Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Abstract

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

Craniofacial dysmorphology in Down Syndrome is caused by increased dosage of Dyrk1a and at least three other genes

“…Primary mouse cranial neural crest explant cultures were performed as previously described (Gonzalez Malagon et al, 2019). Embryos were harvested at E8.5 and immediately placed into ice-cold PBS.…”

GSK3 and Lamellipodin balance lamellipodial protrusions and focal adhesion maturation in mouse neural crest migration

“…This allows systematic assessment of mouse neural crest cells as they delaminate and migrate away from the neural plate border (Figure 2C) 33 . Neural crest cells can then be treated with pharmacological inhibitors at specific time points, for example prior to delamination or during cell migration.…”

Establishing Primary Cultures of Trunk Neural Crest Cells

“…Neural crest cells derived from other animal species or from other axial levels often differ from avian trunk neural crest cells by several features. In mouse for example, neural crest cells are produced in lesser numbers and more slowly, and their migration behavior can be examined only after 24 hr in culture (Gonzalez Malagon et al, 2019). Xenopus cranial neural crest cells do not migrate individually and randomly, instead they form highly cohesive structures migrating collectively by a mechanism driven by contact-inhibition of movement as well as co-attraction (Carmona-Fontaine et al, 2008;Carmona-Fontaine et al, 2011).…”