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Journal of Neuroscience Methods 239 (2015) 206–213

Contents lists available at ScienceDirect

Journal of Neuroscience Methods

jo ur nal home p age: www.elsev ier .com/ locate / jneumeth

asic Neuroscience

he combination of limb-bud removal and in ovo electroporationechniques: A new powerful method to study gene function in

otoneurons undergoing lesion-induced cell death

eronica La Padula ∗,1, Sophie Koszinowski, Kerstin Krieglsteinnstitute of Anatomy and Cell Biology, Department of Molecular Embryology, Albertstr. 17, 79104 Freiburg im Breisgau, Germany

i g h l i g h t s

A method combining induced motoneuron death and gene overexpression is described.The method is performed on chicken embryos developing in ovo.The electroporation rate of motoneurons is 40–50%.The survival rate one day after is 95% and decreases to 80% three days after.

r t i c l e i n f o

rticle history:eceived 9 September 2014eceived in revised form 24 October 2014ccepted 24 October 2014vailable online 3 November 2014

eywords:otoneuron

hickenn ovo electroporationimb-bud removalesion-induced cell death

a b s t r a c t

Background: The chicken embryo is an important model organism for developmental biology studies. Atpresent, many techniques on this model have been set up, from surgical procedures to molecular biologymethods, to answer capital questions of cell biology. The study of the genes involved in motoneurons(MNs) survival and cell death is critical for a better understanding of the molecular mechanisms lead-ing to MNs degenerative diseases, such as amyothophic lateral sclerosis (ALS) and motor peripheralneuropathies.New method: Here, we describe the combination of a well known surgical procedure able to induce MNscell death, the limb-bud removal (LBR), with a very popular method used in molecular biology to testgene function in living organisms, the in ovo electroporation (IOE). The aim of this work is to providean effective method for the investigation of genes involved in MNs survival and cell death under lesionconditions.Results: Our method allows the successful electroporation of the 40–50% of MNs on the side of LBR witha high survival rate early and late after procedure.Comparison with other methods: This modified LBR technique combined with IOE allows a higher MN

expression efficiency compared to an already published method.Conclusions: Our work opens the possibility of screening a multitude of genes involved in MNs survival orcell death in vivo with high reproducibility and efficiency on a flexible and inexpensive animal model. TheLBR/IOE technique opens a new way for the optimization of subsequent studies on mammalian modelsof diseases affecting MNs survival.

© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND

∗ Corresponding author. Tel.: +49 7612035108.E-mail addresses: veronicalapadula@gmail.com

V. La Padula), sophie.koszinowski@anat.uni-freiburg.de (S. Koszinowski),erstin.krieglstein@uniklinik-freiburg.de (K. Krieglstein).1 Tel.: +0049 761 203 5087.

ttp://dx.doi.org/10.1016/j.jneumeth.2014.10.022165-0270/© 2014 The Authors. Published by Elsevier B.V. This is an open access article un

license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Motoneuron (MN) degeneration is a feature common to manydiseases affecting the motor system, such as the Charcot-Marie-Tooth disease type 2 (CMT2) (Saporta and Shy, 2013) andamyotrophic lateral sclerosis (ALS) (Chen et al., 2013) and spinal

muscular atrophy (SMA) (Edens et al., 2014). Many gene mutationsare known to be involved in the pathogenesis of such diseases,but the molecular mechanisms underlying these pathologies arestill not clearly understood. The symptoms of such diseases have

der the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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V. La Padula et al. / Journal of Neu

enerally a late onset in human patients and their gravity pro-resses in time. More precisely, the age of onset can vary on aong time frame, being late especially for ALS affected patients, for

hich the first symptoms appear between 50 and 60 years of ageeven if very rare forms of juvenile ALS exist) (Hentati et al., 1998;eng et al., 2011; Al-Saif et al., 2011), whereas for CMT2 and SMAatients there are more variations accordingly to the genetic muta-ions causing the disease: in particular, in CMT2 the onset can varyrom childhood (e.g. CMT2A1) to around the second–fourth decadef life (e.g. CMT2F) depending on the gene mutation involved andimilarly for SMA the onset varies from the early infancy (SMA types

and II) to adulthood (SMA type IV) based on the expression levelf the survival motor neuron protein (SMN). To better understandhe molecular and cellular mechanisms involved into the beginningnd the progress of these diseases, mammalian transgenic mod-ls have been developed. Indeed, animal models like the mutantouse for the neurofilament light chain as a model of CMT2E (Lee

t al., 1994), the mutant mouse for superoxide dismutase 1 (e.g.he mouse carrying the G93A mutation) as a model of ALS (Gurneyt al., 1994), or the Smn1 null mouse for SMA (Frugier et al., 2000)epresent valuable tools to study these MN degenerative diseasesor several reasons: first of all, their phenotype can be studied atifferent time points during the animals’ development; second,ammalian transgenic strains are genetically pure and the internal

ariability is virtually absent. On the other side, transgenic animalsresent some disadvantages: obtaining transgenic animals is notasy, time consuming and expensive, delaying the study of a geneutation on an in vivo model. Moreover, the animal phenotype

an sometimes be milder than the human one, be different fromhe expected one, or even be absent due, for example, to partialr complete genetic redundancy, to the genetic background of theouse strain used, or to the lack of specific environmental con-

itions in which the animal lives (Pearson, 2002). Therefore, weelieve that our method of combined limb-bud removal (LBR) and

n ovo electroporation (IOE) (LBR/IOE technique) is a way to prelim-nary study the genes related to MN degeneration on an alternativenimal model, the chicken embryo.

The chicken embryo presents many experimental advantages:t is cheap and easy to obtain, the readout of the experiments is rel-tively fast, the embryo is accessible in ovo (meaning in vivo insidehe egg) all along its development for morphological examinations well as for additional experimental procedures. Importantly,he chicken embryo can undergo surgical interventions in ovo (e.g.ransplantation of somites or limbs, LBR) (Aoyama and Asamoto,988; Oppenheim et al., 1978) and can be pharmacologicallyreated with specific drugs at given times during its develop-

ent (Krieglstein et al., 2000). Moreover, the availability of moredvanced techniques – such as IOE – allows the overexpression orhe inhibition of specific genes in vivo on restricted cell populationsnd in a time-dependent manner (Takeuchi et al., 1999; Odani et al.,008).

For these reasons, we designed the method described here,imed to provide an effective tool to overexpress specific genes byOE in the neural tube of chicken embryos on which lumbar MNsre induced to die in consequence of the removal of the lumbarimb-bud by means of a surgical procedure, the LBR technique.

Notably, the LBR causes MN apoptosis first of all because of theack of trophic factors provided by the limb-bud and second byhe absence of synaptic contacts between the MNs and their target

uscles. The cell death leading to neurodegenerative diseases isainly due to apoptotic events (Ghavami et al., 2014), and thene consider that the LBR procedure can be a valuable model to

eproduce the cell death occurring in MN degenerative diseases,n which the axon is the cell primarily affected by degeneration.ndeed, after LBR, the only cell undergoing cell death is the MN, andhis is particularly useful to study cell-autonomous events related

ce Methods 239 (2015) 206–213 207

to the MN survival/death molecular pathways and to better isolateevents occurring in the degenerating MNs overexpressing a genein its wild-type (WT) or in its mutated form.

A wide variety of plasmids is available to perform IOE, bothfor transient and stable expression. Among the plasmids used fortransient gene expression the mostly adopted is the pCAGGS vec-tor (Dale et al., 2003) or its variant pCIG (Megason and McMahon,2002), used for this work. The combination of the cytomegalovirus(CMV) enhancer with the chicken beta-actin promoter ensuresa strong expression starting already 3 h after electroporation,remaining stable up to the 72 following hours. Such plasmids areindistinctly expressed by any cell in the experimental organism,lacking a cell-type specific promoter. On the other hand, for stableexpression of a foreign gene specifically in avians (such as chickenor quail), the usage of the replication-competent avian sarcoma(RCAS; Hughes et al., 1987) virus is the election method. At present,vectors for transient expression (i.e. pCAGGS and pCIG) have beenused for several studies (Yan et al., 2014; Le Dréau et al., 2012) andtheir relatively small size (around 5 and 7 kb, respectively) allowthe cloning of complete genes, such as human ADAM10 (2658 basepairs) and Smad 1, 5, and 8 (1127, 1397, 1070 base pairs, respec-tively), analyzed in the cited works.

Furthermore, the chicken genome is fully sequenced and thecomparison of its genome sequence with the Human one revealedthat about the 60% of the coding genes in chickens present ahuman orthologue (International Chicken Genome SequencingConsortium, 2004). In addition, a bank of chicken gene expressionprofiles exists (http://geisha.arizona.edu/geisha/).

Here, we describe a procedure consisting in the combinationof two distinct techniques: the first is a well-known and estab-lished model of lesion-induced cell death for lumbar MNs, the LBR(Oppenheim et al., 1978). The LBR technique consists in the surgicalablation of the chicken limb-bud at an early stage during embryonicdevelopment, at Hamburger and Hamilton stages 16–17 (HH16-17,corresponding to embryonic day 3, E3) (Hamburger and Hamilton,1951). As already mentioned, following this procedure, nearly allthe lumbar MNs will die because of the lack of the trophic factorsprovided by their final innervation target, the leg muscles, and theabsence of neuromuscular synapses. The second technique is theIOE. The electroporation is a widely used technique employed totransfer plasmidic DNA into target cells by the application of anelectric field. By electroporation it is possible to induce the expres-sion of specific genes on dissociated primary cell cultures (in vitro)(Zeitelhofer et al., 2007), organotypic cultures (ex vivo) (Pakan andMcDermott, 2014) or in living organisms (in utero or in ovo, whentreating mammalian embryos in the uterus or animals developingin egg, respectively) (Bony et al., 2013; Odani et al., 2008). Previousstudies have explored the role of many proteins involved in MNsfundamental cellular process, such as axon pathfinding (Winningand Krull, 2011), differentiation (Roy et al., 2012), and survival(Choi et al., 2006,2013) employing IOE to overexpress the probegenes.

This procedure of combined LBR and IOE (LBR/IOE) is aimed tooverexpress a plasmid at the side of LBR in the lumbar MN pop-ulation before the beginning of the induced cell death, caused bythe lack of trophic factors due to the LBR itself, in the developingchicken embryo.

In conclusion, we have designed a method that will allow theinvestigation of the effect of genes involved in MNs survival and celldeath in conditions of lesion and growth factor deprivation. Addi-tionally, a very important advantage of our method is the possibilityof testing many genes on an animal model that provides fast andreproducible results, allowing a rapid and reliable in vivo pheno-typic screening before to approach transgenic mammalian models

of diseases in which the MNs are the primary target of neuronaldegeneration.

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. Materials and methods

.1. Fertilized eggs and embryo handling

White Leghorn (Gallus gallus domesticus) fertilized eggsere obtained from a local producer (Produits agricoles Haas,altenhouse, France) and then incubated lying on their long side

n a humidified incubator at 38 ◦C. At the third day of incubationhe eggs were wiped with 70% ethanol, 5–6 mL of albumen wereemoved with a sterile syringe and then a stripe of porous tapeDurapore, 3M) was applied on the eggshell to proceed with theindowing by using dissection fine scissors (Fine Science Tools).

mbryos were staged on the base of the Hamburger and HamiltonHH) classification (Hamburger and Hamilton, 1951). Stage HH16-7 embryos were selected for limb-bud removal (LBR) followed by

n ovo electroporation (IOE).

.2. Limb-bud removal

The protocol we used for the limb-bud removal (LBR) differsubstantially from the one previously published by Chu-Wang andppenheim in 1978, so we will describe our method in deep detail.

First of all, the embryo was rinsed with sterile PBS and 20–50 �Lf black Indian ink (diluted 1:8 in sterile PBS) were injected underhe embryo to better visualize it. The vitelline membrane over theimb-bud area was carefully removed under a stereomicroscopeMZ80, Leica Mycrosystems) by the help of a self-made tungsteneedle. The right limb-bud was dissected by cutting it using a pairf Vannas-Tübingen spring scissors with blades angled up (Finecience Tools) and the cut limb was sucked up by pipetting it awayith a sterile yellow tip and discarded in a tube containing sterile

BS. This step is done to verify that the limb-bud has been success-ully removed.

Only those embryos with a sharp removal of the limb-bud andhich were not excessively bleeding were kept for the following in

vo electroporation.

.3. In ovo electroporation

Immediately after the LBR, the in ovo electroporation (IOE)as performed. To better manipulate the embryo, sterile PBSas added into the egg to make the embryo reach the top of the

ggshell. The pCIG plasmid (a pCAGGS plasmid with an insertionf IRES2-GFP, Megason and McMahon, 2002) was injected athe final concentration of 5–7 �g/�L supplemented with 1% Fastreen (Sigma–Aldrich) into the chicken embryo neural tube using

glass needle connected with a mouth pipet (Sigma–Aldrich). Theeedle was inserted at the thoracic level and the DNA was injected

n rostro-caudal direction to fill the neural tube. Glass needlesith a sharp tip at the edge were obtained by pulling borosilicate

lass capillaries (Sutter Instrument, Borosilicate Glass, OD-1.5 mm,D-1.10 mm, 10 cm length) with a Narishige PC-10 puller (tem-erature 66.6 ◦C, four weights, single pull mode). As soon as theeural tube was filled with DNA, IOE was carried out by using theonfiguration described by Linn and Krull (Linn and Krull, 2008)Fig. 1F). Platinum self-made electrodes were manually obtainedy bending a platinum wire (0.5 mm thick) with an angle of 90◦ and

solating it with nail polish on the opposite side of the current flow,etting a free surface of approximately 4 mm. The electrodes werelaced at the two sides of the embryo on a diagonal configurationt around 2 mm from each side of the embryo, positioning theositive electrode below the embryo at the side of the LBR (right)

nd the negative over the embryo at the level of the remainingind limb (left). Five pulses (30 mV, 50 ms of duration, 100 ms of

nterval) were applied (IntraCel, TSS20 Ovodyne electroporatorlus, EP21 current amplifier). After electroporation, the embryos

ce Methods 239 (2015) 206–213

were rinsed with sterile PBS and the PBS used to elevate theembryo (5–6 mL) was removed. The eggs were sealed with poroustape (Durapore, 3 M) and placed back into the incubator to letthe embryos reach the appropriate developmental stage. The dayafter the procedure (E4), the embryos were checked regarding theelectroporation efficiency and their morphology at a fluorescencestereomicroscope (Leica MZ 10F, equipped with a Lumen 200Fluorescence Illuminating System, Prior Scientific), representativeimages were acquired with a digital camera (Leica MC 120HD)and then the embryos were sacrificed for further analysis orplaced back to the incubator. Under our experimental conditions,the embryos continue expressing the plasmid at least until E6(not shown).

2.4. Histology and immunohistochemistry

Chicken embryos were collected one day after (E4) the LBR/IOEprocedure, the membranes and blood vessels were removed andthen the embryos were immediately fixed by immersion in 4%paraformaldehyde (PFA) in 1.0 M phosphate buffer saline (PBS) overnight (ON) at 4 ◦C. The day after, samples were washed several timesin PBS, impregnated in 10% sucrose in PBS 6 h at room tempera-ture (RT) on a rocking plate and then submerged in 30% sucrose inPBS ON at 4 ◦C. The samples were finally embedded in OCT com-pound (TissueTek) and stored at −20 ◦C until usage. Transverseserial thin sections (10 �m) were cut with a Leica CM1520 cryostatand collected on superfrost slides (Mänzel-Gläser) and then stainedwith Isl1/2 antibody for lumbar MNs quantification (39.4D5, 1:50,Developmental Studies Hybridoma Bank). Thin transverse sectionsat the lumbar level were stained with the N-cadherin antibody(6B3, 1:100, Developmental Studies Hybridoma Bank) to check theneural tube morphology and structure. Immunostainings were per-formed using standard protocols. Briefly, cryosections were leftto air dry, washed in PBS and cooked 2 min in cytrate buffer at95 ◦C and again washed in PBS. Antigen blocking was performedincubating the sections with 1.5% Normal Donkey Serum in PBT(0.2% Triton X-100 in PBS) 1 h at RT. After this, the blocking bufferwas removed and the primary antibodies were applied ON in ahumid chamber at 4 ◦C. The day after, the slides were washedin PBS on a rocking plate and the appropriate secondary anti-bodies (Goat anti-Mouse AlexaFluor 568, Life Technologies) wereapplied 2 h at RT in a dark humid chamber. Finally, the slides werewashed with PBS, mounted with DAPI Fluoromount-G (SouthernBiotech) and let to air dry ON at RT in the dark and then stored at4 ◦C.

Sections were imaged with an epifluorescence microscope(Nikon Axioplan 2) for quantification at 40× magnification andrepresentative images of examined samples were taken with aconfocal microscope (Leica SP8) at 10× and 40× magnificationobjectives.

2.5. Quantification and statistical analysis

For MN quantification, images were taken every 5th section atan epifluorescence microscope (Nikon Axioplan 2) at 40× mag-nification and Isl1/2 positive cells were manually counted (n = 3).To calculate the electroporation efficiency, GFP/Isl1/2 double pos-itive cells were manually counted (n = 3). The multi-point tool ofthe ImageJ free software was used for cell counting. Survival rateshave been calculated on three independent experiments for everyexperimental condition.

Statistical analyses were performed using GraphPad software(Graphpad). The statistical significance of the data was determinedby unpaired T test. Values are expressed as average percent-age ± standard error of the means.

V. La Padula et al. / Journal of Neuroscience Methods 239 (2015) 206–213 209

Fig. 1. Combination of limb-bud removal (LBR) with in ovo electroporation (IOE) (LBR/IOE) experimental procedure. Chicken embryos at the third day of development (E3)were selected (A–D). The right limb-bud (B, arrowhead) is removed (C, arrow) and the plasmidic vector pCIG is injected in the neural tube at the thoracic level (D, *′) in caudaldirection (D, *′′). IOE using a diagonal configuration of the electrodes (F, schematic representation of the neural tube and electrodes positioning: dark green, pCIG plasmid;light green, electroporation area; dark grey, somites; light grey, notochord; black dot, negative electrode; red dot, positive electrode). E4 embryos express the pCIG vectoron the LBR side (E, GFP-expressing tract between the asterisks, the arrow indicates the LBR side). A schematic representation of a transversal section of an E4 embryo withLBR/IOE is depicted in G (green area, electroporated hemitube; arrow, LBR). Scale bars: A and E = 1 mm; B–D = 0.1 mm. (For interpretation of the references to color in thisfi

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gure legend, the reader is referred to the web version of this article.)

.6. Molecular biology

The pCIG plasmid was amplified by cloning it in DH5-lpha supercompetent E. coli (ZymoResearch) following standardrotocols and purified by using a Macherey-Nagel midi-prepit following manufacturer’s instructions. Plasmidic DNA was

eluted in sterile water and its concentration was quanti-fied with a Thermo Scientific Nanodrop 2000 spectropho-

tometer. The plasmid was then diluted in sterile water ata final concentration of 5–7 �g/�L mixed with Fast Green(Sigma–Aldrich, 1% final concentration) and stored at −20 ◦C untilusage.

210 V. La Padula et al. / Journal of Neuroscience Methods 239 (2015) 206–213

Fig. 2. The combination of limb-bud removal with in ovo electroporation (LBR/IOE) is an efficient and conservative technique to electroporate MNs undergoing lesion-inducedcell death. Limb-bud removal (LBR) does not change MNs localization (Islet1/2, E and E′) neither neural tube structure (N-cadherin, F and F′) compared to not lesioned samples(A, A′ and B, B′ , respectively). The combination with in ovo electroporation (IOE) does not affect these parameters either with or without LBR (C, C′ versus G, G′ and D, D′ versusH, H′ , respectively for Islet1/2 and N-cadherin). The electroporation rate of MNs does not change in presence (black bar) or absence (white bar) of LBR (I). The embryonics fter (Mt rmiti4

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urvival one day after LBR/IOE is very high (L), while slightly decreases three days ahe one with IOE only. The association of LBR and IOE can induce morphological defo0×.

. Results

We established a method that combines two distinct tech-iques: the limb-bud removal (LBR) (Fig. 1C) and the neuralube in ovo electroporation (IOE) (Fig. 1D) at the same sidef the LBR (LBR/IOE) on chicken embryos at the third dayf development (E3, Hamburger and Hamilton stage 16–17)Fig. 1A–D). The aim of this method is to overexpress a plas-

idic vector into MNs in which cell death is induced by the LBRFig. 1E and G).

.1. Survival rate

First of all, we observed the survival rate of embryos undergo-ng LBR/IOE compared to the one of electroporated-only embryos,xamined as a control group for the effects of the electroporationn embryo survival and morphology. Interestingly, there is no sig-ificant difference in the percentage of embryos surviving one day

fter the electroporation (E4) (Fig. 2L) combined or not with LBRwith 90.38%, n = 47 and without 90.91%, n = 60, p > 0.1). Prolong-ng the incubation time until E6 (three days after the procedure)he number of dying embryos increases but, importantly, there is

) but there is no significant difference between the group undergoing LBR/IOE andes (N). Scale bars: A–H = 500 �m, magnification 10×; A′–H′ = 100 �m, magnification

no significant difference in the percentage of surviving embryosbetween the two groups (with LBR 75.29%, n = 51 and without LBR69.77%, n = 36, p > 0.1) (Fig. 2M).

We stopped our observations at E6 because after this stagethe MN programmed cell death is occurring (Chu-Wang andOppenheim, 1978) and this would interfere with the evaluationof cell death and survival due to the electroporation of a candidategene having a role in these processes.

3.2. Morphological modifications

Changes in morphology are not very evident in ovo at E4, so it isnecessary to dissect the embryo to better see if some gross modifi-cations happened to the embryo. On the contrary, at E6 the embryois much more developed and at this stage the possible deforma-tions caused by the electrical field applied on the side of the LBRare more evident. Indeed, the percentage of E6 embryos presentingdeformations (such as bent or missing tail, bent spinal cord) is sig-

nificantly higher in embryos on which LBR/IOE has been performedcompared to electroporated-only embryos (with LBR 50.42%, n = 51and without LBR 21%, n = 36, p ≤ 0.01) (Fig. 2N). Such an event canbe due to the fact that on the side of LBR the lack of the limb can

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ause an unbalance in the electrical field diffusion, resulting in aigher stress for the lesion side.

.3. Electroporation efficiency

We then wanted to know if the efficiency of the electropora-ion in MNs was influenced by the LBR. To do this, we sacrificedlectroporated embryos with or without LBR one day after electro-oration (E4) and we embedded them for cryosectioning. Serial thisections (10 �m) were transversally cut and stained for the MNsarker Isl1/2 (Yamamoto and Henderson, 1999) (Fig. 2C and G)

nd Isl1/2 positive cells expressing the pCIG plasmid (GFP positive)ere counted. Interestingly, there is no difference in the percent-

ge of MNs expressing the plasmid in presence or absence of LBRFig. 2I, with LBR 50.54% and without 42.17%, n = 3, p > 0.1).

The pCIG vector is not a plasmid specifically designed for MNxpression, not presenting any MN specific promoter sequence, sohere is a not selective localization of the GFP signal in MNs butnstead all the cells in the electroporated hemitube can be equallyransfected (Figs. 1G and 2C, D, G, H and C′, D′, G′, H′). Impor-antly, even if the LBR already drastically reduced the number of

Ns in LBR/IOE embryos (22.09% of surviving MNs on the LBR sideompared to the contralateral side, used as an internal control, nothown), about half of the remaining MNs express the plasmid.

.4. Anatomical evaluation

Finally, we wanted to check if the LBR/IOE procedure can intro-uce structural modifications in the neural tube. For this purposee stained against N-cadherin transversally cut sections (10 �m)

f E4 lumbar spinal cords from LBR/IOE and electroporated-onlymbryos, comparing them with LBR-only and WT embryos asespective controls. In the spinal cord, N-cadherin is localized onhe apical surface of the ventricular zone (Rousso et al., 2012).

We could not detect any evident alteration at the level of theeural tube structure between not electroporated embryos and thelectroporated ones, without or with LBR (Fig. 2B versus F and Dersus H respectively, and insets at higher magnification B′, F′, D′,′).

. Discussion

Many gene mutations are involved in neurodegenerative dis-ases, and in particular the death of motoneurons (MNs) leads tonvalidating or even lethal diseases, such as Charcot-Marie-Tooth

(CMT2) and amyotrophic lateral sclerosis (ALS), and spinal mus-ular atrophy (SMA).

To date, the usage of mammalian transgenic models have rep-esented a precious help to progress in the knowledge of theechanisms leading to such diseases, but we still lack the complete

omprehension of the cellular processes involved and no cure is yetvailable.

It is worth of remembering that other non-mammalian animalodels have also been integrated to the study of MN degenerative

iseases: invertebrates such as the worm (Coenorabditis elegans)Sleigh et al., 2011; Therrien and Parker, 2014) and the fruit flyDrosophila melanogaster) (Wang et al., 2011; Cherry et al., 2013)nd vertebrates (the zebrafish, Danio rerio) (Babin et al., 2014).

In a recent work (Sreedharan et al., 2008), the wild-type geneoding for TAR DNA binding protein 43 (TDP-43) and its twoutations causing ALS were cloned in a pCI-neo vector and were

lectroporated in the neural tube of HH14 chicken embryos. In this

tudy, the authors were able to show that the mutated forms ofDP-43 affect chicken development and induce MNs death.

At present, any study about the genes and the molecular mech-nisms involved in CMT2 and SMA have been performed using the

ce Methods 239 (2015) 206–213 211

chicken embryo as an experimental model, but we strongly believethat future studies should move in this direction.

With the purpose of studying the role of the genes involved inMN degenerative pathologies we developed a method consistingin the combination of two techniques: the LBR and the IOE on thechicken embryo as experimental model, with the aim of inducingcell death specifically in MNs (by LBR) and to overexpress at thesame time a target gene on the dying MNs (by IOE).

Indeed, a similar method has been used to study the anti-apoptotic function of thymosin-� during the period of programmedcell death of MNs in the developing chicken embryo (Choi et al.,2006) and also more recently to evaluate the effects of the mito-chondrial protein Drp1 (Choi et al., 2013) using the same model ofthe previous study. Nevertheless, our method has the character ofnovelty and can be considered as new, since our procedure differsfrom the one previously described in many aspects. First of all, Choiand colleagues follow the method described in 1978 by Chu-Wangand Oppenheim, while we used a different way to remove the limb-bud (see Section 2.2); second, our procedure starts with the LBRand then ends with the IOE while in the method described by Choithe two methods are performed in an inverted order. We decidedfor a different approach because we needed to make a hole intothe chorioallantoic membrane to create an access for the positiveelectrode at the ventral side of the embryo. Consequently, the LBRwould have been almost impossible to do because the Indian inkinjected beneath the embryo would have covered the embryo itselfin the attempt of removing the limb-bud. Third, we used a diagonalconfiguration for the electrodes, placing the negative pole at thedorsal side and the positive on the ventral with the aim of increas-ing the percentage of electroporated MNs, while in the other studythe electrodes were positioned parallel to the sides of the embryo.Indeed, we constantly obtained almost the 50% of MN electropora-tion efficiency while Choi refers an efficiency varying from 20% to50%. Fourth, the plasmidic concentration we used was higher thanthe one used by the group of Choi (5–7 �g/�L against 1–2 �g/�L),while the electroporation parameters are very similar (5 pulses,30 V, 50 ms duration, 100 ms interval against 5 pulses, 25 V, 50 msduration, 950 ms interval). No data about survival and good mor-phology rate is reported in those papers, but that was not the aimof the works published by Choi and colleagues.

Our method introduces an improvement regarding the combi-nation of LBR with IOE and can be considered as a new effectivemethod to electroporate MNs in conditions of trophic factors depri-vation. The high efficiency of IOE, resulting in about the 50% ofMNs expressing GFP, will allow functional studies of specific genesinvolved in MNs survival and cell death, as well as genes implied inhuman pathologies primary affecting the MNs, such as CMT2, ALSand SMA.

Importantly, in the case of ALS, it is known that not only MNs butalso glial cells (Haidet-Phillips et al., 2011) can play a role into itsprogression while there are strong insights about an involvementof interneuron dysfunction (Turner and Kiernan, 2012).

We therefore suggest that the electroporation of additional celltypes in the developing spinal cord could better reproduce thepathology phenotype in the case of ALS-related genes, creating amore physiological model for studying the effects of other cellu-lar types, compared to the MN-only electroporation condition (seealso Section 5.1).

Interesting possibilities could also be offered by two very impor-tant technologies developed to better dissect gene expression: thefirst is the usage of systems of inducible gene expression, such as theTet-On/Tet-Off system (Gossen and Bujard, 1992), to activate the

WT/mutated gene only in a specific time-frame (Watanabe et al.,2007; Yokota et al., 2011); the second are the optogenetic devices,to manipulate the cellular activity in ovo (Sharp and Fromherz,2011; Egawa et al., 2013; Kastanenka and Landmesser, 2013), since

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t is well known that the MN cellular activity during the period ofrogrammed cell death can influence their survival (Oppenheimt al., 2000, 2003).

. Conclusion

The method of combined limb-bud removal and in ovo electro-oration (LBR/IOE) we developed opens the possibility of studying

n vivo the function of genes involved in MN survival and cell deathn the short (E4) and long (E6) term.

The chicken embryo is an animal model easy to handle ando obtain and its usage does not require any ethical committeepproval, making of it a valid model for screening genes involved inN survival, development and death, in view of additional studies

n mammalian models of diseases caused by MN degeneration.

.1. Perspectives

We envisage further developments for this technique, such ashe co-electroporation at the side of LBR of two different constructsarrying two distinguishable reporter genes (e.g. one with GFP andhe other with DsRed) and coding, for example, for a WT gene withts mutated form, two different mutations on the same gene or twoifferent genes related each other, for protein–protein interactiontudies.

Double IOE, one on the side with LBR and the other on the sideithout lesion could also be an option to simultaneously study the

ame gene in two different conditions on the same animal, reducingrastically the inter-experimental variability.

In addition, the use of plasmidic vectors carrying cell type-pecific promoters (such as HB9 for MNs or BLBP for radial glia)ould improve not only the transfection efficiency, but also the phe-otype observed on the electroporated embryos, especially in thease of ALS-related genes.

cknowledgements

The authors would like to thank Ms. Ute Baur and Ms. Lidiaoschny for their precious technical help for molecular biologynd histology, respectively and Sagar for providing advices for thesage of the electroporation device. We are grateful to the Deutscheorschungsgemeinschaft (DFG; Kr1477/11-3) for the financial sup-ort given to the project.

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