Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices
March 8th, 2024
During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.
During the development of the cerebral cortex, neurons and glial cells are derived from the ventricular zone and migrate toward our brain surface. Many genes are involved in this process, including those responsible for neurodevelopmental and psychiatric disorders. We are addressing their functions on these behaviors in this process.
Recently, we have reported that astrocyte progenitors assume two distinct migration modes, erratic and blood vessel-guided migration. These observations were made using a combination of serotype-specific labeling and time-lapse observation methods introduced in this video. To label cells, we utilized in utero electroporation system, which we develop to visualize individual cells with a high signal-to-noise ratio.
This in vivo gene transfer system also enable us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown vectors. Using this experimental system, we aim to observe the cell behaviors of neurons, glia, and blood vessels and elucidate the crosstalk between them. The findings from these studies will contribute to understanding the pathogenesis of neurodevelopmental disorders.
To begin, place the anesthetized mouse on a 38 degree Celsius plate. After making a midline skin incision, place a sterile plastic sheet on the skin, followed by a piece of sterile tissue paper and create a hole in the center. Wet the paper with sterile PBS and carefully draw out the uterine horn through the holes.
Using a micro pipette attached to an aspirator tube assembly, inject one microliter of the plasmid mix into the left or right lateral ventricle. Through the uterine wall, apply expiratory pressure. Pinch the embryo's head with a tweezer-type electrode and deliver electrical pulses using an electroporator.
After placing the uterine horn back into the abdominal cavity, suture the abdominal wall and the skin using a 5-0 silk thread. To begin, obtain the euthanized mouse administered with the appropriate plasmids. For culture dish preparation, add 1.9 milliliters of culture medium to a glass-based dish and carefully put a cell culture insert on it.
Then add 500 microliters of culture medium onto the filter and place it on ice. Remove the embryos and place them in a Petri dish containing ice-cold PBS. Under a microscope, select embryos based on the expression of green fluorescent protein or other fluorescent markers.
After dissecting the brain, place it in molten agarose gel taken in a cryo mold and roll it several times. Once the agarose solidifies at room temperature, place it on ice. Using a vibrating microtome, slice the brains coronally to a thickness of 350 micrometers and arrange them on the filter.
Then remove 600 microliters of culture medium from both inside and outside the filter cup. After five minutes, add 100 microliters of fresh culture medium to the inside of the cell culture insert. For time-lapse imaging, acquire around 10 Z-stack images using a long working distance 20x lens.
Analysis of time-lapse images of mouse brain provided the trajectories of EGFP-positive migrating cells and RFP-positive migrating cells from 2 to 21 hours.
Overview
This study focuses on the development of the cerebral cortex, highlighting the migration of neurons and glial progenitors from the ventricular zone to the brain surface. By employing time-lapse imaging techniques alongside in utero electroporation for cell labeling, the research investigates the migration modes of astrocyte progenitors and the genes influencing neurodevelopmental and psychiatric disorders.
Key Study Components
Area of Science
- Neurodevelopment
- Cell migration
- Neurobiology
Background
- The study explores the origin of neurons and glial cells during cortical development.
- It addresses the role of specific genes in neurodevelopmental and psychiatric disorders.
- Astrocyte progenitors exhibit distinct migration modes relevant to cortical formation.
- In vivo gene transfer techniques facilitate the observation of these migration patterns.
Purpose of Study
- To visualize and analyze the behaviors of migrating neurons and glial cells.
- To elucidate the functional roles of genes involved in neurodevelopment.
- To investigate cell behavior and interactions between neurons, glia, and blood vessels.
Methods Used
- Utilized in utero electroporation for gene transfer and cell labeling.
- Focused on mouse models to examine neuronal and glial progenitor migration.
- Involved time-lapse imaging of EGFP- and RFP-positive cells.
- The protocol outlined specific surgical and imaging techniques for accurate observations.
- Included the creation of brain sections for analysis via a vibrating microtome.
Main Results
- Identified two distinct astrocyte progenitor migration modes: erratic and blood vessel-guided.
- Presented data showed defined trajectories of migrating cells over time.
- Findings contribute to the understanding of neurodevelopmental disorder mechanisms.
- Demonstrated the effectiveness of electroporation for visualizing individual cells.
Conclusions
- This study provides insights into the mechanisms of neuronal migration during cortical development.
- Findings may enhance our understanding of neurodevelopmental and psychiatric disorders.
- The results underscore the complex interplay between glial and vascular components in brain development.
Frequently Asked Questions
Executive Industry Relevance
Time-lapse imaging of migrating neurons and glial progenitors in embryonic mouse brain slices enables direct visualization of cellular dynamics underlying neurodevelopmental processes. This capability is critical for de-risking target validation and understanding gene function in disease-relevant systems, particularly for neurodevelopmental and psychiatric disorder portfolios. The approach supports predictive confidence in early discovery by linking genetic perturbations to quantifiable cellular behaviors.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables functional interrogation of candidate genes implicated in neurodevelopmental disorders.
- Supports mechanistic de-risking by revealing how genetic modifications alter neuronal and glial migration.
- Facilitates high-confidence target validation through direct observation of cellular phenotypes.
- Provides a platform for gain-of-function and loss-of-function studies in a physiologically relevant context.
Screening & Assay Development
- Establishes validated brain slice systems for quantitative analysis of cell migration dynamics.
- Delivers reproducible, high signal-to-noise imaging outputs suitable for downstream screening workflows.
- Enables standardization of migration assays for comparative evaluation of genetic or pharmacological interventions.
- Supports scalability for multiplexed analysis of multiple gene perturbations.
Translational & Preclinical Research
- Aligns with disease-relevant models by recapitulating in vivo neurodevelopmental processes.
- Provides translational continuity from genetic discovery to preclinical validation of mechanistic hypotheses.
- Enables risk-adjusted advancement decisions based on quantitative migration phenotypes linked to disease genes.
- Supports biomarker identification through dynamic cellular readouts.
Pipeline & Workflow Integration
This method integrates into the discovery continuum from early gene function studies through preclinical model validation, bridging target identification and mechanistic de-risking.
- Discovery Biology: Supports hypothesis testing on gene function in neuronal and glial migration.
- Screening: Provides quantitative, reproducible migration metrics for assay development.
- Analytics: Enables measurement of migration speed, trajectory, and mode for comparative analysis.
- Translational Research: Connects genetic perturbations to disease-relevant cellular behaviors in preclinical models.
- Enterprise Reuse: Offers a reusable imaging and analysis platform for diverse neurodevelopmental gene targets.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation and mechanistic understanding.
- Operational Value: Delivers standardized, scalable imaging workflows for cross-project use.
- Strategic Value: Improves go/no-go decisions by linking gene perturbations to functional cellular outcomes.
- Portfolio Impact: Enables risk-adjusted prioritization of neurodevelopmental disorder targets.
Implementation Considerations
- Requires expertise in embryonic brain dissection, in utero electroporation, and advanced imaging.
- Demands access to high-resolution fluorescence microscopy and time-lapse imaging infrastructure.
- Necessitates standardized protocols for reproducibility across teams and projects.
- Adaptation may be needed for different genetic constructs or model systems.
- Potential limitations include technical complexity and throughput constraints inherent to live tissue imaging.
