Monitoring and elucidating the regulation of material viscoelasticity on neural crest cell migration

Abstract

Collective cell migration holds a pivotal place in a spectrum of biological phenomena, spanning from embryonic development, wound healing to cancer metastasis. A prime example is the coordinated movement and subsequent differentiation of neural crest cells (NCCs), a multifaceted population of embryonic stem cells responsible for forming various tissues such as melanocytes and craniofacial bones. Interestingly, recent studies have convincingly demonstrated that, in addition to biochemical cues, the collective migration of cells is also tightly regulated by the physical characteristics of their micro-environment. For example, the stiffening of the surrounding tissue has been found to trigger and guide the migration of NCCs in vivo. It must be pointed out that most existing studies in this area focused on the influence of material rigidity on the coordinated behavior of cells. In reality, almost all natural tissues or synthetic biological gels exhibit significant viscous response; however, the fundamental question of how surrounding viscosity affects the collective movement of NCCs remains largely unknown.

In this thesis, we report a comprehensive experimental investigation to address this unsettling issue. Specifically, a novel electroporation system was first developed allowing us to deliver fluorescent dyes, plasmids, and drugs into the cell and then subsequently achieve live cell staining and real-time adhesion, cytoskeleton and membrane dynamics monitoring of cells during their migration. After that, by changing the concentration and percentage of different formations and crosslinking molecules, polyacrylamide (PAA) and alginate substrates with distinct viscoelastic properties were fabricated and then the behavior of Xenopus and Chick neural crest cells cultured on them was carefully examined. Interestingly, it was observed that increasing the viscosity of either the culture medium or the substrate leads to a delayed dispersion of NCC clusters. We further showed that such delay is likely caused by the enhanced endocytosis in cells which could modulate their cadherin expression and ultimately inhibit the epithelial-to-mesenchymal transition (EMT).

To test whether the observed influence of material/medium viscosity on maintaining inter-cellular connections and consequently the coherence of migrating cell clusters is important in the in vivo movement of NCCs, the viscosity of major surrounding tissues of neural crest, namely mesoderm and epidermis, in developing Xenopus embryos were carefully measured. Interestingly, although the epidermis was found to possess a relatively constant viscosity level, mesoderm becomes more viscous when the embryo develops into stage 19. Given that stage 19 signifies the imminent closure of the neural plate, a prelude to the onset of neural crest migration, this finding indicates that surround viscosity indeed may play a critical role in regulating the coordinated movement of NCCs in vivo.

Overall, our study demonstrates that changes in the surrounding viscoelasticity of cells could profoundly influence their collective cell migration. Such regulation could originate from the inhibited epithelial-to-mesenchymal transition via viscosity-mediated endocytosis. In addition to significantly advancing our fundamental understanding of embryo development, these findings could also provide clues for the development of future biomaterials and novel strategies in regenerative medicine.