Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Summary

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 Isl^MN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Abstract

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 Isl^MN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Introduction

The ocular motor system provides an elegant system for investigating axon guidance mechanisms. It is relatively uncomplicated, consisting of three cranial nerves innervating six extraocular muscles (EOMs) which move the eye, and the levator palpebrae superioris (LPS) which lifts the eyelid. The oculomotor nerve innervates the LPS and four EOMs - the inferior oblique and the medial, inferior, and superior rectus muscles. The other two nerves, the trochlear and abducens, each only innervate one muscle, the superior oblique and lateral rectus muscle, respectively. Eye movements provide an easy readout, showing if innervation was appropriate, missing, or aberrant. Additionally, there are human eye movement disorders that result from deficits in neuronal development or axon guidance, collectively termed the congenital cranial disinnervation disorders (CCDDs)¹.

Despite these advantages, the ocular motor system is rarely used in axon guidance studies^{2,3,4,5,6,7,8,9,10}, due to technical drawbacks. In vitro axon guidance assays have many disadvantages¹¹. Co-culture assays, in which neuronal explants are cultured together with explants of target tissue¹² or transfected cells¹³, depend on both symmetry of the explant and precise positioning between the explant and target tissue. Stripe assays^14,15, in which two cues are laid down in alternating stripes and axons are assessed for preferential growth on one stripe, only indicate that one substrate is preferable to the other, not that either is attractive or repulsive, or physiologically relevant. Microfluidics chambers can form precise chemical gradients, but subject growing axons to shear stress^16,17,18, which can affect their growth. Moreover, in each of these approaches, collecting explants or dissociated cells requires that outgrowing axons be axotomized and thus these assays actually examine axon regeneration, rather than initial axon outgrowth. Finally, these in vitro approaches remove the microenvironment that influences axons and their responses to cues along different points of their course, and traditionally only test one cue in isolation. Compounding these disadvantages, the small size of each nucleus in the ocular motor system makes dissection technically challenging for either explants or dissociated cultures. Additionally, primary cultures of ocular motor neurons are usually heterogeneous, have significant cell death, and are density dependent, requiring pooling of cells from multiple embryos (Ryosuki Fujiki, personal communication). In vivo methods, however, including knockout mouse models, are inappropriate to use for screening, given the time and expense required.

Methods developed to culture whole embryos¹⁹ allow labeling of migrating cells²⁰ or blockade of specific molecules²¹, but whole embryo cultures require incubation in roller bottles which precludes real-time imaging of labeled structures. Surgical techniques that allow manipulation of the embryo and then subsequent further development either in the uterus or in the abdomen of the mother (maintaining the placental connection)²² are also possible, but these also do not allow time-lapse imaging.

To overcome the obstacles of in vitro assays and allow rapid screening of signaling pathways, an ex vivo embryonic slice culture technique was developed²³, adapted from a previously published protocol for peripheral nerve outgrowth²⁴. Using this protocol, the developing oculomotor nerve can be imaged over time in the presence of many of the surrounding structures along its trajectory, including EOM targets. By adding small molecule inhibitors, growth factors, or guidance cues to the culture media, we can assess guidance perturbations at multiple points along the axon trajectory, allowing more rapid assessment of potential growth and guidance factors.

Protocol

All animal work described here was approved and performed in compliance with the Boston Children's Hospital Institutional Animal Care and Use Committee (IACUC) protocols.

1. Timed matings

1. Place ISL^MN:GFP (Islet Motor Neuron Green Fluorescent Protein; MGI: J:132726; Jax Tg(Isl-EGFP*)1Slp/J Stock No: 017952) male and female mice together overnight. Weigh the females and record weights prior to mating.
   NOTE: ISL^MN:GFP specifically labels motor neurons with a farnesylated GFP that is not cytotoxic, localizes to the cell membrane of motor neurons and their axons, and allows the nerves to be visualized during development²⁵. Other fluorescently labeled lines could also be used.
2. Check for vaginal plugs in early morning. The date a plug is identified is designated as E0.5.
3. Confirm pregnancy using weight gain and/or ultrasound at E10.5. Pregnant dams should have gained at least 1 g. Embryos can be seen on ultrasound at E10.5.

2. Preparation of reagents and vibratome for slice culture

1. Prepare 500 mL of slicing buffer: add 5 mL of HEPES and 5 mL of penicillin/streptomycin to 500 mL of Hank’s Balanced Salt Solution (HBSS) without Ca²⁺ and Mg²⁺.  Chill to 4 °C.
   NOTE: Extra slicing buffer should be stored at 4 °C and can be used for future slice culture experiments.
2. Prepare 50 mL of culture media: In a sterile hood, add 12.5 mL of HBSS, 12.5 mL of Fetal Bovine Serum, 250 µL of glucose, 250 µL of L-glutamine, 125 µL of HEPES to 24.4 mL of Fluorobright DMEM. (Final concentrations: FlouroBright DMEM with 25% HBSS, 25% FBS, 0.5% glucose, 1 mM glutamine, and 2.5 mM HEPES.).  Warm the culture media to 37 °C in a sterile water bath.  
   NOTE: Culture media can be stored at 4 °C for up to 3 weeks.
   1. In a sterile hood, add 1.5 mL of culture media to each well of a 6-well plate.  Add a cell culture insert (Table of Materials) to each well.  Place in a sterile 37 °C and 5% CO₂ incubator.
3. Prepare 4% low-melting temperature agarose: dissolve 2 g of low-melting temperature agarose in 50 mL of sterile PBS. Microwave in 30-60 s intervals until fully dissolved. Place in a 40 °C water bath to keep liquid.
   NOTE: Extra agarose can be stored at room temperature (RT) and melted for future slice culture experiments.
4. Set up vibratome: Place a new blade on vibratome. Check settings: thickness 400-450 µm. Pre-chill the vibratome stage. Place some ice in the outer chamber. Use a chamber and stage dedicated to live slices. Do not use the same vibratome chamber for fixed tissue as residual fixative could be damaging to slices.
5. Prepare the microscope stage top incubator to 37 °C and 5% CO₂.
6. Prepare for dissection: Clean surgical instruments and spray with 70% ethanol. Fill two Petri dishes with HBSS, place on ice. Open a 12 well tissue culture plate and place the lid on ice with the underside facing up. Open a 6 well tissue culture plate and place cell culture membrane inserts and 1.5 mL cell culture media in each well. Pre-warm the plate in a 37 °C and 5% CO₂ incubator.

3. Harvesting E10.5 embryos and preparing slices

NOTE: All steps from this point should be done as quickly as possible. Keep the embryos on ice at all times.

1. Euthanize the pregnant dam (E10.5) in a CO₂ chamber. Perform cervical dislocation.
2. Spray the abdomen with 70% ethanol. Cut open the abdomen with scissors, remove the uterus and place it in a Petri dish with ice cold HBSS to quickly wash away blood. Move the washed uterus to a second Petri filled with dish ice cold HBSS.
3. Under a dissecting scope, remove the embryos from the uterine horn and individual amniotic sacs. Place the embryos on the underside of the lid of a 12 well plate. Keep on ice.
4. Under a dissecting scope, use filter paper to remove any liquid surrounding each embryo.
   NOTE: Embryos will stick to the filter paper if touched.
5. Embed embryos in agarose. Pour melted agarose over each embryo to cover it. Keep on ice. As soon as the agarose has hardened, flip each embryo and pour additional agarose on the other side. Keep on ice.
6. Using a fluorescent dissecting scope, trim the agarose around each embryo so it will be oriented properly when glued to the vibratome stage. The oculomotor nucleus and early axon outgrowth are fluorescent. Align the embryo so that the nucleus, outgrowing axons, and eye form a line, and trim the agarose with a razor blade cut parallel to this line (dorsal to the embryo, see Figure 1A).
   NOTE: This will be the side glued to the vibratome stage (the embryo will be positioned on its back, head closest to the vibratome blade). A line between the oculomotor nucleus and eye should be parallel to the vibratome blade.
7. Fill the vibratome chamber with ice-cold slice buffer. Superglue the embryo to the vibratome stage so that the blade will be parallel with the oculomotor nucleus and eyes. Once the superglue is dry, submerge the vibratome stage so the embryo is oriented facing away from blade.
8. Slice 400-450 µm slices.  Collect each slice with a sterile transfer pipet. Place into cold slicing buffer.
9. Under the dissecting scope, choose the slice containing the oculomotor nuclei and eyes. Using a sterile transfer pipet, place it on the cell culture insert in the 6 well plate. Return the plate to the 37 °C incubator. Alternatively, have one person slicing and another placing the slices. Minimize the time between slicing and placing into incubator.
   NOTE: Slices should be oriented in a way that the maximum fluorescence emitted from the nuclei and axons is closest to the imaging microscope objective. On an inverted microscope, the slices should be placed on the membrane with the nuclei and axons closest to the objective underneath the plate.
10. Remove the residual agarose from the vibratome stage, superglue the next embryo to the stage and repeat steps 3.7-3.9 until all embryos have been sliced and plated.
11. Add inhibitor or recombinant molecule of choice to media in each well to create a dose-response curve.  Dilute in appropriate solvent.
    NOTE: If using DMSO, use only tissue culture grade DMSO. Alternatively, protein-eluting beads can be placed in specific locations on the slice.
12. Place on the microscope in the 37 °C and 5% CO₂ chamber.  Set the microscope to take phase contrast and fluorescent photographs of each slice every 30 min (or more often if desired). Slices can be maintained for 48-72 h.

Results

Normal Results: Figure 1 provides a schematic of the experiment. Starting as early as E9.5 in mouse, the first axons begin to emerge from the oculomotor nucleus²⁶. By E10.5, a fasciculated oculomotor nerve, which contains the early pioneer neurons, can be seen in the mesenchyme. There is significant variability between embryos at E10.5 (even within the same litter) in how far the nerve has progressed towards the orbit, likely due to developmental differences of a few ...

Discussion

This ex vivo slice culture protocol provides significant advantages over traditional axon guidance assays²³. The size of each cranial motor nucleus is not a limiting factor, and no difficult dissection is necessary. The endogenous microenvironment through which the axons travel is maintained, allowing modification of one signaling pathway while maintaining other signaling pathways. Additionally, effects can be assessed at different points along the axon trajectory. Since axon guidance requires mul...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-Well Tissue Culture Plate	Genesee Scientific	25-107
6-Well Tissue Culture Plate	Genesee Scientific	25-105
Disposable Pasteur Pipet (Flint Glass)	VWR	14672-200
Fine Forceps	Fine Science Tools	11412-11
Fluorobrite DMEM	Thermo Fisher Scientific	A1896701
Glucose (200 g/L)	Thermo Fisher Scientific	A2494001
Hank's Balanced Salt Solution (1X)	Thermo Fisher Scientific	14175-095
Heat Inactivated Fetal Bovine Serum	Atlanta Biologicals	S11550H
HEPES Buffer Solution (1M)	Thermo Fisher Scientific	15630106
L-Glutamine (250 nM)	Thermo Fisher Scientific	25030081
Loctite Superglue	Loctite
Low Melting Point Agarose	Thermo Fisher Scientific	16520050
Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um)	Millipore Sigma	PICM03050
Moria Mini Perforated Spoon	Fine Science Tools	10370-19
Penicillin/Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
Petri Dish (100 x 15mm)	Genesee Scientific	32-107G
Phosphate Buffered Saline (1X, pH 7.4)	Thermo Fisher Scientific	10010049
Razor Blades	VWR	55411-050
Surgical Scissors - Blunt	Fine Science Tools	14000-12
Ti Eclipse Perfect Focus with TIRF	Nikon
Vibratome (VT 1200S)	Leica	1491200S001
Vibratome Blades (Double Edge, Stainless Steel)	Ted Pella, Inc.	121-6