Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Summary

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Abstract

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Introduction

The overall goal of this protocol is to describe how to perform live imaging of neural progenitor mitosis in embryonic brain slices. Using live imaging of brain slices in culture, this protocol provides a simple method to assay multiple aspects of mitosis in neural progenitors in an environment highly similar to an in vivo setting. It can be applied to brains of mutant animals and/or brains that have been manipulated with in utero electroporation)^1-5. This technique is also ideal to test the effect of pharmacological agents on neural precursors' mitosis, by simply adding an agent to the culture medium. In sum, this article will make a ....

Protocol

1. Preparation of Media (Figure 1, Step 1)

1. Slice Culture Medium
   1. 25 ml of slice culture medium is sufficient to prepare 5 glass bottom dishes with 2 slices per well.
   2. In a 50 ml conical tube, add 250 µl of a 100x N2 solution and 500 µl of a 50x B27 solution without vitamin A. Add DMEM/F12 to a volume of 22.5 ml.
   3. Filter sterilize solution and subsequently add 1.25 ml of heat inactivated horse serum and 1.25 ml of fetal bovine serum.
   4. Incubate solution in a water bath at 37 °C for the duration of the preparation of the slices.
   5. Add growth factors (FGF and EGF, final concentration of....

Results

The success of this assay and the observation of multiple mitotic cells during one live imaging session largely depend upon both the integrity and anatomical level of the slice where acquisitions are made. As discussed below, the anatomical level of a slice is an important factor. Figure 3A shows rostro-caudal and medial-lateral locations where we have been most successful. For additional discussion of this subject please see Noctor²⁶. The integrity of the slice can vary in different regions o.......

Discussion

The major advantage of the protocol we have described is that it provides a dynamic temporal resolution of mitosis of neural progenitors. Typically, assays to visualize mitosis in the developing brain are performed using immunofluorescence of fixed tissue sections. But this approach only provides a snapshot of mitosis at one time point.

There are several steps that are most critical for imaging mitosis in brain slices: 1) The brain slices should remain attached to the agarose, to preserve the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100X N2	Life technologies	17502048	for culture medium
50X B27 without vitamin A	Life technologies	12587010	for culture medium
DMEM/F12	Life technologies	11320033	for culture medium
Heat-inactivated horse serum	Sigma Aldrich	H1138-500ml	for culture medium
Heat-inactivated calf serum	Sigma Aldrich	F4135-500ML	for culture medium
FGF	R&D Sytems	3139-FB-025	for culture medium
EGF			for culture medium
10X HBSS	Life technologies	14065-056	for the dissection of the embryos
Hepes Free Acid	Sigma Aldrich	H4034	dilute to 1 M (pH 7.4)
2.5 M D-Glucose	Sigma Aldrich	G8769	for the dissection of the embryos
0.9 M NaHCO3	Life technologies	25080-094	for the dissection of the embryos
Low-melting agarose	Fisher	BP165-25	for generating slices
Syto11	Life technologies	S7573	Make 5µl aliquots
3 mg/ml collagen type I	Life technologies	A1048301	for culturing slices
Glass bottom dish	MatTek	P35G-1.5-14-C	for culturing slices
Petri dishes			for dissection of the embryos
Digital thermometer			to measure the temperature of the agarose
Spatula			to transfer brains
Paintbrush			alternative to transfer brains
Vibratome	Leica	VT1000s	for generating slices
Dissecting microscope			dissecting out embryos
Imaging microscope			A confocal microscope is required, it needs to be equipped with an incubation chamber