1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg
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Heermann, S., Krieglstein, K. Analyzing Murine Schwann Cell Development Along Growing Axons. J. Vis. Exp. (69), e50016, doi:10.3791/50016 (2012).
The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.
1. Preparation of Collagen Gels
2. Dissection of Embryonic SCGs
3. Treatment of SCG Explants
4. Time-lapse Imaging
Use an inverted microscope for analyses. Various objectives can be used, defining the field of view and magnification. One important aspect, however, is the working distance of the used objective, as imaging of the SCG explants is performed through the glass slide and the collagen gel. The recording frame rate of 1/10-30 minutes showed good results (2). However, this aspect has to be adjusted to the scientific question. A normal CCD camera can be used for image acquisition. For time lapse imaging a cell culture incubation chamber has to be attached to the stage of the microscope. Incubate the tissue during imaging at 37 °C, with 5% CO2 and humid conditions. Start the incubation system one hour before the start of imaging. This allows the microscope parts (e.g. objective) including the chamber slide to adjust to the temperature and prevents temperature induced drifts. Define specific areas of analyses (within on explant and between different explants) with the help of the microscope software and a software-controlled motorized stage (multiposition setup) for simultaneous analyses of different applied conditions to the SCG explants. For time-lapse imaging wildtype tissue as well as tissue from transgenic animals can be used marking the SCs (s100b:GFP) (3). For imaging fluorophores a fluorescent light source has to be implemented in the micropscope setup. Use standard filters.
5. Quantification of SC migration distances
Axonal growth is facilitated from SCG explants upon treatment with NGF (4) (movie scheme S1 Figure 1 scheme). This process is easily visible by any inverted microscope and can be followed by time-lapse imaging (movie S2). If a scientist is new to dissecting SCG from mouse embryos we strongly recommend a validation of the technique by a simple anti- tyrosine hydroxylase (TH) immunohistochemistry. TH is a common marker for catecholaminergic neurons (in this case sympathetic neurons) and does also label axons (Figure 2B). By this means axonal lengths can be analyzed in this system (2).
Importantly, after axonal growth has been induced, a wave of migrating cells can be observed (movie S2 and S3). Migrating cells were validated as S100 immuno positive SC (2). In addition a transgenic mouse line, in which GFP is under the control of the human s100b promoter fragment (3) can be used to directly analyze the labeled SC population (2) (movie S4 and S5). With the help of this transgenic line these experiments can also be performed with confocal- or light sheet microscopy (not performed here).
For analyses of SC during migration, we recommend an area of analysis with a low axonal density (Figure 1 close-up DIV3 and DIV4) and thereby a small population of SC per area of analysis. This facilitates best possibilities to see cell morphology and cellular behavior (movie S3).
SC migration distances can be measured at the end of an experiment (e.g. DIV4). To this end DAPI nuclear labeling can be performed on fixed tissue and distances from the leading SC nuclei to the border of the explant can be measured with the help of Fiji (NIH software) (Figure 2 scheme and DAPI labeling) (2). In addition also SC proliferation or SC death can be analyzed. To this end immunohistochemistry for pHH3 or for activated Caspase 3 can be performed for example, identifying mitotic or apoptotic cells respectively (2).
Figure 1. A: Scheme showing axonal growth from an explanted SCG over time (DIV0 -DIV4). B/C: brightfield images, recorded during time-lapse imaging showing close ups of one region of an SCG explant at DIV3 (B) and DIV4 (C) (scale-bar = 100 μm).
Figure 2. A: Scheme showing SC (blue) along extended axons (grey), grown from an explanted SCG (light blue) at DIV3 and DIV4. Migration distances can be measured on DAPI nuclear-labeled samples by measuring the distance from the nucleus of the leading SC to the border of the explanted SCG at multiple locations (C). TH immunohistochemistry (DIV4) should be performed if a scientist is new to SCG dissection. A positive labeling clearly identifies the explanted ganglion as a sympathetic ganglion (B). TH immunohistochemistry also enables analysis of axons grown from explanted SCGs.
Movie S1. scheme showing axonal growth from an explanted SCG over several days in vitro. Click here to view movie.
Movie S2. Axonal growth and SC migration from an NGF treated SCG explant. Imaging started at DIV2. Recording frame rate was 1/30 min. Scale-bar = 100 μm. Click here to view movie.
Movie S3. Close up of a peripheral area of an SCG explant. Due to a low axonal density single SC can easily be analyzed. Imaging start was DIV3. Recording frame rate was 1/10 min. Scale- bar = 100 μm. Click here to view movie.
Movie S4. The combination of brightfield and fluorescence enables visualization of S100 GFP positive SCs and the axons. Recording frame rate was 1/10 min. Scale- bar = 100 μm. Click here to view movie.
Movie S5. SC migration at the border of an SCG explant. Here only the fluorescence channel is shown enabling easier interpretation of S100 GFP positive SCs. Recording frame rate was 1/10 min. Scale- bar = 100 μm. Click here to view movie.
The development of the peripheral nervous system is an exciting process. When the development is completed, axons are ensheathed by SCs along the entire length, which can, in humans, often be over 100 cm. To this end the correct number of the required SCs has to be established during development and the SCs also have to move along extending axons to the periphery to ensure the complete axonal coverage. This holds true for myelinated but also for unmeylinated axons. In both cases all axons are in contact with SCs and depend on their support. To study SC development, assays are needed which take the axonal compartment into account and therefore mimic the in vivo development to a better extent than scratch assays (5), Boyden assays (6) or chamber assays can do. In some assays the axonal compartment was taken into account already. For example sections of sciatic nerves were used as routes for the SCs to migrate along (7, 8), or SCs were co-cultured with neurons along which axons they were observed to migrate (9).
The SCG explant SC migration assay, however, has even more advantages. It is especially interesting because SCs are developing along their own physiological axons, and these are themselves still growing. Furthermore the technique is easy to learn and requires only a bit of technical dissection skills and does not require a complex co-culture system of neurons and SCs. A quite similar setup, however not using SCGs but rather DRGs, was proposed by Gumy and colleagues (10). However, to exploit the full possibilities of such assay, an inverse microscope setup is needed enabling time-lapse imaging. It is important to have the possibility to do "multi- position experiments", in order to be able to analyze differentially treated SCG explants, situated in a chamber slide, at the same time, enabling the work with optimal controls side by side with the factors of interest (2). For analyzes we only used conventional light- and conventional fluorescence (for transgenic s100b:GFP mice) microscopy. The technique, however, can also easily be used with confocal- or even light sheet microscopy (not performed here). Time-lapse recordings can be used to identify specific cellular properties/behaviors during migration. So far only wildtype and transgenic mice have been used (2). However, transfection of SCs and thereby alteration of pathways with RNAi for example should be feasible. With this technique it could even be possible to analyze the SC-axon interplay of normal and altered SC on the same or neighboring axon. Regarding migration distances, SC proliferation and SC survival, end point analyses can be easily performed by immunohistochemistry with DAPI nuclear labeling and Fiji (NIH) software. However, eventually even life-reporter for proliferation (11) and cell death (12,13) could be used, thereby enabling a direct readout during imaging.
No conflicts of interest declared.
We want to thank Urmas Arumae for sharing a collagen protocol and Jutta Fey and Ursula Hinz for excellent technical assistance. Furthermore we want to thank Christian F. Ackermann, Ulrike Engel and the Nikon Imaging Center at the University of Heidelberg and also Joachim Kirsch for kindly help for the video shoting. The work was partially funded through the Deutsche Forschungsgemeinschaft (SFB 592).
|Sodium Bicarbonate (7.5%)||Gibco||25080|
|syringe needle||Braun||BD # 300013|
|8 well chamber slide||Lab tek||177402|