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

Summary

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.

Abstract

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 (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 endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization – 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 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 °C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 °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.

Introduction

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 elongation^1,2. 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 β (TGF-β) family members. It directs BMP9 binding on the cell surface to regulate downstream ....

Protocol

All animal experiments described were approved by the Research Ethics Committee of Zhejiang University school of medicine.

1. Whole-mount in situ hybridization

1. Zebrafish line husbandry and reproduction
   1. Feed and raise all adult zebrafish at 26-28 °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.
   2. Put a layer of pebbles in the bottom of the breeding box as .......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
µ-Slide Angiogenesis	ibidi	81506	Cell culture
Amp 1-FL	ACD	SDS 320852	Signal Amplification
Amp 2-FL	ACD	SDS 320853	Signal Amplification
Amp 3-FL	ACD	SDS 320854	Signal Amplification
Amp 4 Alt B-FL	ACD	SDS 320856	Signal Amplification
Corning Matrigel Matrix	Corning	354234	Growth factor-reduced Matrigel
DEPC	Sigma	D5758	RNAase-free Water
Human Endothelial-SFM	Thermofisher	11111044	Cell culture
Paraformaldehyde	Sigma	30525-89-4	Fixed embryos
Paraformaldehyde	Sigma	30525-89-4	Fixed Cells
Protease K	ACD	SDS 322337	Digest tissue
Sodium Citrate	Sigma	6132-04-3	SSCT solution: Wash Buffer
VEGF-165	STEMCELL Technologies	78073	Growth factor