Journal of Neuroscience Methods 85 (1998) 21–26

Drosophila nerve cord culture: a tool for studying neural development

Y.E. Wang a,b,*, R. Chandler a,c, P. Lau a,b, A.J. Bieber a,d

a Department of Biological Sciences Purdue Uni6ersity, West Lafayette, IN 47907, USAb Department of Molecular and Cell Biology, Uni6ersity of California, Berkeley, CA 94720, USA

c Department of Biology, Union College, Barbour6ille, KY 40906, USAd Department of Neurology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA

Received 9 March 1998; received in revised form 8 June 1998; accepted 14 June 1998

Abstract

The culture of explanted neural tissues has been a useful tool for the study of cellular and molecular neurobiology invertebrates. We have developed a technique for the culture of explanted ventral nerve cords from Drosophila embryos. We haveexamined the morphology and dynamic behaviour of the growth cones that extend from these nerve cords, and the effect ofcalcium deprivation on the bundling of axons that are regenerated from the explanted tissue. This technique offers a uniqueopportunity to combine in vitro techniques for neuronal cell culture with the powerful techniques of genetic analysis that areavailable with Drosophila. © 1998 Published by Elsevier Science B.V. All rights reserved.

Keywords: Drosophila ; Neuronal tissue culture; Neurite outgrowth; Growth cone; Microtubule; Calcium; Axon bundling

1. Introduction

In the past decade, Drosophila has become an impor-tant model organism for the study of the cellular andmolecular mechanisms of neural development. The ner-vous systems of insect embryos are relatively simple andthe patterns of cell proliferation, cell migration andaxon outgrowth that occur during embryonic neuraldevelopment in Drosophila have been extensively char-acterized (Goodman and Doe, 1993). This simplicity,combined with the powerful techniques for geneticanalysis that are available with Drosophila, have madeit a valuable experimental system in developmentalneurobiology.

Techniques for the growth and maintenance of disso-ciated neurons or intact neuronal tissues in culture,have been of great value for the cellular analysis ofneuronal function in vertebrates (Cestelli et al., 1992).Although most vertebrate systems lack the anatomical

simplicity and elegant genetics of Drosophila, they oftenhave well developed systems for the culture of neuronsavailable for cellular biology studies. In Drosophila,techniques for the primary culture and differentiationof neuroblasts have been described (Furst and Ma-howald, 1985; Salvaterra et al., 1987) and a techniquehas been developed for the culture of whole dissectedDrosophila embryos (Broadie et al., 1992). Here, wedescribe a method for the culture of explantedDrosophila embryonic nerve cords. This tissue is rela-tively easy to culture on plain glass microscope slides ina simple culture medium.

2. Methods

Drosophila embryos were collected for four hourintervals on apple juice-agar medium (Ashburner,1989a) and allowed to continue development to embry-onic stages 12–15 (Campos-Ortega and Hartenstein,1985). It is during this window of developmental timethat most neurons extend their processes and make

* Corresponding author. Tel.: +1 510 64339961; e-mail: ye-wang@uclink.berkeley.edu

0165-0270/98/$ - see front matter © 1998 Published by Elsevier Science B.V. All rights reserved.PII S0165-0270(98)00107-1

Y.E. Wang et al. / Journal of Neuroscience Methods 85 (1998) 21–2622

connections with their target cells. A mass of embryoswas transferred to a piece of double-stick tape mountedon a microscope slide, and the embryos were rolledwith forceps to mechanically remove the chorion. Then,10–15 dechorionated embryos were aligned in rows ona clear area of the tape and this area was excised witha razor blade and transferred to a culture chamber.Culture chambers consisted of a 4 cm×2 cm×0.5 mmthick piece of adhesive backed magnetized rubber(available at craft stores or from Master Magnetics,Castle Rock) with a 1 cm×3 cm hole cut from itscenter. The adhesive backing was used to apply thisrubber rectangle to a clean glass slide to produce ashallow 1 cm×3 cm well on the slide. The dechorion-ated embryos aligned on the tape were transferred tothe center of the well and covered with phosphatebuffered saline (PBS: 2 mM NaH2PO4, 8 mMNa2HPO4, 170 mM NaCl, pH 7.4). All subsequentmanipulations including, dissection, culture, fixationand antibody staining, were carried out inside the well.The use of thin sheets of adhesive backed magnetizedrubber is a useful and easily modified design for theproduction of chambers for the dissection and cultureof Drosophila tissues.

Embryo dissections were performed essentially asdescribed by Ashburner (Ashburner, 1989b). The em-bryos were covered with PBS, a dorsal incision wasmade in the vitelline membrane with a sharpened tung-sten needle, and the embryo was removed from themembrane and placed ventral side down on the glassslide. In PBS, embryos will stick tightly to a clean glassslide. Using the tungsten needle, an incision was thenmade along the length of the dorsal surface of theembryo and the right and left body walls were presseddown onto the glass. The gut and remaining yolk sacwere then removed, resulting in a flat embryonic prepa-ration similar to that shown in Fig. 1A. Fig. 1A showsa dissected embryo at developmental stage 15, that hasbeen fixed and stained with a monoclonal antibody(BP104, Hortsch et al., 1990) that stains nervous tissue.The brain (Br), ventral nerve cord (VNC), and theaxons and cell bodies of the peripheral nervous system(PNS) are labeled. Embryos of this stage were routinelyselected for explant cultures and gave good results.

A sharpened tungsten needle was used to lift theventral nerve cord out of the dissected embryo, startingat the posterior end and carefully lifting the cord andsevering its connections with the underlying epidermaltissue and with the PNS axons. When the nerve cordhas been dissected to the level of the brain, the brain issevered from the nerve cord and the cord is lifted onthe tip of the needle and moved to a clean area of theslide where it is placed dorsal side down; this orienta-tion is important, as ventral side down nerve cords donot exhibit good neurite outgrowth. Fig. 1B shows aventral nerve cord that was fixed and stained immedi-

ately after excision and before neurite outgrowth hasoccurred. The severed nerve roots of the PNS arevisible along the sides of the ventral nerve cord (arrowsin Fig. 1B), and it is from these stumps that outgrowthof axons will occur. The PBS solution in the chamber isthen replaced with Hepes buffered saline (HBS: 10 mMHepes, 55 mM NaCl, 40 mM KCl, 15 mM MgSO4, 20mM glucose, 50 mM sucrose, pH 6.95) supplementedwith CaCl2 (5 mM), insulin (0.2 IU/ml, Sigma Chemi-cal) and 20-hydroxyecdysone (1 mM from a 10 mMstock in ethanol, Sigma Chemical). The nerve cordswere allowed to develop overnight at room tempera-ture. During this time neurite regeneration and out-growth occurs from the severed ends of the peripheralnerves.

Fig. 1. Drosophila nerve cord culture. (A) A dissected Drosophilaembryo (stage 15) stained with an anti-neuroglian antibody (BP104)to visualize the nervous system. The brain (Br), ventral nervous cord(VNC) and peripheral nervous system (PNS) are visible. (B) A nervecord immediately after explant and prior to neurite regeneration.Arrows indicate the severed stumps of the peripheral nerve roots. (Cand D) Nerve cords after neurite outgrowth either on a Cell-Taksubstrate (C) or on plain glass (D). Scale in A: 60 mm.

Y.E. Wang et al. / Journal of Neuroscience Methods 85 (1998) 21–26 23

3. Results

3.1. Time course and quality of neurite outgrowth

Outgrowth from explanted tissues is visible within 1h and is complete by 12–15 h. Fig. 1C and 1D showtwo examples of nerve cords that have completed neu-rite outgrowth. The magnification of the nerve cords inFig. 1C and 1D are the same as in Fig. 1A, demonstrat-ing that the lengths of the regenerated processes arecomparable to the lengths of the processes in the intactembryonic PNS prior to removal. Initial axon out-growth appears to result from the extension of axonsthat are severed from the original segmental and inter-segmental nerves during the dissection process, thusrepresenting regeneration of these axons. It is possiblethat outgrowth also arises from neurons whose axonsare uncut and from neurons that initiate axonogenesislater in development. The most extensive neurite out-growth was generally observed from nerve cords dis-sected from embryos that were in later stages ofdevelopment.

The cultures in Fig. 1(B–D) are stained with anti-HRP, but most of the neurites extending from thesecultures also stain with the monoclonal antibody 1D4(anti-fasciclin II; data not shown) which stains predom-inantly motor neurons, suggesting that a majority ofthe extending neurites are motor axons.

3.2. Effects of substrate on neurite outgrowth

Neurite outgrowth will occur on untreated glassslides, but is more robust and consistent if the slides arefirst coated with polylysine, and then overlayered with‘Cell-Tak’ cell and tissue adhesive (CollaborativeBiomedical Products). Polylysine (75–100 kD, SigmaChemical) is first spread over clean slides at a concen-tration of 0.2 mg/ml, allowed to stand for 30 min,rinsed three times with distilled water and air dried.Cell-Tak is then applied according to the manufacturersrecommendations. The nerve cord in panel Fig. 1C wascultured on polylysine/Cell-Tak, while the cord in fig.1D was cultured on uncoated glass. Neurite outgrowthis extensive in both preparations. Neurites tend toassociate in large neuronal bundles; this is most pro-nounced in nerve cords cultured on uncoated glass. Inprinciple, any substrate could be tested for its affect onneurite outgrowth including putative components of theextracellular matrix such as laminin and fibronectin, ormonolayers of cells such as Drosophila S2 cells ortransfected cells expressing particular genes of interest.

3.3. Growth cone morphology and beha6ior

The growth cones on regenerating neurites appearsimilar in morphology to those previously described on

Fig. 2. Individual growth cones imaged with antibody labeling andconfocal microscopy. Growth cone plasma membranes were labeledwith anti-HRP antibody (red) and microtubules were labeled withanti-tubulin antibody (green). Scale: 5 mm.

other isolated neurons from both insects and verte-brates (Sabry et al., 1991; Tanaka and Kirschner, 1991).Fig. 2 shows examples of two growth cones that werelabeled with anti-horseradish peroxidase (HRP) anti-body to visualize plasma membrane (red), labeled withanti-tubulin antibody (kindly provided by W.Z. Cande,University of California) to visualize microtubules(green), and then examined with confocal microscopy.On a plain glass substrate, individual growth conesgenerally take a form that exhibits a small number offilopodial projections. Microtubules form a tight bundlewithin the axon, and form packed loops within thecentral domain of the growth cone with single micro-tubules invading the leading edge.

The dynamic behavior of growth cones and theirassociated filopodia can be examined in sequential im-ages of live growth cones on regenerated neurites. Ex-planted nerve cords from stage 15 embryos wereallowed to extend processes for 5 h and then contactlabeled with DiI (1,1%-dihexadecyl-3,3,3%,3%tetramethy-lindocarbocyanine perchlorate; Molecular Probes, Eu-gene, OR), visualized and processed as previously de-

Y.E. Wang et al. / Journal of Neuroscience Methods 85 (1998) 21–2624

scribed (O’Connor et al., 1990). Fig. 3 shows the dy-namic extension and retraction of individual filopodiaover a 39 min period.

3.4. Effects of calcium on neurite outgrowth

Calcium is an important regulatory element for manyneuronal processes including growth cone function andcalcium dependent neural cell adhesion. Campenot andDraker (1989) used a compartmented culture system todemonstrate that neurite outgrowth from sympatheticnerve fibers in culture does not require extracellularcalcium ions. We examined the effects of Ca2+ depriva-tion on the outgrowth of neurites from explanted nervecords.

Fig. 4 shows two nerve cords that were cultured inthe presence of calcium (5mM CaCl2; panel A) or in theabsence of calcium (0 mM CaCl2, 5 mM EGTA; panelB). Both nerve cords were cultured on uncoated glass.Unlike rat sympathetic neurons, which die if the cellbodies are deprived of calcium (Campenot and Draker,1989), the Drosophila nerve cords survive and showextensive neurite outgrowth in the absence of Ca2+.Resistance to calcium deprivation has also been ob-served in amphibian spinal neurons which not onlysurvive in the absence of calcium, but extend neurites(Bixby and Spitzer, 1984) and form at least some

Fig. 4. Effects of calcium on neurite outgrowth and axon bundling.Nerve cords were explanted onto a plain glass substrate in a mediumcontaining either 5 mM calcium (A) or 0 mM calcium, 5 mM EGTA(B). In the absence of calcium, neurites do not form the tight axonalbundles that are seen in the presence of calcium.

Fig. 3. The dynamic behavior of filopodia from Drosophila growthcones can be imaged in culture. This growth cone was labeled withDiI and imaged on a high resolution (6.8 mm pixels) cooled CCDcamera. Time given in minutes.

functional synapses (Henderson et al., 1984). Culturedchick sympathetic neurons have been shown to requireonly trace amounts of calcium for survival and neuriteextension (Wakade et al., 1995).

Although the time course of neurite outgrowth andthe lengths of the extended fibers are largely unaffectedby changes in calcium concentration, the large neuronalbundles that are formed in the presence of Ca2+ areabsent under conditions of calcium deprivation. Fig.4A shows a nerve cord cultured in the presence of 5mM calcium; large bundles of axons extending from thenerve cord are clearly apparent. In the absence ofcalcium, Fig. 4B, large numbers of neurites are ex-tended, but they tend not to bundle, extending individ-ually over the surface of the substrate instead. Verysimilar effects of calcium have been observed on chicksympathetic neurons. In the presence of calcium, theseneurons aggregate in culture and extend thick bundlesof neurites but in the absence of calcium remain assingle cells which extend thin branching neurites(Wakade et al., 1995).

What is the mechanism of the observed effects ofcalcium? Calcium influx has been shown to be animportant element in the directed outgrowth of individ-ual growth cones (Harper et al., 1994) and the observed

Y.E. Wang et al. / Journal of Neuroscience Methods 85 (1998) 21–26 25

effects could be due to its effect on the function ofextending growth cones. However, culture in the pres-ence of calcium channel blockers, such as Co2+ ions(100 mM CoCl2) and verapamil (500 mM verapamilHCl), did not produce this effect (data not shown).Another possible explanation is that calcium ions maybe necessary for calcium dependent cell adhesion func-tions which bind axons together during neurite out-growth. Such calcium dependent adhesion events,mediated by the cadherin and integrin family ofmolecules, have been shown to play roles in axonpathfinding and neurite outgrowth in vertebrates(McKerracher et al., 1996; Redies, 1997). Other reportssupport a role for N-cadherin in axon fasciculation(Drazba and Lemmon, 1990; Honig and Rutishauser,1996). Members of the cadherin and integrin genefamilies have now been identified in Drosophila (Broweret al., 1995; Qwai et al., 1997), and mutations affectingone of these, DN-cadherin, appear to result in defectsin axonal bundling and the directional migration ofgrowth cones (Iwai et al., 1997). A final possibility isthat calcium deprivation of the neuronal cell bodiesresults in more generalized effects on cellular biochem-istry, such as changes in gene expression, which resultin the altered patterns of neurite outgrowth and axonalbundling. Regulation of neuronal transcription in re-sponse to calcium flux has previously been described invertebrates (Morgan and Curran, 1986, 1989).

4. Discussion

We have described a technique for the culture ofexplanted Drosophila nerve cords. Neurites vigorouslyregenerate from explanted embryonic nerve cords andwe have shown that the morphology and behavior ofindividual growth cones can easily be examined. Wehave also demonstrated that calcium has a sign)ficanteffect on the quality of neurite outgrowth and axonalbundling. Many of these neuronal properties have beenstudied in vertebrate neuronal cultures but the directapplication of genetic analysis to such cultures has beenproblematic. It should now be possible to combine thisDrosophila culture technique with Drosophila geneticsto help answer some of the questions raised by thevertebrate studies.

Studies with cultured vertebrate neurons suggest thatcell adhesion molecules, extracellular matrix compo-nents and their receptors, cytoskeletal proteins, calciumchannels, phosphatases and kinases, and many othercellular components, play important roles in generatinggrowth cone structure and function. Genes that areinvolved in many of these functions in Drosophilagrowth cones have now been identified and the mutantphenotypes have been characterized. For example,Drosophila neuroglian, fasciclin II, and fasciclin III are

all members of the immunoglobulin superfamily andtheir embryonic phenotypes suggest that they play rolesin growth cone guidance and target recognition (Linand Goodman, 1994; Chiba et al., 1995; Hall andBieber, 1997). The same is true for the neural receptortyrosine phosphatases DPTP69D, DPTP99A andDLAR (Desai et al., 1997), and for a variety of otherdevelopmentally regulated neural proteins. Althoughmany of these mutations are embryonic lethals, devel-opment proceeds until the nervous system is well estab-lished and well past the point at which explant andculture of the nerve cord becomes feasible. It shouldtherefore be possible to use this culture system to beginto combine neuronal culture technique with geneticanalysis to directly address questions relating to thestructure and function of the proteins expressed bygrowth cones or on the substrates over which theyextend.

Acknowledgements

This work was supported by a grant from the Bur-roughs Wellcome Fund of the Appalachian CollegeAssociation to R.C., by grants from National ScienceFoundation (IBN-9410068) and National Institute ofHealth (NS 09074) to David Bentley, and by grantsfrom the Showalter Foundation and the National Sci-ence Foundation (IBN-9120981) to A.J.B. We alsothank Mr and Mrs Eugene Applebaum for their gener-ous financial support.

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