Probing Single Cell Gene Expression in Tissue Morphogenesis and Angiogenesis

Abstract

The fascinating capability of cellular self-organization during tissue development and repair is a central question in developmental biology and regenerative medicine. Understanding the dynamic morphogenic and regenerative processes of biological tissues will have important implications in biology and medicine. Nevertheless, the elucidation of the cellular self-organization processes is hindered by a lack of effective tools for monitoring the spatiotemporal gene expression distribution and a lack of ability to perturb the self-organization processes in living cells and tissues. Multimodal modularities that allow both single cell perturbation and gene detection are required to enable a new paradigm in the investigation of complex tissue morphogenic processes. To address this critical challenge in the field of developmental and regenerative medicine, we are developing a multimodal gold nanorod-locked nucleic acid (GNR-LNA) composite for single cell gene expression analysis in living cells and tissues at the transcriptional level. Using antisense RNA sequences, we design LNA probes for detecting specific molecular targets in living cells. The LNA probes bind to the GNR spontaneously due to the intrinsic affinity between the GNR and LNA. In close proximity, the fluorescent probes are effectively quenched by the GNR. Therefore, a fluorescent signal is only observed when the specific target thermodynamically displaces the LNA probe from the GNR. Furthermore, the GNR also serves as a transducer for photothermal ablation. Thus, we established a novel modularity for imaging the spatiotemporal gene expression distribution in living cells and tissues. The single cell analysis capability of our techniques enables us to adopt a unique approach to study the tissue regenerative processes during normal development and diseases, and this will have a profound impact on regenerative medicine and disease treatment in future. Moreover, we applied this GNR-LNA probe to explore the endothelial cell mRNA dynamics during capillary morphogenesis. Three different types of cells were identified due to their different roles during endothelial cell capillary-like formation process. Our findings indicated that the endothelial cell behavior is directly related to the Dll4 mRNA expression, and Dll4 expression in ECs determine the cell fate. Our GNR-LNA probe enable us to investigate the correlations between Dll4 mRNA expression and cell behavior during capillary morphogenesis. Experimental results indicated that: (1) When the endothelial cells aggregate, the cells migrate with certain displacement, the Dll4 mRNA expression decreases. (2) When the endothelial cells sprout, the cells migrate with small displacement but the cell shape changes to an ellipse shape, the Dll4 mRNA expression begin to increase. (3) When the endothelial cells elongate and form cell-cell contract with adjacent cells, the Dll4 expression decreased to a certain level and keep stable until the cell activity change to another stage. Furthermore, it has been demonstrated endothelial cells compete for the leader cell position during wound healing, collective cell migration, and tip cell formation during angiogenic process. It has been demonstrated that endothelial cells compete for the tip cell formation through Notch signaling pathway. However, how the mechanical force regulates tip cell formation is still unclear, and if mechanoregulation of tip cell formation through Notch pathway still unknown. Mechanical and chemical regulations of tissue morphogenesis and angiogenesis are being investigated in both in vitro capillary-like network formation assay and in vivo mice retina angiogenesis assay. Here, we investigated the mechanoregulation of mechanotransduction of tissue morphogenesis and angiogenesis using both in vitro endothelial cell tube formation model and in vivo mice retina blood vessel development model. Our results demonstrated that (1) Notch pathway negatively regulates tip cell formation: inhibition of Notch pathway (DAPT) enhances tip cell formation, induces Dll4 and Notch1 activity, activation of Notch pathway (Jag1 peptide) inhibits tip cell formation, suppresses Dll4 and Notch1 activity. (2) Mechanical force negatively regulate tip cell formation: (a) Decrease mechanical force via Rho kinase inhibitor Y-27632, myosin II inhibitor Blebbistatin, or laser ablation, enhances tip cell formation and induces Dll4 activity through mediation of Dll4-Notch1 lateral inhibition, (b) increase mechanical force via traction force inducer Nocodazole and Calyculin A, suppresses tip cell formation and inhibits Dll4 activity through activation of Notch pathway. (3) Mechanical force negatively regulates tip cell formation partially via mediation of Notch pathway. Mechanical force is necessary for tip cell formation and negatively regulate tip/stalk selection via Dll4-Notch1 lateral inhibition. Interruption of mechanical force enhance tip cell formation via suppression of Dll4-Notch1 lateral inhibition, thus resulting the increase of Dll4 expression. Enhance of mechanical force inhibits tip cell formation via activation of Dll4-Notch1 lateral inhibition, thus resulting the decreases of Dll4 expression. All these finding wills have great significance for various biomedical applications, such as tissue engineering, cancer, and drug screening.