We would like to thank Sharon Boast for many insightful discussions and B. Novitch for the kind gift of the antibodies to Foxp1. This work as funded by a studentship from the Wellcome Trust to KCT and a project grant from the Wellcome Trust to SRP (Grant reference number 094399/B/10/Z).

1. Make Required Solutions
 <ol>
 <li>1 M Phosphate buffer: Weigh out 109.4 g Na2HPO4 and 32 g NaH2PO4.H20, mix and dissolve in water to total volume of 1 liter.</li>
 <li>Phosphate Buffered Saline (PBS): Prepare 0.1 M Phosphate buffer, 0.15 M NaCl. 100 ml of PBS should suffice for the processing of slices from 10 embryos.</li>
 <li>Agarose: Heat PBS solution to around 70 &deg;C and add solid low melting temperature agarose slowly whilst stirring continuously. Make the solution to a final concentration of 4% (w/v) agarose. Make a stock of 50 ml of this solution. Leave this solution in a waterbath kept at 48 &deg;C until required. Any agarose solution unused after the experiment can be stored at 4 &deg;C for several weeks.</li>
 <li>Antibody block solution: Add foetal calf serum and triton X-100 to a final concentration of 1% (v/v) foetal calf serum, 0.1% (v/v) Triton X-100 in PBS. 10 ml of block solution will suffice for the immunostaining of 5 slides of cryostat sections.</li>
 <li>Fixative: Prepare 0.75 mm NaOH, 4% (w/v) paraformaldehyde (CAUTION). To prepare fixative heat required amount of water to 70 &deg;C, add concentrated NaOH to required final concentration, add solid paraformaldehyde and mix until dissolved. Cool on ice to 4 &deg;C. 10 ml of this solution will suffice for 20 wells of embryo slices.</li>
 <li>Sucrose Solution: Prepare 10 ml of a 30% (w/v) sucrose solution containing 0.1 M Phosphate buffer. 10 ml of this solution will suffice for 10 wells of embryo slices.</li>
 <li>70% (v/v) Ethanol. Dilute 70 ml of absolute ethanol with 30 ml of water.</li>
 <li>Culture Medium: Prepare a solution of 1% chicken embryo extract, 1% Penicillin/ Streptomycin, 0.35% L-Glutamine, 0.1% 2-mercaptoethanol, 4% chicken serum, 2% B27 supplement and 100 ng/ml ciliaryneurotrophic growth factor (CNTF) all diluted in neurobasal medium; prepared on ice and used the same day. 10 ml of culture medium is required for 20 wells of embryo slices.</li>
 </ol>
 2. Chick Embryo Preparation
 <ol>
 <li>Place fertilized hens eggs with the longest side horizontally in a forced draft incubator at 38 &deg;C until they develop to stage15 24. This usually requires approximately 4 days of incubation.</li>
 <li>On the second day of incubation, sterilize the egg shell by wiping with a tissue paper soaked in 70% ethanol. Carefully pierce the flat end of the egg shell using a 21 gauge needle attached to a 5 ml syringe and remove 5 ml of albumin. This lowers the embryo away from the shell so the embryo is not damaged when the shell is opened. Return the eggs to the incubator and incubate them for a further 2 days.</li>
 <li>Using blunted dumont #5 forceps carefully pierce the top middle of the shell and remove around 9 cm2 of shell. Cut around the embryo and carefully lift out the embryo and place in HBSS during dissection. The embryos should be staged according to Hamburger and Hamilton (1992)15. All embryos used should be at close to stage 24. Any embryos that show any signs of abnormality should be discarded.</li>
 <li>Remove the amnion membrane, chorio-allantoic membrane, the head, and allantois using two dumont #5 forceps. Eviscerate the embryo to remove all internal organs. To do this, cut open the ventral midline of the embryo with micro dissecting scissors, hold the embryo around the rostral trunk region with one pair of forceps, grab the rostral part of the gut and the heart and pull caudally. It is important that this is done cleanly. You should be able to see the embryo somites clearly. Cut the embryo in half, transversely between the upper and lower limb buds.Now trim the thoracic body wall using microdissection scissors.</li>
 </ol>
 3. Embryo Slice Preparation
 <ol>
 <li>Remove the agarose solution from the water bath. Cut 5 cm from the bottom of a disposable 3 ml plastic Pasteur pipette.</li>
 <li>Gently remove the lumbar half of the dissected embryo using two forceps to cradle the embryo out of the dissection solution and place in a dry Petri dish. Using the Pasteur pipette, remove around 2 ml of agarose, pipette approximately 0.5 ml on top of the embryo and then suck up the embryo slice into the pipette. Transfer the embryo and agarose to a peel away plastic mould take care not to introduce any bubbles in the agarose. Gently lower the embryo to the bottom of the agarose in the plastic mould with the thoracic section facing downwards. The embryo should be positioned to be straight using a 21 gauge needle. The slices will also contain part of the developing limb and it is important that the orientation of the embryo in the agarose will allow this. Place the boat on ice to set the agarose. Take care that the embryo does not change orientation during this period (around 2 min).</li>
 <li>The Leica VT1000S vibratome (Speed 3, Frequency 4, 400 &mu;m thick slices) should be set up with the slicing chamber filled with HBSS and surrounded by ice-cold water. The agarose block is then stuck to the vibratome platform with super glue. The agarose block is trimmed with a razor blade to leave the embryo piece with 1-3 mm of agarose surrounding it. The slices that include limb bud sections with a clear apical ectodermal rigde are selected and these slices are then placed in HBSS. Generally, there are only one to two suitable slices per embryo. Gently remove the agarose around the tissue slices using two needles. It is essential that this is done thoroughly and carefully.</li>
 </ol>
 4. Culturing Embryo Slices
 <ol>
 <li>Add 500 ml of culture solution (outlined above) to the required number of wells of a 24-well ultra low attachment treated tissue culture plate. Transfer the slices carefully from the HBSS solution into the well. In general, we try to have two to three slices in each well. Place the 24-well plate in the tissue culture incubator at 37 &deg;C with 5% CO2 for 24 hr.</li>
 </ol>
 5. Sectioning the Slices for Immunostaining
 <ol>
 <li>Following the culture period, add 500 &mu;l of unbuffered fix to each well of embryo slices and leave on ice for 20 min. The total solution is then removed carefully and three PBS washes are carried out, with 5-min intervals. The slices are then left in sucrose solution at 4 &deg;C, until the slices sink, around 6 hr but can be left for longer (e.g. overnight).</li>
 
 <li>Remove the slices and dab off extra sucrose solution and equilibrate in around 1 ml O.C.T for around 5 min at 20 &deg;C. Mount each slice flat in an O.C.T.-filled peel away plastic mold. After mounting, place the molds vertically on dry ice to solidify.</li>
 
 <li>Mount the O.C.T. blocks into the cryostat and equilibrate the blocks at -24 &deg;C for around 20 min. Cut each slice with 15 mm sections mounted on Superfrost-Plus slides. Slides can be stored at -80 &deg;C until they are needed.</li>
 </ol>
 6. Immunofluorescence
 <ol>
 <li>Pipette 1-2 ml of PBS on the horizontal slides held in a humidified chamber with the slides raised from the bottom of the chamber (we use 1 or 2 ml plastic pipettes fixed with autoclave tape). Incubate the sections in PBS for 5 min at 20 &deg;C to remove excess O.C.T. Remove the PBS solution from the slide and add 500 ml of block solution over the sections, incubate for 30 min at 20 &deg;C. Remove the block solution and immediately add 500 ml of diluted primary antibody added to the slides, incubate at 4 &deg;C for 18-22 hr.</li>
 <li>Remove primary antibody solution and immediately wash the slides with 1 ml of PBS for 5 min, three times at 20 &deg;C to remove excess primary antibody solution. Add 500 ml of diluted secondary antibody over the sections. The slides are then left at 20 &deg;C for 30 min. The secondary antibody solution is then removed and the slide washed 3 times 5 min in PBS at 20 &deg;C.</li>
 <li>Following these washes, remove the final PBS wash, dab the edge of the slide to remove excess PBS, add two drops of vectashield on the slides and gently lay a glass coverslip over the sections, avoiding bubble formation. Remove excess Vectashield by blotting from the edge of the slides before imaging.</li>
 </ol>
 7. Imaging
 <ol>
 <li>Image the immunostained sections. We use a Nikon Eclipse E80i fluorescence microscope equipped with a Hamammatsu ORCA ER digital camera and 10 X, 20X and 40 X objective lenses.</li>
 </ol>

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None of the authors have competing interests or conflicts of interest.

The protocol described here allows a chicken embryo slice to be incubated for more than one day whilst keeping motor neurons alive and continuing to migrate during the culture period. In elucidating the conditions to keep motor neurons alive, we have identified several key features required. It seems that up to 4% chick serum is critical to the survival of motor neurons and its replacement with serum from a different species is not tolerated. Interestingly, we found that the presence of higher concentrations of serum were detrimental to the slices as they promoted overgrowth of the tissue leading to gross distortions in the slice shape. More importantly, the presence of cilliary neurotrophic factor also proved critical. It remains a distinct possibility that the slices may be able to be cultured for considerably longer periods than just 24 hr, perhaps even allowing the visualization of sensory afferent input to the spinal cord. Additionally, the culture conditions here may also prove useful to slice cultures of the developing brainstem and midbrain/ cerebellum.
	Perhaps the biggest potential benefit in the use of these slice culture conditions is that they facilitate the possibility for pharmacological manipulation of protein function whilst minimizing the potential for adverse effects on the rest of the embryo, such as the heart. A large number of inhibitors and agonists of different signalling pathways can be used to assess the migration of different spinal neuron subtypes and, potentially, their effect of the formation of local spinal circuits during development. Additionally, some genetic perturbations of protein expression, such as those that make use of siRNAs and morpholino oligonucleotides, suffer from the fact that they can only be introduced early in development (owing to limitations in in ovo electroporation procedures) and the effects only last for a short period of time (typically one day). Our slice culture is started at later stages and affords the possibility to use this technology later in development at the slicing stage to view their effects during the period of culture of the slice.
	The slice culture protocol could also allow the visualization of both spinal motor neuron as well as dorsal interneuron migration in real time via time-lapse video microscopy. This could be achieved using phase contrast microscopy under white light or through the use of fluorescence imaging. If the latter technique were to be used then the introduction of fluorescent labels is needed. This could be achieved either by introduction of fluorescent dyes such as DiI or DiO via either retrograde labelling from the limb or anterograde labelling from the ventricle or in ovo electroporation of constructs to drive fluorescent proteins in a subset of neurons.

During normal embryo development, many different cell types are generated which must migrate from their point of origin to where they will eventually function in the mature organism. Within the developing spinal cord, progenitor cells are located in the ventricular zone at the midline and generate many subtypes of postmitotic neurons and glial cells which must migrate to integrate into functional neuronal circuits required for sensory processing and motor outputs1. Spinal motor neurons that control the contraction of limb muscles have been extensively studied and much is known of the molecules and mechanisms that drive the formation of their different subclasses. However, much less is known of the mechanisms by which motor neurons migrate, segregate and receive synaptic inputs.
	Motor neuron subtype identity is acquired during development in a hierarchical fashion. Motor neuron subtypes can be broadly classed into distinct columns, divisions and pools which are defined by their axon projections. Motor columns project axons to either axial, visceral or limb muscles. Motor divisions subdivide the limb projecting motor neurons of the lateral motor column (LMC) into those projecting to ventral or dorsal muscles2. Within the divisions, clusters of motor neurons termed motor pools project to individual muscles in the limb2, 3. Limb-projecting motor neurons are defined by their expression of the transcription factor Foxp14, 5, whereas divisional identity is characterized by the expression of the homeodomain transcription factors Islet-1 (ventrally projecting) or Lhx-1 (dorsally projecting)6. Indeed, Lhx-1 expression is required for the appropriate dorsal projections of the motor neurons7. Each of these subtypes occupy distinct positions within the spinal cord; ventral projecting motor neurons come to reside medial to dorsally projecting motor neurons. This results in the LMC being segregated into so called LMCmedial (LMCm) and LMClateral (LMCl) divisions. Divisional and motor pool organization is acquired in two phases8. In the first phase, divisional segregation is achieved via a migration of motor neurons in a characteristic medial to lateral fashion. The LMCl cells are generated after the LMCm cells and so LMCl migrates through the LMCm to achieve its final settling position. The second phase takes motor neurons within a division and clusters them into motor neuron pools, presumably via a dorsal-ventral sorting of the motor neurons. Members of the catenin-dependent classical cadherin family have been shown to be crucial to both phases of motor neuron organization8-10. However, how cadherin function controls cell movement is poorly understood.
	The acquisition of columnar, divisional and pool identities requires extrinsic signals that pattern intrinsic expression of transcription factors11. Additionally, around 50% of all motor neurons die via apoptosis during normal development in a process that requires extrinsic signals from the limb, largely through neurotrophic support of motor neuron survival. Thus, motor neuron development requires the appropriate environment to be maintained over a relatively long timecourse. For this reason, the practicalities of generating spinal cord slice cultures to maintain motor neuron survival require the elucidation of appropriate culture conditions. Previous embryo slice cultures have been used to follow the behavior of extrinsically added purified neuronal populations12-14. However, to our knowledge culture conditions that facilitate in situ motor neuron survival and migration within the slice itself have not been published. We modified a standard culture condition that facilitates the survival of purified, dissociated cranial motor neurons to facilitate motor neuron development within an embryo slice culture system. We also monitored the positioning of the surviving motor neurons and demonstrate that largely normal positions of the motor neuron divisions is acquired during the culture period.

<table><tbody><tr><td>Eggs</td><td>Henry Stewart Farms, UK</td><td></td><td>Fertilised Brown Bovan Gold Hen&#039;s eggs (Henry Stewart Farms, UK), stored at 14 &amp;deg;C until incubation</td></tr><tr><td>21 Gauge needle</td><td>BD-Microlance-3</td><td>304432</td><td></td></tr><tr><td>5 ml syringe</td><td>BD Plastipak</td><td>302187</td><td></td></tr><tr><td>#5 Dumont forceps</td><td>Roboz</td><td>RS 5045</td><td></td></tr><tr><td>Micro Dissecting scissors</td><td>Roboz</td><td>RS 5910SC</td><td>(3.5 in straight)</td></tr><tr><td>Embryo embedding moulds: Peel away moulds</td><td>Agar Scientific</td><td>G3764</td><td>Pyramidal shaped 22 mm to 12 mm</td></tr><tr><td>Super glue</td><td></td><td></td><td>Power Flex, Loctite</td></tr><tr><td>Ethanol</td><td>SIGMA</td><td>32221</td><td></td></tr><tr><td>Sodium di-hydrogen Phosphate, mono-hydrate</td><td>BDH</td><td>102454R</td><td></td></tr><tr><td>Di Sodium mono-hydrogen Phosphate</td><td>VWR/ BDH</td><td>102494C</td><td></td></tr><tr><td>Sodium Chloride</td><td>VWR/BDH</td><td>27810.295</td><td></td></tr><tr><td>Low melting temperature agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>Triton X-100</td><td>SIGMA</td><td>T8787</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>FLUKA</td><td>71689</td><td></td></tr><tr><td>Paraformaldehyde</td><td>VWR/BDH</td><td>28794.295</td><td></td></tr><tr><td>Plastic disposable pasteur pipettes</td><td>ElKay Precision laboratory consumables</td><td>127-P503-000</td><td>Liquipette 3 ml volume</td></tr><tr><td>Hanks Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14170</td><td></td></tr><tr><td>Sucrose</td><td>SIGMA</td><td>S0389</td><td></td></tr><tr><td>Sodium Hydrogen Carbonate</td><td>SIGMA</td><td>S5761</td><td></td></tr><tr><td>Chicken Embryo Extract</td><td>Seralabs</td><td>CE650-J</td><td></td></tr><tr><td>Penicillin/ Streptomycin solution</td><td>Gibco</td><td>15070</td><td></td></tr><tr><td>L-Glutamine solution</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>2-mercapt&amp;#8211;thanol</td><td>Gibco</td><td>21985</td><td></td></tr><tr><td>Horse serum</td><td>Southern Group Labs</td><td>9028B</td><td></td></tr><tr><td>B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>Cat ~ 2103</td><td></td></tr><tr><td>CilliaryNeurotrophic factor (CNTF)</td><td>R and D Systems</td><td>557-NT-010</td><td></td></tr><tr><td>Chick Serum</td><td>SIGMA</td><td>C5405</td><td></td></tr><tr><td>Primary antibodies</td><td></td><td></td><td>were purchased from Developmental Studies Hybridoma Bank, with the exception of anti-foxP1 antibodies, which were a kind gift from Bennett Novitch of UCLA. Primary antibodies dilutions were Rabbit anti-FoxP1 (1/32000), Mouse anti-Lhx1/2 (4F2) (1/50), Guinea Pig anti-FoxP1 (1/1000).&lt;br/&gt; All antibodies were stored at 4 &amp;deg;C.</td></tr><tr><td>Secondary antibodies</td><td></td><td></td><td>Donkey anti-Guinea Pig (cy3) (1:1000) purchased from Jackson Immunoreseach Laboratories Inc. Donkey anti-Mouse (Alexa Fluor 488) (1:1000), Donkey anti-Rabbit (Alexa Fluor 594) (1:1000) purchased from Invitrogen&lt;br/&gt; All antibodies were stored at 4 &amp;deg;C.</td></tr><tr><td>24-well plate</td><td>Corning COSTAR</td><td>3473</td><td>ultralow attachment 24-well plate</td></tr><tr><td>O.C.T.</td><td>Tissue Tek, Sakura</td><td>4583</td><td>Optimum Cutting Temperature</td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H1000</td><td>Vectashield fluorescent mounting medium</td></tr><tr><td>Slides</td><td>Thermo Scientific</td><td></td><td>Superfrost-Plus positively-charged slides, 25 x 75 x 1.0 mm</td></tr><tr><td>Cover Glass</td><td>VWR International</td><td>631-0167</td><td>25x60 mm</td></tr><tr><td>Forced draft incubator: </td><td>Lyon Electric Company</td><td></td><td>kept humid by ensuring water levels always sufficient, maintained at 38 &amp;deg;C</td></tr><tr><td>Cryostat</td><td>Brite, Huntingdon, UK</td><td></td><td>-36 &amp;deg;C</td></tr><tr><td>Tissue Culture Incubator</td><td>DH Autoflow</td><td></td><td>Nuaire 38 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Vibratome</td><td>Leica</td><td></td><td>Leica VT1000-S</td></tr><tr><td>Microscope</td><td>Nikon</td><td></td><td>Nikon Eclipse E80i fluorescence microscope</td></tr><tr><td>Digital Camera</td><td>Nikon</td><td></td><td>Nikon DS5M and Hamammatsu ORCA ER</td></tr></tbody></table>

chicken embryo spinal cord slice culture protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

The use of cultured slices to follow the development of interneuron and projection neurons has been influential in progressing our understanding of the generation of organization within the cortex16. In the spinal cord, motor neuron development has been followed using cultures of whole zebrafish embryos17. However, spinal motor neuron organization in zebrafish is relatively simple (owing to the lack of large limb muscles in fish). The molecular mechanisms that drive a hierarchy of spinal motor neuron organization during the development of higher vertebrates are currently poorly understood. Culture conditions that facilitate neuron survival and cell body migration in embryo slice cultures of higher vertebrates are thus required. Currently, spinal cord slice cultures of higher vertebrates have been used to seed dissociated neurons on top of the slices12-14, investigate fluorescently labelled axons in the periphery of the slice18, or of progenitor cell behavior in early spinal cord development19. Slice culture conditions for later spinal cords, particularly those that maintain motor neuron development are currently lacking.
To attempt to follow spinal neuronal development, particularly that of spinal motor neurons, we investigated various culture conditions that might promote motor neuron survival and the acquisition of initial order in motor organization through lateral migration of the neurons. Initial trials using stage 18 to stage 20 chick embryo spinal cord slices proved fruitless; we were not able to keep motor neurons alive for longer than a few hours and were unable to demonstrate the generation of substantial numbers of LMCl cells over the culture period (data not shown). We therefore investigated slice cultures of stage 24 embryos, a timepoint when the majority of LMCl and LMCm neurons have been generated, but when LMCl neuron migration is still in its infancy (Figure 1a-c). This lateral migration of LMCl neurons is largely complete by stage 27, around 24 to 36 hr later (Figure 1 d-f). Our assay could thus be simplified to being able to keep the neurons alive and to facilitate their migration laterally into the ventral horn. We started our experiments with relatively simple culture conditions containing just Hank's balanced salt solution with or without chicken serum or chicken embryo extract. In all cases, although we were able to maintain LMC neurons in the slice, we found little evidence of LMCl neurons being maintained (at least by expression of Lhx-1). Further, we found inappropriate expression of genes such as HB9 in the dorsal spinal cord, a situation that never occurs in vivo (data not shown). Thus, these simple culture conditions are not suitable for the investigation of the acquisition of motor neuron organization.
We thus sought out more complex conditions and as a base used a formulation that has been published to facilitate the survival of dissociated embryonic chicken cranial motor neurons. This condition20 (1% chick embryo extract, 1% Pen Strep, 0.35% L-Glutamine, 0.1% 2-mercaptoethanol, 2% horse serum, 2% B27 supplement and 50 ng/ml ciliaryneurotrophic growth factor (CNTF) in neurobasal medium (here called Guthrie medium) did keep more motor neurons alive than the more simple media. Additionally, it did not result in spurious expression of transcription factors in the dorsal spinal cord. However, the survival of LMCl neurons was poor (Figure 1 g-i, p). We therefore varied what we considered to be key factors in the Guthrie medium, namely the animal of origin of the serum, the concentration of the serum and the concentration of CNTF in the medium. We found that changing the serum from Horse serum to Chick serum and an increase in concentration of CNTF from 50 ng/ml to 100 ng/ml substantially increased the survival of the LMCl and LMCm cells and also allowed their migration into the ventral horn over the 24 hr of the culture conditions (Figure 1 j-l, p). The total number of motor neurons, the ratio of LMCm to LMCl and, indeed, the migration of the LMCl cells into the ventral horn appeared very similar to sections of embryos that had been allowed to develop in ovo rather than in the slice culture (Figure 1 k-o, p). Thus, we believe that based on transcription factor expression, cell number and position of motor neurons, our slice culture conditions recapitulate normal spinal motor neuron development at least over a 24 hr period.
<img alt="Figure 1" fo:content-width="2in" fo:src="/files/ftp_upload/50295/50295fig1highres.jpg" src="/files/ftp_upload/50295/50295fig1.jpg" /> 
Figure 1. a-c. Status of generation of LMC (Foxp1 staining in b) and LMCl (Lhx-1 staining in a) at stage 24. c is a merge of the two channels. The midline is shown as a dotted line in a and D, V shows the orientation of the dorsoventral (D, V) axis of the spinal cord. d-f. Status of LMC (Foxp1 staining in d) and LMCl (Lhx-1 staining in e) organization at stage 27. f is a merge of the two channels. g-i. Lhx-1 (g) and Foxp1 (h) expression in sections of a slice cultured in a medium that supports cranial motor neuron cell survival (Guthrie medium, our "initial medium" see reference 20). Lhx-1 is no longer expressed in motor neurons, although there is some survival of LMC cells evidenced by Foxp1 expression. D, V in (g) shows the orientation of the dorsoventral (D, V) axis of the spinal cord. M-L shows the orientation of the mediolateral (M, L) axis of the spinal cord j-l. LMCl cells (Lhx-1 in the ventral horn in j) and the LMC in general (Foxp1 in k) can be observed in slices cultured in medium containing 4% chick serum and 100 ng/ml CNTF. Note that some LMCl cells are found in a lateral position in the ventral horn. The arrow in (j) shows the lateral position of some of the the LMCl cells. (l) is a merge of the two channels. m-o. Status of expression of Lhx-1 (m) and Foxp1 (n) in sections of an embryo kept in ovo during the slice culture of the embryo shown in j-l. (o) is a merge of the two channels. p. Quantitation of the percentage of LMCl (Foxp1+ve/ Lhx1+ve) cells versus LMC (Foxp1+ve/Lhx1-ve) cells found in slice cultures in different conditions compared to control embryos kept in ovo (which represent 100%). Student's t-test *** represents p&lt;0.01. <a href="https://www.jove.com/files/ftp_upload/50295/50295fig1large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Chicken Embryo Spinal Cord Slice Culture Protocol at JoVE.com

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

chicken-embryo-spinal-cord-slice-culture-protocol

Research

JoVE Journal

Neuroscience

22.0K Views.  University College London. Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Video: Chicken Embryo Spinal Cord Slice Culture Protocol

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biology

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Developmental Biology

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...

Chicken Embryo Spinal Cord Slice Culture Protocol

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Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits