The method presented in this article arose from work funded by the BBSRC BB/I021507/1 (TB, RJTW) and an MRC doctoral studentship (MH).

Note: All experiments were performed with accordance to King&#39;s College London, UK and the UK Home Office animal care guidelines.
1. Dissection of e14 Cerebellum
<ol>
	<li>Incubate brown fertilised hens&#39; eggs at 38 &#176;C to embryonic Day 14.</li>
	<li>Using egg scissors decapitate chick embryo in ovo and remove head to a Petri dish containing ice cold PBS (Figure 1A).</li>
	<li>Using standard forceps, make incisions behind each eye all the way through the t...

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 rodents43-47. Such analysis is not currently possible in vivo due to the extend...

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 phylogeny5-10.
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 cerebellum12-14. The ventricular zone gives rise to the inhibitory GABAergic cerebellar neurons, most prominently the large Purkinje neurons14,15. 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 pia20-22 and granule cells express the CXCL12 receptor CXCR4. Their tangential migration is thus reminiscent of that of neocortical tangentially migrating inhibitory interneuron populations23-25. 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 neurons27,28. Proliferation is dependent upon mitogenic SHH signalling from underlying Purkinje neurons29-32. 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 vivo34,35. 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 exit38-42, 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 rodents43-48. 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.

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	<tbody>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">McIlwain tissue chopper</td>
			<td style="width:71px;">Mickle Laboratory Engineering Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Cut at 300&#956;m for best results.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Basal Medium Eagle (Gibco)</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:119px;">41010-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4&#956;m culture insert</td>
			<td style="width:71px;">Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSS20 Ovodyne electroporator&#160;</td>
			<td style="width:71px;">Intracel</td>
			<td style="width:119px;">01-916-02</td>
			<td>Use 3x10v, 10ms pulses for electroporation.</td>
		</tr>
	</tbody>
</table>

ex vivo culture of chick cerebellar slices and spatially targeted electroporation of granule cell precursors

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.

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...

Watch this Scientific Journal Video about Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors at JoVE.com

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

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.

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

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

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JoVE Journal

Developmental Biology

9.2K visualizações.  King's College London.  A camada externa de grânulo cerebelar é o local da maior amplificação de trânsito no cérebro em desenvolvimento. Aqui, apresentamos um protocolo para direcionar modificação genética a esta camada no pico de proliferação utilizando ex eletroporação vivo e cultura de fatias de cerebelo a partir do dia 14 embriões de galinha embrionárias. 