Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination

Abstract

Epithelial to mesenchymal transition (EMT) is a cellular process that converts epithelial cells to mesenchymal cells with migratory potential in both developmental and pathological processes. Although originally considered a binary event, EMT in cancer progression involves intermediate states between a fully epithelial and a fully mesenchymal phenotype, which are characterized by distinct combinations of epithelial and mesenchymal markers. This phenomenon has been termed epithelial to mesenchymal plasticity (EMP), however, the intermediate states remain poorly described and it's unclear whether they exist during developmental EMT. Neural crest cells (NCC) are an embryonic progenitor cell population that gives rise to numerous cell types and tissues in vertebrates, and their formation is a classic example of developmental EMT. An important feature of NCC development is their delamination from the neuroepithelium via EMT, following which NCC migrate throughout the embryo and undergo differentiation. NCC delamination shares similar changes in cellular state and structure with cancer cell invasion. However, whether intermediate states also exist during NCC EMT and delamination remains unknown. Through single cell RNA sequencing, we identified intermediate NCC states based on their transcriptional signature and then spatially defined their locations in situ in the dorsolateral neuroepithelium. Our results illustrate the progressive transcriptional and spatial transitions from premigratory to migratory cranial NCC during EMT and delamination. Of note gene expression and trajectory analysis indicate that distinct intermediate populations of NCC delaminate in either S phase or G2/M phase of the cell cycle, and the importance of cell cycle regulation in facilitating mammalian cranial NCC delamination was confirmed through cell cycle inhibition studies. Additionally, transcriptional knockdown revealed a functional role for the intermediate stage markerDlc1in regulating NCC delamination and migration. Overall, our work identifying and characterizing the intermediate cellular states, processes, and molecular signals that regulate mammalian NCC EMT and delamination furthers our understanding of developmental EMP and may provide new insights into mechanisms regulating pathological EMP.

Cell extrusion - a novel mechanism driving neural crest cell delamination

“…S1P2 activates Rho signaling through ARHGEF1 to target the localization of microtubules, actin and myosin within the neighboring cells (Slattum, McGee et al 2009, Gu, Forostyan et al 2011). To determine if these Piezo1 signaling components are expressed during NCC delamination, we analyzed our previously acquired single cell RNA-sequencing data of E8.5 mouse embryos which covered NCC development from pre-migratory to migratory stages (Falcon, Watt et al 2022, Zhao, Moore et al 2023). Piezo1 , S1p2 and Arhgef1 are expressed throughout NCC development, including during delamination (Supplemental Figure 5), suggesting a potential role for Peizo1 mediated mechanotransduction in NCC delamination.…”

“…While our data indicates that NCC can delaminate by two different mechanisms, our previously published single cell RNA-seq data suggests the type of delamination does not restrict their capacity for differentiation. We previously identified two intermediate transition populations of NCC during their delamination, which were denoted as clusters 10 and 2 (Zhao, Moore et al 2023). Piezo1 is specifically expressed in cluster 2, indicating cluster 2 contains the extruding NCC population (Supplemental Figure 5E).…”

“…Piezo1 is specifically expressed in cluster 2, indicating cluster 2 contains the extruding NCC population (Supplemental Figure 5E). Pseudo-time trajectory analysis performed on this single cell RNA-seq data determined that these intermediate transition populations are not lineage restricted (Zhao, Moore et al 2023). Therefore, it is unlikely that the extruding cell population is limited to a certain fate at this developmental point.…”

“…Bioinformatic analyses of single-cell RNA sequencing of E8.5 on Mef2c-F10N-LacZ and Wnt1-Cre;R26R-eYFP embryos were performed as previously described (Falcon, Watt et al 2022, Zhao, Moore et al 2023).…”

Retinoic acid, an essential component of the RP organizer, promotes the spatio-temporal segregation of dorsal neural fates

“…These comprised SOX10, SASH1, CALD1, NPR3, MEF2C, LOXL1 , etc. Additionally, it is worth mentioning DLC1 , recently found to mediate cranial NC delamination and migration 44 , and CCN2 , a glial ECM protein with EMT properties 45 . Thus, emergence of the bridge cluster in the treated sample confirms the maintenance of a link between DRG progenitors and melanocytes in absence of RA activity.…”