Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Podsumowanie

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

Streszczenie

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.

Protokół

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 tamoxifen per 40 g of body weight into pregnant females at embryonic 6.5 (E6.5) to induce Cre expression and dsRed labeling in a small subset of cells.
3. 48 hr later dissect embryos, identify dsRed positive embryos using the fluorescent microscope, transfer them to the culture dish and observe single cell divisions during ex vivo imaging (see below).

2. Whole Mouse Embryo Culture

1. Dissect E8.5 embryos in pre-warmed wash medium containing DMEM/F12 (1:1) supplemented with 10% newborn calf serum and 1% Penicillin/Streptomycin (P/S) ⁹.
2. Directly after dissection, place embryos on the 37 °C heating stage under the fluorescent microscope and identify as GFP and/or dsRed positive (see below).
3. Transfer up to 2 embryos into a 500 μl drop of pre-equilibrated culture media containing 50% Rat Serum from a Sprague-Dawley male and 50% DMEM/F12 (1:1) without phenol red supplemented with L-Glutamine and 1% of 1 M HEPES in 0.85% NaCl and P/S ⁹.
4. Apply a thin layer (0.1 cm) of equilibrated light mineral oil over the medium to prevent evaporation and transfer the culture dish containing the embryos to the 37 °C, 5% CO₂ incubator.

3. Viral Infection

1. In order to label cilia in the neuroepithelium, add 5-10 μl of Sstr3-GFP lentivirus, approximately 2 million virions, to a 500 μl equilibrated drop of culture medium containing an E8.5 embryo.
2. After 18 hr of culture, transfer infected embryo into several drops of wash medium to wash out the virus and prepare neural tube slice.

4. Neural Tube Slice Preparation for Live Imaging

1. Dissect neural tube of the E8.5 embryo in pre-warmed wash medium using a micro-knife size 0.025 mm, on a 1% agar coated dish.
2. Place the isolated neural tube ventral side down in a 150 μl drop of equilibrated culture medium without phenol red on the 35 mm poly-L-lysine coated glass bottom dish.
3. Put small amounts of a 1:1 mixture made from 100% pure petroleum jelly and melted candle wax (candle from IKEA) around the mounted neural tube, and gently press by a narrow piece of glass coverslip in order to immobilize the sample.
4. Cover the dish with a thin layer (~0.1 cm) of equilibrated light mineral oil (Figure 2).

5. Live Imaging and Time-lapse Confocal Microscopy

1. Place dish under the Nikon A1R Laser Scanning Confocal Inverted Microscope equipped with an environmental chamber that regulates temperature, set to 37 °C, and 5% CO₂.
2. Use 60x oil-immersion objective to record GFP labeled cilia and dsRed positive cells, while the 40x oil-immersion objective use to monitor Olig2-GFP and dsRed positive cells.
3. Open the NIS Elements software to set up time lapse imaging conditions. Every 10 min acquire z-stacks of up to 25 μm with a spacing of 1.5 μm (40x objective) and up to 8 μm with a spacing of 0.4 μm (60x objective). Use 488 and 561 nm the excitation wavelengths and transmitted channel if needed. Acquire images at 512 x 512 size. Set up multiple user-defined regions of interest to perform simultaneously recording.
   Optimal exposure to laser power and brightness of the image was adjusted by use low laser intensity (up to 11% for mCherry 561; up to 4.5% EGFP 405/488), scan speed 1, line average 2 and size of pin hole (61 μm for dsRED; 28.1 μm for Olig2; 72 μm for SSTR3GFP; 61.3 μm for dsRED and SSTR3GFP; 58 μm for dsRED and Olig2). The use of genetically encoded fluorescent reporters enabled us to detect the brightness with low laser power.
4. Analyze recorded data using Imaris 3D reconstruction software.

6. Immunofluorescence

1. Fix embryos or isolated neural tubes in 4% paraformaldehyde / 0.1 M Phosphate Buffer on ice (4 °C) for 1 hr in a glass dish.
2. Wash samples 2 hr in PBS on ice (4 °C) (change PBS a few times) and put in 30% sucrose / 0.1 M Phosphate Buffer over the night or until embryos sink.
3. Embed in OCT, freeze on dry ice and store in -80 °C. Perform sectioning on cryostat (10 mm).
4. Wash slides for 10 min in wash solution containing 1% Heat-inactivated sheep serum and 0.1% Triton X-100 in PBS.
5. Dilute primary antibodies in wash solution at following concentrations: Rat monoclonal anti-RFP (5F8,) 1:200; Rabbit anti-Arl13b serum 1:1500 and mouse monoclonal anti-Arl13b 1:5 (295B/54); Rabbit anti-Olig2 1:300; Mouse monoclonals Pax6, Shh and Nkx2.2- all 1:10; and Rabbit polyclonal Ki67 1:500. Add around 150 μl per slide in flat humidified chamber, cover with parafilm to avoid drying out and leave over the night at 4 °C.
6. Wash slides in wash solution three times, 20 min each time at room temperature.
7. Dilute secondary antibodies Alexa Fluor 488, 568, 350 in wash solutions at 1:200 concentration. Use Hoechst 1:3,000 or TO-PRO-3 1:500 to stain nuclei. Add 150 μl per slide, leave 1 hr at room temperature in humidified chamber protected from light.
8. Wash slides in wash solution two times, 30 min each time at room temperature.
9. Mount slides in 80% ProLong Gold anti-fade reagent and view within 24 hr.

Wyniki

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...

Dyskusje

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...

Materiały

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