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>

<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. 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=Autophagy+contributes+to+BMP+type+2+receptor+degradation+and+development+of+pulmonary+arterial+hypertension.">Autophagy contributes to BMP type 2 receptor degradation and development of pulmonary arterial hypertension.</a> The Journal of Pathology. 249 (3), 356-367 (2019).</li><li>Kim, S. K., Henen, M. A., Hinck, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+of+betaglycan+and+endoglin,+membrane-bound+co-receptors+of+the+TGF-beta+family.">Structural biology of betaglycan and endoglin, membrane-bound co-receptors of the TGF-beta family.</a> Experimental Biology and Medicine. 244 (17), 1547-1558 (2019).</li><li>Dakou, E., Vanbekbergen, N., Corradi, S., Kemp, C. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromogenic+in+situ+hybridization+compared+with+other+approaches+to+evaluate+HER2/neu+status+in+breast+carcinomas.">Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas.</a> Brazilian Journal of Medical Biology Research. 46 (3), 207-216 (2013).</li><li>Hauptmann, G., Lauter, G., Soll, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+signal+amplification+in+zebrafish+RNA+FISH.">Detection and signal amplification in zebrafish RNA FISH.</a> Methods. 98, 50-59 (2016).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> The Journal of Molecular Diagnostics. 14 (1), 22-29 (2012).</li><li>Gross-Thebing, T., Paksa, A., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+detection+of+multiple+transcripts+combined+with+localization+of+proteins+in+whole-mount+embryos.">Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos.</a> BMC Biology. 12 (1), 55 (2014).</li><li>Fang, Z., 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=EXPRESS:+Autologous+correction+in+patient+iPSC-ECs+to+identify+a+novel+pathogenic+mutation+of+Hereditary+Hemorrhagic+Telangiectasia.">EXPRESS: Autologous correction in patient iPSC-ECs to identify a novel pathogenic mutation of Hereditary Hemorrhagic Telangiectasia.</a> Pulmonary Circulation. , (2019).</li><li>Kubota, Y., Kleinman, H. 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. E., 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=Induced+pluripotent+stem+cell-derived+endothelial+cells+promote+angiogenesis+and+accelerate+wound+closure+in+a+murine+excisional+wound+healing+model.">Induced pluripotent stem cell-derived endothelial cells promote angiogenesis and accelerate wound closure in a murine excisional wound healing model.</a> Bioscience Reports. 38 (4), (2018).</li><li>Rufaihah, A. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cells+for+Cardiovascular+Disease+Modeling+and+Precision+Medicine:+A+Scientific+Statement+From+the+American+Heart+Association.">Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association.</a> Clinical Genomics and Precision Medicine. 11 (1), e000043 (2018).</li><li>Wang, L. 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=Apelin+attenuates+TGF-&#946;1-induced+epithelial+to+mesenchymal+transition+via+activation+of+PKC-&#949;+in+human+renal+tubular+epithelial+cells.">Apelin attenuates TGF-&#946;1-induced epithelial to mesenchymal transition via activation of PKC-&#949; in human renal tubular epithelial cells.</a> Peptides. 96, 44-52 (2017).</li><li>El Hallani, S., 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=Tumor+and+Endothelial+Cell+Hybrids+Participate+in+Glioblastoma+Vasculature.">Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature.</a> Biomed Research International. , 1-9 (2014).</li><li>Anderson, C. M., 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=Fully+Automated+RNAscope+In+Situ+Hybridization+Assays+for+Formalin-Fixed+Paraffin-Embedded+Cells+and+Tissues.">Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.</a> Journal of Cellular Biochemistry. 117 (10), 2201-2208 (2016).</li><li>Sun, X., Tan, G., Liew, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+challenges+for+patient-specific+induced+pluripotent+stem+cells+in+cardiovascular+medicine.">Future challenges for patient-specific induced pluripotent stem cells in cardiovascular medicine.</a> Expert Review of Cardiovascular Therapy. 10 (8), 943-945 (2012).</li></ol>

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 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 ...

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

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

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 ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

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 ...

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 and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

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 in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 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 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 ...

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 genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

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 developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

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. 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. ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...