEGFR2+signalling.">Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling.</a> Nature Cell Biology. 19 (6), 639-652 (2017).</li><li>Sugden, W. W., 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=Endoglin+controls+blood+vessel+diameter+through+endothelial+cell+shape+changes+in+response+to+haemodynamic+cues.">Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues.</a> Nature Cell Biology. 19 (6), 653-665 (2017).</li><li>Zhang, 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=Endoglin+is+a+conserved+regulator+of+vasculogenesis+in+zebrafish+-+implications+for+hereditary+haemorrhagic+telangiectasia.">Endoglin is a conserved regulator of vasculogenesis in zebrafish - implications for hereditary haemorrhagic telangiectasia.</a> Bioscience Reports. 39 (5), (2019).</li><li>Lawera, A., 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=Role+of+soluble+endoglin+in+BMP9+signaling.">Role of soluble endoglin in BMP9 signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (36), 17800-17808 (2019).</li><li>Gallardo-Vara, E., Tual-Chalot, S., Botella, L. M., Arthur, H. M., Bernabeu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+endoglin+regulates+expression+of+angiogenesis-related+proteins+and+induction+of+arteriovenous+malformations+in+a+mouse+model+of+hereditary+hemorrhagic+telangiectasia.">Soluble endoglin regulates expression of angiogenesis-related proteins and induction of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia.</a> Disease Models &amp; Mechanisms. 11 (9), (2019).</li><li>Gomez-Puerto, M. 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R., Leyns, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Mount+In+Situ+Hybridization+(WISH)+Optimized+for+Gene+Expression+Analysis+in+Mouse+Embryos+and+Embryoid+Bodies.">Whole-Mount In Situ Hybridization (WISH) Optimized for Gene Expression Analysis in Mouse Embryos and Embryoid Bodies.</a> Methods in Molecular Biology. 1211, 27-40 (2014).</li><li>Xiao, C., Gao, L., Hou, Y., Xu, C., Xiong, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin-remodelling+factor+Brg1+regulates+myocardial+proliferation+and+regeneration+in+zebrafish.">Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish.</a> Nature Communications. 7, 13787 (2016).</li><li>Isogai, S., Horiguchi, M., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+anatomy+of+the+developing+zebrafish:+an+atlas+of+embryonic+and+early+larval+development.">The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.</a> Developmental Biology. 230 (2), 278-301 (2001).</li><li>Rosa, F. E., Santos, R. M., Rogatto, S. R., Domingues, M. 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K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> Journal of Cell Biology. 107 (4), 1589-1598 (1988).</li><li>Clayton, Z. 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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=Endothelial+Cells+Derived+From+Human+iPSCS+Increase+Capillary+Density+and+Improve+Perfusion+in+a+Mouse+Model+of+Peripheral+Arterial+Disease.">Endothelial Cells Derived From Human iPSCS Increase Capillary Density and Improve Perfusion in a Mouse Model of Peripheral Arterial Disease.</a> Arteriosclerosis Thrombosis &amp; Vascular Biology. 31 (11), e72-e79 (2011).</li><li>Musunuru, K., Sheikh, F., Gupta, R. M., Houser, S. R., Wu, J. 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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 w...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-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 islands<sup class...

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 ...

The whole-mount, in situ hybridization, WISH, provides a high resolution and a low background result compared to traditional methods. Also, U-slide plates improve the focus at the same focal length by using less WISH can be applied to mouse embryos, Drosophila embryos and other tissue difficult to handle. The tube formation assay can be applied to stem cell differentiated androgen cells.WISH makes it easy to understand the functional meaning of mutation correction. The person who will demonstrate the procedure is Yong Wang, a research assistant from my laboratory. Working under a microscope, use the FemtoJet system to inject two nanograms of Morpholinos and 500 picograms of MRNA into one-cell embryos.Practice the microinjection of Morpholinos many times until you can inject it into cells successfully and keep the integrity of the eggs. Incubate the zygotes until they're dechorionated and reach the state of development that corresponds to the intended experiment. As described in table one of the manuscript.Next, use a plastic pipette to transfer 20 to 40 embryos to a 1.5 milliliter tube. Add one milliliter of freshly prepared 4%PFA solution at room temperature. After fixing, washing and dehydrating the embryos as described in the manuscript, place the embryos in a sieve with nylon mesh at the bottom.Hydrate the embryos by sequentially washing them in 75%50%and 25%methanol for five minutes each. After washing the embryos with PBST, add 50 microliters of proteinase K into the tube with the embryos. After digestion, remove the proteinase K and wash the embryos with PBST three times for five minutes each time.Add 50 to 100 microliters of mixed target probes into each tube with the embryos. Incubate the embryos overnight at 40 degrees Celsius. After washing the embryos as described in the manuscript, use 4%paraformaldehyde to fix the embryos for 30 minutes at room temperature.Then wash the embryos in 2x SSCT solution three times for 15 minutes each time at room temperature. Remove the SSCT solution and replace it with 50 microliters of Amp 1. Then incubate the embryos at 40 degrees Celsius.After 30 minutes, wash the embryos in 2x SSCT solution three times for 15 minutes each time at room temperature. Remove the SSCT solution and add 50 microliters of Amp 2. After incubating for 15 minutes and washing the embryo as done previously, add 50 microliters of Amp 3 and tap the tube gently.Incubate the embryos at 40 degrees Celsius. After 30 minutes wash the embryos as described previously. Then add 50 microliters of Amp 4 dropwise and carefully tap the tube.Then incubate the embryos at 40 degrees Celsius for 15 minutes and wash them three times with SSCT. After preparing the embryos for imaging as described in the manuscript, use a DAB peroxidase substrate kit to stain the specimen. Add 50 microliters each of color reagents A, B and C to one milliliter of distilled water.And mix well to obtain a complete DAB working fluid. Add the fluid to the specimen and cover for ten minutes. After thoroughly washing the specimens, use an optical microscope to image them.To begin culturing and control of HHT iPSCs, add basement membrane matrix to six well cell culture plates to cover the bottom of the wells. Incubate the plates for one hour at 37 degrees Celsius. Next, remove the used basement membrane matrix solution from the wells, and add two milliliters of mTeSR 1 medium into each well.Then plate the iPSCs harvested from the last passage at approximately one times ten to the six cells per well. After culturing the iPSCs for approximately four days, exchange the cell culture medium with supplemented BEL medium. Grow the cells for three days to generate mesoderm cells.To expand vascular endothelial cells, replace the medium with supplemented BEL medium for four days. And then treat the cells with the same medium and culture for another three to four days. Purify the mature vascular endothelial cells using CD31 Dynabeads according to kit instructions.Then collect the CD31 positive cells by elution buffer. Maintain and expand the purified endothelial cells in EC-SFM medium containing VEGF165, bFGF and FBS. To plate the endothelial cells, begin by adding 10 microliters of basement membrane matrix per well to the angiogenesis plates.Incubate the plates at 37 degrees Celsius for 30 minutes. Harvest the endothelial cells and resuspend them in endothelial growth medium 2 by pipetting repeatedly. Add 50 microliters of this cell suspension to the solidified matrix in each well and then incubate the cells at 37 degrees Celsius.After three to five hours of incubation, use a high resolution microscope to assess endothelial tube formation. Capture images of at least ten areas for each group. Inspect the overall tube length, tube number and branch points.CISH and WISH were performed to determine the expression of endoglin in 24 hour post-fertilization embryos. A hemogenic endothelial marker and an endothelial progenitor marker were used to examine the effect of endoglin silencing. The red arrows indicate regions where the expression of these markers, aplnrna and npr1a, significantly decreased.The endothelial tube formation assay was performed on the endoglin mutant and control iPSC-derived endothelial cells. Tube formation was assessed and photographed after three hours. Tube length, tube number and branching were quantified using ImageJ software.Mutant endothelial cells formed fewer branches than control endothelial cells. And branches in mutant endothelial cells significantly increased after stimulation with vascular endothelial growth factor. This method also can be used for iPS generation from peripheral blood.And by the method clarifies the role of endocrine in vascular formation in vitro and in vivo. Knowing that correcting the mutation can make a difference in angiogenesis, it is possible to transplant the cells back into patients for potential stem cell therapy.

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Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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

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 ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

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 ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

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. ...