Glial-dependent clustering of voltage-gated ion channels in Drosophila precedes myelin formation

  1. Simone Rey
  2. Henrike Ohm
  3. Frederieke Moschref
  4. Dagmar Zeuschner
  5. Marit Praetz
  6. Christian Klämbt  Is a corresponding author
  1. Institut für Neuro- und Verhaltensbiologie; Röntgenstraße, Germany
  2. Max-Planck Institut für molekulare Biomedizin; Wissenschaftliches Service-Labor für Elektronenmikroskopie; Röntgenstraße, Germany
9 figures, 1 table and 1 additional file


Figure 1 with 1 supplement
Localization of Paralytic (Para) voltage-gated ion channels in the larval nervous system.

(A) Schematic view on the para gene. Alternative splicing at the circled exons results in the generation of more than 60 Para isoforms. All isoforms share a common N-terminus. Here, the MiMIC insertion MI08578 allows tagging of the endogenous para gene. The peptide sequence AEHEKQKELERKRAEGE (positions 33–49) that was used for immunization is indicated by a green star, the MiMIC insertion is indicated by a magenta star. (B) Homozygous paramCherry, paraApex2 or paraST76 flies were tested for temperature-induced paralysis. The recovery time is indicated. (C) Third instar larval paramCherry nervous system stained for Cherry localization. ParamCherry is detected in the ventral nerve cord (vnc) and diffusely along peripheral nerves (arrows). (D,D’) Affinity purified anti-Para antibodies detect a protein in the CNS of dissected 24-hr-old wild type first instar larvae. (E,E’) No protein is found in the CNS of dissected age-matched para mutant animals. (F) Third instar larval nervous system stained with the pre-immune control. (G) Third instar larval nervous system stained with affinity purified anti-Para antibodies. Scale bars are as indicated.

Figure 1—figure supplement 1
Ensheathing- / wrapping glia in Drosophila and characterization of Para protein.

(A–C) Schematic representation of the larval (A,B) and the adult Drosophila nervous system (C). The ensheathing glia is labeled in blue, the ensheathing/wrapping glia is labeled in green, the wrapping glia is shown in red. The tract glia of the adult nervous system is shown in green and red stripes. The tract glia likely corresponds to the ensheathing/wrapping glia but the exact lineage relationship is not known. (D) Western blot of protein lysates of adult heads. Purified anti-Paralytic (Para) antibodies detect a band of 105 kDa and a band of >250 kDa in size. The size of the >250 kDa protein band increases in paramCherry heads compared to wild type control as well as paraMiMIC heads, indicating that this band corresponds to the Para protein. Note that elevated levels of the endogenous Para::mCherry fusion protein are detected. Anti-dsRed antibodies detect only the ParamCherry fusion protein.

Figure 1—figure supplement 1—source data 1

Four images of western blots, with and without marker bands, are provided.
Differential localization of voltage-gated ion channels in Drosophila.

(A) Third instar larvae with the genotype [paramCherry; OK371-Gal4, UAS-mCherrydsRNA]. paramCherry expression is suppressed in all glutamatergic neurons and thus, ParamCherry localization along axons of cholinergic sensory neurons becomes visible. (B) Third instar larvae with the genotype [paramCherry; Chat-Gal4, UAS-mCherrydsRNA]. Here, expression of paramCherry is suppressed in all cholinergic neurons which reveals Paralytic (Para) localization in motor neurons. Note the prominent Para localization at the CNS/PNS transition point (arrowheads). (C) Third instar larval shakerGFP nervous system stained for GFP localization. Shaker is found in the neuropil (dashed areas). (D) Third instar larval shabGFP nervous system stained for GFP localization. Shab is distributed evenly along all peripheral axons. (E) Third instar larval shalGFP nervous system stained for GFP localization. Shal localizes similar as Para on motor axons. Scale bars are 100 µm. (F) Adult paramCherry ventral nerve cord stained for Para localization. ParamCherry localizes prominently along segments of peripheral nerves (arrow) as they enter thoracic neuromeres. Note that some axons entering the CNS neuropil show only a weak Para signal (open arrowhead). (G) Control (Oregon R) adult ventral nerve cord stained for Para protein localization using purified anti-Para antibodies. Note the differential localization of Para along axons entering the nerve (arrow, open arrowhead). (H) Ventral nerve cord of an adult fly with the genotype [paraFlpTag-GFP; Ok371-Gal4, UAS-flp]. The boxed area is shown enlarged in (H’). The arrowheads point to high density of Para. (I) Ventral nerve cord of an adult fly with the genotype [paraFlpTag-GFP; Chat-Gal4, UAS-flp]. The boxed area is shown enlarged in (H’). Note that Para localization is reduced as soon as axons enter the neuropil (arrows). Scale bars are as indicated.

Clustered localization of Paralytic (Para) along motor axons.

(A) High-resolution Airyscan analysis of ParamCherry and (B) HRP localization in an adult nerve. The boxed area is shown in higher magnification below (A’,B’). Note the clustered appearance of ParamCherry, clusters are about 0.6–0.8 µm apart (arrowheads). (C,C’) Primary wild type neural cells cultured for 7 days stained for Repo (magenta) to label glial nuclei, HRP (cyan) to label neuronal cell membranes and anti-Para antibodies (green). The Para protein localizes in a dotted fashion. (D) Ventral nerve cord of a third instar larva with the genotype [paraFlpTag-GFP; 94G06-Gal4, UAS-flp]. The arrow points to a single neuronal cell body found in every hemineuromer. (E) Higher magnification of single ParaGFP expressing axons. Note the dotted arrangement of ParaGFP along motor axons (arrowheads). (F,F’) Ventral nerve cord of a third instar larva with the genotype [paraFlpTag-GFP; 94G06-Gal4, UAS-flp] imaged with super-resolution. The dashed box is shown in high magnification in (F’). Arrows point to clusters of Para protein. Scale bars are as indicated. (G) Quantification of Para cluster distance using super-resolution imaging (ParaFlpTag::GFP, average distance is 620 nm, n=91 clusters on 3 axons, 2 larvae) or electron microscopy (ParaApex2, average distance is 706 nm, n=64 clusters on 8 axons 4 animals, Mann-Whitney U-test, p=0.0747, two-tailed). Scale bars are as indicated.

Localization of Paralytic (Para) along sensory axons.

(A,A’) Ventral pickpocket expressing sensory neuron (v’ada) of a third instar larva with the genotype [paraFlpTag-GFP; ppk-Gal4, UAS-flp, UAS-tdTomato] stained for GFP (green), HRP (magenta), and tdTomato (white). The dashed boxes are shown in higher magnification in (B,C). The asterisk denotes the position of the neuronal cell soma. The filled arrows indicate localized Para along some of the dendritic processes. The open arrowhead points to a dendritic process lacking Para localization. Note that Para localization along the descending axon becomes prominent only after about 50 µm (open arrow). (B,B’) Magnification of the neuronal soma attached dendrites. (C,C’) Descending axon of the v’ada neuron. Note that the strong Para signal starts 50 µm distal to the cell soma and fades out after 100 µm (open arrows). Scale bars are as indicated.

A glial lacunar system surrounds the axon initial segment.

(A) Weak Paralytic (Para) expression can be detected on paraApex2 expressing small axons (arrowheads) running in fascicles within the nerve. (B–D) Cross sections through the same axon at various positions. Distance between individual sections (B,C) is 15 µm, distance between (C,D) is 6.5 µm. Note the intense labeling of the axonal membrane is changing between the different sections. (E) Cross section, to determine the staining intensity along the membrane (below the blue line), a corresponding ROI was defined and (F) quantified using Fiji. (G) Surface plot of ParaApex2 distribution along 16 consecutive axonal cross sections. For details, see Materials and methods. The intensity of diaminobenzidine (DAB) precipitates is transformed to different colors. Note that Para clusters are organized in two longitudinal lines across the axonal membrane surface. (H) Longitudinal section of a paraApex2 expressing axon. The staining intensity along the membrane (above the blue line) was quantified using Fiji. (I) Staining intensity of the membrane stretch shown in (H). Note the regular increase in staining intensity every 0.6–0.8 µm. For quantification see Figure 3G. Scale bars are as indicated.

Figure 6 with 1 supplement
Organization of the lacuna forming tract glial.

(A,B) Apex2 expression directed by 75H03-Gal4. Axons (asterisks) are engulfed by lacunar structures that are largely formed by the tract glia. (C) Maximum projection of a confocal image stack. 75H03-Gal4 directed expression of GFP labels the ensheathing/wrapping or tract glia. Note that GFP expression ends proximal to the dissection cut (white dashed circles). (D–F) multicolor flipout (MCFO2) analysis of the nrv2-Gal4, R90C03-Gal80 positive wrapping glia. Note that glial cells tile the nerve roots with no gaps in between. Scale bars are as indicated.

Figure 6—figure supplement 1
Glial cell types involved in lacuna formation.

(A–C) The tract glial cells as defined by 75H03-Gal4 UAS-tdTomato activity also express the CNS ensheathing glia marker 83E12-LexA LexAop-CD8::GFP. (D) The PNS wrapping glia marker nrv2-Gal4 90C03-Gal80 UAS-mCherry labels cells that overlap in their expression domain with the tract glial cells. HRP (blue) labels neuronal membranes. Scale bar is 100 µm. (E) Schematic summary of central and peripheral wrapping glial cells in Drosophila. The neuropil is covered by the ensheathing glia. The peripheral axons are wrapped by the peripheral wrapping glia. The 75H03-Gal4 positive glial cells are located in between these two glial cell populations.

Figure 7 with 5 supplements
Drosophila wrapping glia form myelin.

(A) Drosophila leg of a 3-week-old fly with wrapping glial nuclei in green, the cuticle is stained by autofluorescence, the genotype is [nrv2-Gal4, UAS-lamGFP]. (B–K) Electron microscopic images of sections taken from 3-week-old female flies. (B) Section at the level of the femur. (C) Electron microscopic section at the level of the coxa. In some areas, an increased amount of glial membranes can be detected close to large caliber axons (box with white dashed lines, enlarged as an inlay). (D,E,G) Cross sections through a 2 weeks adult leg of a fly with the genotype [75H03-Gal4, UAS-Myr-Flag-Apex2-NES]. Glial cell processes are stained by the presence of Apex2 which generates an osmiophilic diaminobenzidine (DAB) precipitate. (D) Small caliber axons (ax) are engulfed by a single glial process as fascicle. Larger axons are individually wrapped (asterisk). (E) Large caliber axons are surrounded by glial membrane stacks. The asterisk denotes an axon engulfed by a few glial wraps (red dots). (F) Up to 15 densely packed membrane sheets are found (see inlay for enlargement). (G) Darkly stained tract glia membrane stacks (black arrowhead) can be found next to unlabeled membrane stacks (white arrowhead), suggesting that myelin-like structures can be derived from both, central and peripheral wrapping glial cells. (H) High-pressure freezing preparation showing a single axon covered by myelin-like membrane sheets in a lacunar area (asterisks). (I) Note the bulged appearance of the growing tip of the glial cell processes that form the myelin-like structures (arrowheads). The inlay shows a highly organized membrane stacking. (J,K) High-pressure freezing preparation of prefixed samples to reduce tissue preparation artifacts. Note the compact formation of membrane layers. The white dashed area is shown in (K). Scale bars are as indicated.

Figure 7—figure supplement 1
Count of axons in Drosophila leg nerves and measurement of glial cell process width.

(A) About 760 axons innervate the leg. The majority is smaller than 0.5 μm in diameter, very few ones are larger than 2 μm. (B) The width of glial cell processes is about 28 nm and very regular.

Figure 7—figure supplement 2
Extent of the lacunar system.

(A) CNS/PNS boundary of the mesothoracic neuromere of an adult ventral nerve cord with the genotype [75H03-Gal4, UAS-tdTomato, 83E12-lexA, lexAop-CD8GFP] imaged for tdTomato, GFP, and HRP expression. (B) Schematic representation of the image shown in (A) with the position of the lacunar region indicated. (C–F) Examples of a serial section series taken every 5 µm for 40–65 µm. The green shading indicates the lacunar region. The numbers in circles show relative distances to the first distal section with lacunar structures. Scale bars are as indicated.

Figure 7—figure supplement 3
Quantification of myelin distribution in the leg nerve.

(A–I) Examples of myelin-like structures of the leg nerve. (A) Myelin-like sheets can be found separating the lacunar region from small caliber axons. (B) Myelin-like sheets separate large caliber axons from small caliber axons. (C) Myelin-like sheets can be found toward the blood-brain barrier. (D) Myelin-like sheets are rarely found in the lacunar region without close contact to axons. (E,F) Myelin-like sheets can partially wrap larger axons. (G–I) Myelin-like sheets can be found with different complexity around single large caliber axons. (J) Quantification of the number of myelin-like stacks detected in a specific section plane (see Figure 3). The value set as 0 corresponds to the distal most point where lacunar structures were detected. Progression of sections is toward the CNS (proximal). (K) Quantification of the number of axons contacting the myelin-like stacks.

Figure 7—figure supplement 4
Multilayered myelin-like structures are formed around single axons in the adult nervous system.

(A) Loosely wrapped glial membranes around one single axon (asterisk). The spacing of the glial membranes resembles the glial lacunae. (B) Wrapping around a single axon. The green shaded glial cell process wraps spirally around the central axon. The ends are denoted by the asterisk and the circle. (C) Simple wrapping around single axons. The shading indicates the different glial cell types present in the nerve: wrapping glia, WG; perineurial glia, PG; subperineurial glia SPG. (D) Tight wrapping around a single axon. Unlike the image shown in (A) a close apposition of glial membranes is noted. Scale bars are as indicated.

Figure 7—figure supplement 5
Formation of myelin-like structures in the adult CNS of Drosophila.

(A) High-pressure freezing preparation. Spiral growth of a glial cell process. (B) Membrane stack formed by a wrapping glial cell. Note the bulbed growing tips of the glial membrane sheets (arrowheads). (C–F) Myelin-like membrane sheets can be connected by comb-like structures. (C) Overview of a multilayered membrane stack around several axons, red shading highlights some of the glial membrane sheets. The arrowhead indicates a bulb structure at the end of the glial membrane sheet (D). In some cases, the ends of the membrane sheets are connected by comb-like structures (asterisks) (E). Growing tip of a wrapping glial cell process that navigated around an axon (ax) (F). Scale bars are as indicated.

Figure 8 with 2 supplements
Localization of the voltage-gated sodium channel depends on glia.

(A,A’) Third instar larval filet preparation with the genotype [nrv2-Gal4, UAS-CD8-GFP; R90C03-Gal80] showing the localization of Paralytic (Para) as detected using the anti-Para antibody in a control larva. (B,B’) Third instar larval filet preparation with the genotype [nrv2-Gal4, UAS-hid; R90C03-Gal80] showing the localization of Para as detected using the anti-Para antibody in a wrapping glia ablated larva. The white dashed boxes were used for quantification of Para fluorescence intensity in the CNS/PNS transition zone in relation to its expression in the muscle field area. The yellow boxed areas are shown in higher magnification (A’,B’). Note the increased localization of Para along the peripheral nerve at the level of the muscle field (asterisks). Scale bars are as indicated. (C) Quantification of Para fluorescence intensity in the CNS/PNS transition area and the muscle field area in control and wrapping glia ablated larvae (n=10 larval filets, 3 nerves/filet). To exclude a possible influence seen in individual animals, the average fluorescence intensities along nerves of each individual were compared. Note, Para distributes more evenly along the axon in the absence of wrapping glia (p=0,0003; Mann-Whitney U-test). (D) Quantification of para mRNA expression using qRT-PCR in control and wrapping glia ablated larvae (n=7, with 15–20 brains each). para ct-values were normalized to ct-values of control gene, RPL32. Note, the significant increase in para mRNA expression upon wrapping glia ablation (p=0,0006, Mann-Whitney U-test). Scale bars are as indicated.

Figure 8—figure supplement 1
Glia ablation does not affect the localization of the voltage-gated potassium channel Shal.

(A) Control larva with an endogenously tagged Shal potassium channel. Shal predominantly localizes to the axon initial segment. (B) Upon ablation of the ensheathing glia, no change in Shal localization is detected. (C) Control larva. The inlay shows co-staining for wrapping glial cell processes (magenta) and HRP to detect neuronal membranes. (D) Upon ablation of wrapping glia, no change in Shal localization is detected. Scale bars are as indicated.

Figure 8—figure supplement 2
Ablation of central ensheathing glia does not affect positioning of Paralytic (Para) at the axon initial segment (AIS).

CNS preparations of third instar larvae of the genotypes indicated are shown. (A) Control larva, expressing CD8GFP under the control of the split Gal4 driver [83E12-Gal4AD, repo-Gal4DBD, UAS-CD8GFP] specific for ensheathing glial cells stained for Para protein expression. (B) Upon ablation of the ensheathing glia following expression of the proapoptotic gene hid, no change in the Para expression levels is detected. (C) Quantification of the Para fluorescence intensity in control and ensheathing glia ablated larvae (n=5 larval brains, 10–16 nerves/brain). To exclude influence of individual animals 10–16 nerves per individual were measured and analysis was performed on the mean fluorescence intensity of all values from one animal (p=0,8016, Mann-Whitney U-test). (D) Control larvae for ensheathing glia ablation using the FlpTag approach. The GFP encoding exon was flipped in all motor neurons using [vGlut-lexA, lexAop-Flp]. Note, the pronounced localization of ParaGFP at the AIS-like domain of the nerve. (E) Upon ablation of the ensheathing glial cells, no change in Para localization in motor axons can be detected. (F–H) Filet preparations of third instar larvae stained for Para localization. Control larva (F). Upon expression of activated FGF-receptor Heartless no change in Para localization is noted (G). Upon expression of dominant negative Heartless, no change in Para localization is noted (H). Scale bars are as indicated.

Organization of the axon initial segment (AIS) in Drosophila motor axons.

Voltage-gated sodium channels are preferentially positioned at the AIS of the motor axon. (A) In the larval nervous system positioning is mediated by the peripheral wrapping glia. (B) In adults these cells form myelin-like structures, which fray out in the lacunae which represent a reservoir possibly needed for ion homeostasis during sustained action potential generation.


Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)y[1] w[*] Mi{MIC}MI08578a Mi{MIC}MI08578bBloomington Drosophila Stock CenterBDSC51087
Genetic reagent (Drosophila melanogaster)y[1] w[*] Mi{FlpStop}para[MI08578-FlpStop.D]/FM7cBloomington Drosophila Stock CenterBDSC67680
Genetic reagent (Drosophila melanogaster)Para-mCherryThis studyFigures 1, 2
Genetic reagent (Drosophila melanogaster)Para-ApexThis studyFigures 1, 5
Genetic reagent (Drosophila melanogaster)Para-FlpTag GFP/Fm7iFendl et al., 2020
Genetic reagent (Drosophila melanogaster)y[1] w[*]; Mi{PT-GFSTF.1}Shal[MI00446-GFSTF.1]Bloomington Drosophila Stock CenterBDSC60149
Genetic reagent (Drosophila melanogaster)y[1] w[*] Mi{PT-GFSTF.2}Sh[MI10885-GFSTF.2]/FM7j, B[1]Bloomington Drosophila Stock CenterBDSC59423
Genetic reagent (Drosophila melanogaster)y[1] w[*]; Mi{PT-GFSTF.1}Shab[MI00848-GFSTF.1]/TM6C, Sb[1] Tb[1]Bloomington Drosophila Stock CenterBDSC60514
Genetic reagent (Drosophila melanogaster)y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=VALIUM20-mCherry}attP2Bloomington Drosophila Stock CenterBDSC35785
Genetic reagent (Drosophila melanogaster)UAS-CD8GFP II; R90C03Gal80 IIIKottmeier et al., 2020
Genetic reagent (Drosophila melanogaster)UAS-Hid/CyOw; R90C03Gal80 IIIKottmeier et al., 2020
Genetic reagent (Drosophila melanogaster)UAS-lacZ NLS IIY. Hirmoi
Genetic reagent (Drosophila melanogaster)UAS-lambda-HtlGisselbrecht et al., 1996
Genetic reagent (Drosophila melanogaster)UAS-htlDN IIBloomington Drosophila Stock CenterBDSC5366
Genetic reagent (Drosophila melanogaster)UAS-CD8GFP IIBloomington Drosophila Stock CenterBDSC5137
Genetic reagent (Drosophila melanogaster)UAS-CD8mCherry IIBloomington Drosophila Stock CenterBDSC 27391
Genetic reagent (Drosophila melanogaster)UAS-Hid IIIgaki et al., 2000
Genetic reagent (Drosophila melanogaster)UAS-FlpBloomington Drosophila Stock CenterBDSC 4539
Genetic reagent (Drosophila melanogaster)UAS-Myr-Flag-APEX2-NES86Fb IIIThis studyFigures 6, 7
Genetic reagent (Drosophila melanogaster)pBPhsFLP2::pEST/I;; UAS HA, FLAG, V5, OLLAS/IIINern et al., 2015
Genetic reagent (Drosophila melanogaster)ChAT-Gal4Salvaterra and Kitamoto, 2001
Genetic reagent (Drosophila melanogaster)OK371-Gal4Mahr and Aberle, 2006
Genetic reagent (Drosophila melanogaster)GMR94G06Gal4 IIIBloomington Drosophila Stock CenterBDSC40701
Genetic reagent (Drosophila melanogaster)Nrv2Gal4 IISun et al., 1999
Genetic reagent (Drosophila melanogaster)Nrv2Gal4 II; R90C03Gal80 IIIKottmeier et al., 2020
Genetic reagent (Drosophila melanogaster)Nrv2Gal4 II; R90C03Gal80, UAS-CD8Cherry IIIKottmeier et al., 2020
Genetic reagent (Drosophila melanogaster)GMR83E12_AD II; Repo4.3_DBD IIIBittern et al., 2021
Genetic reagent (Drosophila melanogaster)GMR75H03-Gal4 IIIBloomington Drosophila Stock CenterBDSC39908
Genetic reagent (Drosophila melanogaster)ppkGal4, AUS-tdTomato IIIHerzmann et al., 2017
Genetic reagent (Drosophila melanogaster)lexAop-Flp IIIBloomington Drosophila Stock CenterBDSC55819
Genetic reagent (Drosophila melanogaster)w[*]; TI{2A-lexA::GAD}VGlut[2A-lexA]/CyOBloomington Drosophila Stock CenterBDSC84442
Genetic reagent (Drosophila melanogaster)ParaST76Bloomington Drosophila Stock CenterBDSC26701
Genetic reagent (Drosophila melanogaster)Oregon RBloomington Drosophila Stock CenterBDSC5
AntibodyAnti-Para N-term (rabbit, polyclonal)This studyIF (1:1000), WB (1:1000) 
Figures 1, 2
AntibodyAnti-dsRed (rabbit, polyclonal)TakaraCat#632496
IF (1:1000)
AntibodyAnti-GFP (chicken, polyclonal)AbcamCat#ab13970
AntibodyAnti-GFP (rabbit, polyclonal)InvitrogenCat#A-11122
AntibodyAnti-Rumpel (rabbit, polyclonal)Yildirim et al., 2022IF(1:1000)
AntibodyAnti-Repo (mouse, monoclonal)Hybridoma BankCat#8D12
RRID: AB_528448
AntibodyAnti-V5 (rabbit, polyclonal)Sigma-AldrichCat#V8137-.2MG
AntibodyAnti-HA (mouse, monoclonal)Covance
AntibodyAnti-Flag (rat, monoclonal)Novus BiologicalsCat#NBP1-06712SS
AntibodyFluoTag-X4 anti-GFP (Alpaca, monoclonal)NanoTag BiotechnologiesCat#N0304
AntibodyFluoTag-X4 anti-RFP (Alpaca, monoclonal)NanoTag BiotechnologiesCat#N0404
AntibodyAnti-rabbit Alexa 488 (goat, polyclonal)Thermo FisherCat#A-11008
AntibodyAnti-rabbit Alexa 568 (goat, polyclonal)Thermo FisherCat#A-11011
AntibodyAnti-chicken Alexa 488 (goat, polyclonal)Thermo FisherCat#A-11039, RRID:AB_2534096IF(1:1000)
AntibodyAnti-mouse Alexa 488 (goat, polyclonal)Thermo FisherCat#A-11001, RRID:AB_2534069IF(1:1000)
AntibodyAnti-HRP Alexa 647 (goat, polyclonal)Thermo FisherCat#123-605-021, RRID:AB_2338967IF(1:500)
AntibodyAnti-rabbit HRP (goat, polyclonal)InvitrogenCat#31460
Recombinant DNA reagentpBS-KS-attB1-2-PT-SA-SD-0-mCherryDrosophila Genome Research CentreDGRC#1299
Recombinant DNA reagentpBS-KS-attB1-2-PT-SA-SD-0-Apex2This studyGeneration of transgenic fly
Sequence-based reagentBamH1 Apex2 fwdThis studyPCR primerAAGGATCCGGAAAGTCTTACCCAACTGT
Sequence-based reagentBamH1 Apex2 revThis studyPCR primerAAGGATCCGGCATCAGCAAACCCAAG
Sequence-based reagentMiLFVenken et al., 2011PCR primerGCGTAAGCTACCTTAATCTCAAGAAGAG
Sequence-based reagentMiLRVenken et al., 2011PCR primerCGCGGCGTAATGTGATTTACTATCATAC
Sequence-based reagentmCherry-Seq fwdPCR primerACGGCGAGTTCATCTACAAG
Sequence-based reagentmCherry-Seq revPCR primerTTCAGCCTCTGCTTGATCTC
Sequence-based reagentApex253_rev1This studyPCR primerAGCTCAAAATAGGGAACTCCG
Sequence-based reagentApex286_fwd1This studyPCR primerTACCAGTTGGCTGGCGTTGTT
Sequence-based reagentPara qPCR PrimerThermo Fisher ScientificCat#4331182
Sequence-based reagentRPL32 qPCR PrimerThermo Fisher ScientificCat#4331182
Commercial assay or kitRNeasy KitQIAGENCat#74104
Commercial assay or kitQuantitect Reverse Transcription KitQIAGENCat#205313
Commercial assay or kitTaqman gene expression assay, Universal Master Mix II, with UNGThermo Fisher ScientificCat#4440038
Software algorithmGraphPad PRISMGraphPad Software, USAVersion 6.0
Software algorithmFiji
Software algorithmZEN SoftwareZeissBlack version
Software algorithmAffinity PhotoSerif (Europe)
Software algorithmMATLABThe MathWorks, Inc
Software algorithmPhotoshop CS6Adobe

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  1. Simone Rey
  2. Henrike Ohm
  3. Frederieke Moschref
  4. Dagmar Zeuschner
  5. Marit Praetz
  6. Christian Klämbt
Glial-dependent clustering of voltage-gated ion channels in Drosophila precedes myelin formation
eLife 12:e85752.