PNAS: Adjacent cell labeling technology based on membrane-permeable fluorescent proteins

On January 3, the international academic journal PNAS published the research results "Genetic dissection of intercellular interactions in vivo by membrane-permeable protein"
cooperated by Zhou Bin Group of the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences and Professor Wang Lixin of Zhongshan Hospital affiliated to Fudan University 。 Using mice, a genetic tool expressing membrane-permeable fluorescent protein, this study established a neighboring cell labeling technique in vivo and used this technique to reveal the heterogeneity
of endothelial cells in different regions of the liver.

Cell-cell interactions are essential for processes such as growth and development, homeostasis maintenance, and damage repair in multicellular organisms, but genetic techniques to monitor cellular interactions in vivo have rarely been reported
.
Current genetics basically operate on specific cells themselves, and cannot delve into the interactions between cells
.
Therefore, the establishment of novel neighboring cell labeling techniques is of great significance
for understanding the intercellular interactions and functions in organisms.

sLP-mCh is a fat-soluble tag-linked fused fluorescent protein of mCherry (Ombrato et al.
, Nature 2019).

After sLP-mCh is expressed in donor cells, it is released from donor cells and enters neighboring cells, labeling neighboring cells as ^mCherry+
.
Based on the properties of the sLP-mCh protein, to achieve in vivo neighboring cell markers, the researchers constructed gene knock-in mice: R26-sLP-mCh-GFP and R26-sLP-mCh.

First, mouse hepatocytes were used as donor cells to express sLP-mCh and GFP, and other types of cell markers around hepatocytes were detected
.
The researchers injected the virus AAV2/8-TBG-Cre specifically targeting hepatocytes in R26-sLP-mCh-GFP mice, and when the virus entered the hepatocytes, Cre recombinase expressed and Cre-LoxP recombination occurred, the stop sequence in the middle of the R26-sLP-mCh-GFP site was removed, and the hepatocytes started expressing sLP-mCh and GFP fluorescent proteins and became donor cells
of sLP-mCh.
The researchers found that liver cells were GFP^+mCherry+, and non-parenchymal cells of GFP^–mCherry+ were also detected, with 80% of endothelial cells, 76% of immune cells, and 54% of fibroblasts in the liver being labeled.

The researchers called this Cre-induced intercellular protein labeling technique CILP
.

The basic liver is the hepatic lobule, which can be divided into three zones (zones), and the hepatocytes in each region have different characteristics and molecular markers
.
A zone of hepatocytes surrounds the portal vein of the liver and is highly expressed in cadherin E (E-Cad); Three-zone hepatocytes surround the central vein of the liver and are highly expressed glutamylamide synthetase (GS); The second region of the hepatic lobule is located between zones I and III and is made up
of hepatocytes of E-Cad–GS–.
The capillaries in the liver are a specialized network of blood vessels, called hepatic sinusoidal endothelial cells, which interact closely with liver cells, and liver cells in different areas of the liver may be affected
by endothelial cells to varying degrees.

The researchers then used Mfsd2a⁺ hepatocytes as an example to elucidate the interactions between hepatocytes and their adjacent endothelial cells
.
When tamoxifen induces double genotype adult mouse Mfsd2a-CreER; After R26-sLP-mCh, Mfsd2a⁺ hepatocytes initiate the expression of sLP-mCh and become donor cells
.
The researchers found that there was a clustered mCherry signal around the portal vein and the presence of mCherry⁺CDH5+ cells, indicating that after Mfsd2a⁺ hepatocytes expressed sLP-mCh as donor cells, the surrounding endothelial cells were also labeled mCherry⁺
.
The researchers found that this part of the endothelial cells is mainly distributed around the portal vein, and more than 90% of endothelial cells in the first region of the liver lobule are mCherry+, about 30% of endothelial cells in the second region are mCherry+, and almost no endothelial cells
of mCherry⁺ are detected in the third region 。 It can be seen from the above that the researchers achieved regional labeling of hepatic lobular endothelial cells through CILP technology combined with Mfsd2a-CreER mice, and efficiently labeled endothelial cells
around the portal vein of the liver.

To further analyze differences in epithelial cells in different regions of the liver, the researchers used FACS to sort mCherry-positive and negative endothelial cells and perform transcriptome sequencing
.
Principal component analysis showed that the two groups of endothelial cells were swarmed separately and were quite different
from each other.
The characteristic genes Dll4, Lama4, Msr1 and Ltbp47 of periportal endothelial cells were significantly upregulated in ^mCherry+ endothelial cells, and the characteristic genes Rspo3, Wnt9b, Cdh13 and Thbd7 of central perivenous endothelial cells were significantly upregulated
in mCherry-endothelial cells.
Heat map analysis of differential genes showed that the expression of genes related to angiogenesis, regulating cell adhesion and growth factor stress was significantly upregulated in mCherry+ endothelial cells, while the expression of genes related to extracellular matrix composition, chemophemotaxis and tissue morphogenesis was significantly downregulated
in ^mCherry+ endothelial cells.

In summary, the researchers developed a new technology for in vivo neighboring cell labeling CILP
.
CILP utilizes a membrane-permeable fluorescent protein that, when expressed in donor cells, can be released outside the cell and enter neighboring cells, enabling labeling
of neighboring cells.
The researchers successfully labeled neighboring cells of hepatocytes in mouse livers using CILP technology and used Mfsd2a⁺ hepatocytes as donor cells to label and analyze the characteristics
of periportal endothelial cells in the liver.

Zhang Shaohua, a postdoctoral fellow at the Center for Molecular Cell Excellence, is the first author of the paper, and Professor Zhou Bin of the Center of Excellence for Molecular Cells and Professor Wang Lixin of Zhongshan Hospital affiliated to Fudan University are the co-corresponding authors
of the paper.
This work was strongly supported
by Professor Ailan Lu of the University of Chinese Hong Kong and Professor He Lingjuan of Westlake University.
We would like to thank the Center of Excellence for Molecular Cells for their strong support of this study, and the Chinese Academy of Sciences, the Foundation Committee, the Ministry of Science and Technology, and the Shanghai Municipal Science and Technology Commission for their financial support
.

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Figure: (A) sLP-mCh fluorescent protein enters recipient cells from donor cells
.
(B and C) Genetic tool mouse construction and experimental strategies
.
(D–F) expressed sLP-mCh with hepatocytes as donor cells, and non-parenchymal cells in the liver were labeled mCherry⁺
.
(G and H)sLP-mCh is used to label neighboring cells
in the pancreas and heart.