Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Area of Science:

• Developmental Biology
• Stem Cell Biology
• Neuroscience

Background:

• Neural crest cells (NCCs) are crucial embryonic stem cells known for their remarkable migration capabilities.
• Previous studies utilized transgenic mice to track NCCs, but a robust ex vivo system for detailed analysis was lacking.

Purpose of the Study:

• To establish a novel ex vivo culture system for mouse embryos to study NCC behavior.
• To investigate the role of specific extracellular signals in regulating NCC migration.
• To compare the migratory responses of NCCs from different axial levels.

Main Methods:

• Developed an ex vivo culture method for E9.5 mouse embryos in collagen gel, enabling over 24 hours of development.
• Utilized P0-Cre/CAG-CAT-EGFP transgenic mice for fluorescently labeled NCCs, allowing single-cell resolution analysis.
• Implanted beads with platelet-derived growth factor-AA (PDGF-AA) to assess chemoattraction.
• Performed in vitro assays with isolated NCCs, exposing them to neurotransmitters like 5-hydroxytryptamine (5-HT) and dopamine.

Main Results:

• The ex vivo system maintained NCC migratory potential and responsiveness to extracellular signals.
• Demonstrated that PDGF-AA acts as a chemoattractant for NCCs in developing embryos.
• Showed that dopamine, in addition to 5-HT, stimulates NCC migration in vitro.
• Identified distinct migration velocities and dose-response patterns to 5-HT and dopamine in NCCs from different axial origins.

Conclusions:

• The developed ex vivo mouse model offers a powerful platform for studying NCCs, overcoming limitations of previous avian models.
• This system facilitates detailed analysis of NCC migration, division, differentiation, and apoptosis in a controlled environment.
• The findings highlight novel roles for PDGF-AA and dopamine in regulating NCC behavior, providing insights into developmental processes.