Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Summary

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Abstract

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Introduction

The cerebellum sits at the anterior end of the hindbrain and is responsible for the integration of sensory and motor processing in the mature brain as well as regulating higher cognitive processes¹. In mammals and birds, it possesses an elaborate morphology and is heavily foliated, a product of extensive transit amplification of progenitors during development that produces over half of the neurons in the adult brain. The cerebellum has been a subject of study for neurobiologists for centuries and in the molecular era has likewise received significant attention. This relates not only to its inherently interesting biology, but also to the fact that it is heavily implicated in human disease including developmental genetic disorders such as autism spectrum disorders² and most prominently the cerebellar cancer, medulloblastoma³, which is the most prevalent paediatric brain tumour. Importantly, it is an excellent model system within which to study fate allocation and neurogenesis during brain development⁴. In recent years, it has also been established as a model system for the comparative study of brain development, owing to the huge diversity of cerebellar forms seen across the vertebrate phylogeny^5-10.

The cerebellum develops from the dorsal half of rhombomere 1 in the hindbrain¹¹ and developmentally is comprised of two primary progenitor populations, the rhombic lip and the ventricular zone. The rhombic lip extends around the dorsal region of the neuroepithelium of the hindbrain at the border with the roof plate. It is the birthplace of the glutamatergic excitatory neurons of the cerebellum^12-14. The ventricular zone gives rise to the inhibitory GABAergic cerebellar neurons, most prominently the large Purkinje neurons^14,15. Later in development (from about embryonic day 13.5 in mouse; e6 in chick¹⁶), glutamatergic progenitors migrate tangentially from the rhombic lip and form a pial layer of progenitors: a secondary progenitor zone called the external granule layer (EGL). It is this layer that undergoes the extensive transit amplification that leads to the huge numbers of granule neurons found in the mature brain.

Proliferation in the EGL has long been linked to the sub-pial location that results from tangential migration from the rhombic lip¹⁷, with the switch to cell cycle exit and neuronal differentiation of progenitors being associated with their exit from the outer EGL layer into the middle EGL¹⁸. Extensive tangential migration of post-mitotic granule cells in medial-lateral axis occurs in the middle and inner EGL¹⁹, before final radial migration into the inner granule layer of the mature cerebellar cortex. Migration of cells from the rhombic lip over the cerebellar surface is dependent upon CXCL12 signalling from the pia^20-22 and granule cells express the CXCL12 receptor CXCR4. Their tangential migration is thus reminiscent of that of neocortical tangentially migrating inhibitory interneuron populations^23-25. Intriguingly, electron microscopic studies¹⁷ have suggested that EGL cells with a proliferative morphology maintain pial contact, linking cell behaviour with proliferative capability in a manner reminiscent of the basal progenitors of the mammalian cortex²⁶. This is reflected in the aforementioned stratification of the EGL into three sublayers that are defined by distinct extracellular environments and where granule precursors have distinct gene expression signatures¹⁸.

Proliferation of progenitors in the oEGL occurs with a normal distribution of clone sizes such that when progenitors are individually genetically labelled at the end of embryonic development in the mouse, they give rise to a median average of 250-500 postmitotic granule neurons^27,28. Proliferation is dependent upon mitogenic SHH signalling from underlying Purkinje neurons^29-32. The ability to respond to SHH has been shown to be entirely dependent upon cell autonomous expression of the transcription factor Atoh1, both in vitro³³ and in vivo^34,35. Likewise, cell cycle exit and differentiation has been shown to be dependent upon the expression of the downstream transcription factor NeuroD1³⁶, which is likely a direct repressor of Atoh1³⁷.

Despite this progress, and considerable advancement in deciphering the cell biological basis of cell cycle exit^38-42, the fundamental molecular mechanism(s) that underlie the decision to exit the cell cycle and to transition from a progenitor to a differentiating neuron, and the associated postmitotic tangential migration in the inner EGL as well as the later switch to radial migration, remain incompletely understood. This is to a large extent because of the experimental intractability of the EGL: it is late developing, and difficult to target genetically since many of the same neurogenic molecules are also crucial earlier in the life of granule precursors at the rhombic lip. To overcome this issue, numerous authors have developed in vivo and ex vivo electroporation as a method to target the postnatal cerebellum in rodents^43-48. Here, we pioneer the use of ex vivo electroporation in chick to study the EGL, which represents considerable advantages in terms of cost and convenience. Our method of electroporation and ex vivo slice culture of chick cerebellar tissue uses tissue dissected from embryonic Day 14 chicks at the peak of EGL proliferation. This method allows genetic targeting of the EGL independently of the rhombic lip and will set the stage for genetic dissection of the transition from granule progenitor to postmitotic granule neuron in the cerebellum.

Protocol

Note: All experiments were performed with accordance to King's College London, UK and the UK Home Office animal care guidelines.

1. Dissection of e14 Cerebellum

1. Incubate brown fertilised hens' eggs at 38 °C to embryonic Day 14.
2. Using egg scissors decapitate chick embryo in ovo and remove head to a Petri dish containing ice cold PBS (Figure 1A).
3. Using standard forceps, make incisions behind each eye all the way through the tissue, removing the eyes and upper jaw. Make a second incision all the way through the pharynx removing the lower jaw (Figure 1B).
4. Using standard forceps, remove all skin from surface of skull by peeling it away (Figure 1C) and remove frontal and parietal bones, revealing brain.
5. From a ventral aspect, remove pharyngeal cartilage and auxiliary mesenchyme.
6. From a dorsal aspect, carefully remove mesenchyme dorsal to the hindbrain taking care not to damage pia, and separate entire brain (Figure 1D).
7. Make incision all the way through the tissue between midbrain and hindbrain and separate hindbrain including cerebellum (Figure 1E).
8. By making incisions all the way through the tissue at both lateral junctions of cerebellum and alar plate of the hindbrain, remove entire cerebellum, taking care to maintain the integrity of the pia throughout dissection (Figure 1F). Remove the forming choroid plexus (Figure 1G).
9. Move the dissected cerebellum into ice cold HBSS.

2. Slice Culture of e14 Cerebellum

1. Transfer the whole cerebellum to the sterile platform of a Tissue Chopper using a spatula or a 3 ml Pasteur pipette with the tip cut away to widen the aperture. Remove excess liquid using a pipette.
2. Cut entire whole cerebellum in the required orientation at 300 µm thickness using the tissue chopper set with a cutting speed at 50% of the maximum. Tissue integrity is most easily preserved in sagittal section; however, orientation is also an important consideration for cell analysis (Purkinje cell dendrites are sagittally aligned, while granule cell axons run transversely, perpendicular to the plane of Purkinje cell dendrites).
3. Using a 3 ml Pasteur pipette, cover the sliced cerebellum in ice cold HBSS.
4. Transfer the slices in HBSS to a 60 mm Petri dish containing ice cold fresh HBSS using a 3 ml Pasteur pipette with cut tip.
5. Under a dissecting microscope illuminated with a fiber optic light source, ensure separation of individual slices using watchmaker forceps. Identify slices to be electroporated, based upon their tissue integrity and medio-lateral position.
   Note: Each dissection from embryo through to slice incubation in culture medium takes around 10-20 min depending upon experience. Perform dissections one at a time to ensure that cerebella spend minimum amount of time between decapitation and ex vivo culture.

3. Electroporation of Slices

1. Construct an electroporation chamber by fixing the anode of an electroporator to the base of a 60 mm Petri dish with insulation tape. Add approximately 1 ml of HBSS to cover the electrode.
2. Place a 0.4 µm culture insert on top of the electrode covered in HBSS. Allow the culture insert to rest on the electrode making sure there is always contact between the insert and the electrode.
   Note: In this setup, the culture insert with the slices will rest upon the surface of the solution, maintaining the circuit but allowing spatial targeting of the cathode.
3. Transfer identified slices (up to five per culture insert) onto culture insert using 3 ml Pasteur pipette with the cut tip. Separate and allow to settle onto culture insert in a sagittal orientation.
4. Using a pipette, remove excess HBSS so that slices are no longer bathed in solution.
5. Using a P10 pipette tip, pipette DNA (at a concentration of 1 µg/µl) diluted with 20% fast green over the surface of targeted region of a slice. 20% fast green ensures viscous DNA solution and prevents prohibitively wide dispersal of DNA. Add approximately 5 µl DNA/fast green solution to each slice (Figure 2B).
6. Place the cathode over desired targeted tissue and electroporate with 3 x 10 V, 10 msec duration pulses. Avoid direct contact of the cathode with the tissue. Instead, take advantage of surface tension of the liquid to maintain conductance (place the cathode as close to the tissue as possible without actually touching the tissue).
7. Repeat DNA delivery and electroporation to multiple regions of EGL on each individual cerebellar slice as desired.
8. Upon completion of electroporation, transfer culture insert to 30 mm Petri dish.
9. To each culture, add 1 ml of pre-warmed culture medium (Basal Medium Eagle, 0.5% (w/v) D-(+)-glucose, 1% B27 supplement, 2 mM L-Glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin) underneath the culture insert such that the culture insert is in contact with medium, but slices are not bathed in it.
10. Incubate cultures at 37 °C/6% CO₂ for up to 3 days. Replace all of the culture medium every 24 hr with fresh pre-warmed medium.
11. Following culture, fix slices on culture inserts for 1 hr in 4% paraformaldehyde (or O/N at 4 °C) in a fresh 30 mm dish with no culture media.

4. Imaging of Cerebellar Slices

1. Following fixation, wash slices 3 times for 5 min in PBS.
2. Using a razor blade, dissect each electroporated and cultured cerebellar slice from the culture insert by cutting around the slice and removing the slice and the region of insert it is adhered to. Do not attempt to remove slice from the culture insert surface.
3. Mount slices in approximately 1 ml of mounting medium containing DAPI (if desired) under a coverslip taking care not to introduce bubbles, and image using laser-scanning confocal microscopy.
   Note: Imaging parameters can be varied according to the constructs electroporated, and at the discretion of the individual user.

Results

This section illustrates examples of results that can be obtained using slice electroporation and culture of cerebellum from embryonic Day 14 chick. The dissection of the cerebellum is illustrated in Figure 1 and the electroporation chamber set up is shown in Figure 2. We show that it is possible to electroporate and successfully culture cerebellar slices, which retain their structure and cellular morphologies in vitro (Figure 3A...

Discussion

The protocol reported here describes a method for dissecting, electroporating and culturing slices of embryonic Day 14 cerebellum from the chick. This protocol enables targeting of electroporation to small focal regions of the EGL, including isolated targeting of individual cerebellar lobes. It enables genetic analysis and imaging at a high resolution and convenience, and at a low cost compared to established techniques in rodents^43-47. Such analysis is not currently possible in vivo due to the extend...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
McIlwain tissue chopper	Mickle Laboratory Engineering Ltd		Cut at 300 μm for best results.
Basal Medium Eagle (Gibco)	Life Technologies	41010-026
L-glutamine	Sigma	G7513
penicillin/streptomycin	Sigma	P4333
0.4 μm culture insert	Millipore	PICM0RG50
TSS20 Ovodyne electroporator	Intracel	01-916-02	Use 3 x 10 V, 10 msec pulses for electroporation.