Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Summary

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Abstract

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line ^1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP ⁴. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Protocol

Adult mice are euthanized by mechanical cervical dislocation. All animal procedures were approved by the IACUC and the Biosafety Committee at Emory University.

1. Embryo Generation

1. Cross tamoxifen- inducible Cre line, CAGGCreER, and dsRedCre reporter line (Tg(CAG-Bgeo,-DsRed*MST)1Nagy) (Figure 1) ^5,6. To monitor the relative timing of Shh response in the daughter cells, cross CAGGCreER and dsRed line with the Olig2-eGFP BAC transgenic mice (Tg(Olig2-EGFP)EK23Gsat) (Figure 1) ^7,8.
2. Dissolve tamoxifen in 100% ethanol, and perform intraperitoneal injection of 2.5 mg t....

Results

Here we performed ex vivo live imaging of single cell divisions within the E8.5 mouse neuroepithelium. To label individual cells, we induced Cre recombinase in a subset of cells containing a Cre reporter line that expressed dsRed upon recombination ^5,6(Figure 3A). Thus, 48 hr later we were able to observe single cell divisions during ex vivo imaging (Figures 4A-D). Concomitantly, we monitored when the labeled cells became Shh-responsive by including a Shh tra.......

Discussion

Our ex vivo system enables us to directly observe single cell divisions within the developing neuroepithelium in real time. As an example we examined cell divisions within the mouse embryonic neural tube and monitored either cilium formation or Shh response. We confirmed our imaging results (n = 24) were consistent with results from fixed sections (n = 178) indicating our technique provides physiologically relevant data.

Our technique relies on Cre induction being l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J	Jackson Laboratory	005438
Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd	MMRRC,	010555-UCD
CAGGCreER	Jackson Laboratory	003724
Tamoxifen	Sigma	T5648
DMEM/F12 (1:1)	GIBCO	21041-025
Newborn calf serum	Lonza	14-416F
Penicillin/Streptomycin	Invitrogen	15140-122
Rat Serum SD male	Harlan Bioproducts	4520
1M Hepes	BioWhittaker	17-737E
L-Glutamine	GIBCO	21041-025
Light mineral oil	Sigma	M8410
Sstr3-GFP lentivirus	Emory Viral Core
Micro-knife, size 0.025 mm	Electron Microscopy Sciences	62091
35 mm poly-L-lysine coated glass bottom dish	MatTek	P35GC-0-10-C
100% Petroleum Jelly	Kroger	FL9958c
A1R Laser Scanning Confocal Inverted Microscope	Nikon
NIS Elements software	Nikon
Imaris 3D software	Bitplane AG	Imaris 7.2.3
OCT	Tissue-Tek	4583
Cryostat	Leica	CM1850
Heat-inactivated sheep serum	Invitrogen	16210-072
Triton X-100	Fisher Scientific	BP151
Parafolmaldehyde	Sigma	P6148
Phosphate Buffer	Lab made
Rat monoclonal anti-RFP (5F8)	Chromotek	110411
Rabbit anti-Arl13b serum	NeuroMab
mouse monoclonal anti-Arl13b 1:5	NeuroMab
Rabbit anti-Olig2	Chemicon	AB9610
Mouse monoclonals Pax6	Developmental Hybridoma Bank	Pax6
Mouse monoclonalsShh	Developmental Hybridoma Bank	5E1
Mouse monoclonals Nkx2.2	Developmental Hybridoma Bank	74.5A5
Rabbit polyclonal Ki67	Abcam	AB15580
Alexa Fluor 488	Molecular Probes	A11029
Alexa Fluor 568	Molecular Probes	A11031
Alexa Fluor 350	Molecular Probes	A11046
H–chst	Fisher	AC22989
TO-PRO-3	Invitrogen	T3605
ProLong Gold anti-fade reagent	Invitrogen	P36934