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

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.