XB-ART-50155
J Comp Neurol
2015 Aug 01;52311:1639-63. doi: 10.1002/cne.23761.
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Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.
Mundell NA
,
Beier KT
,
Pan YA
,
Lapan SW
,
Göz Aytürk D
,
Berezovskii VK
,
Wark AR
,
Drokhlyansky E
,
Bielecki J
,
Born RT
,
Schier AF
,
Cepko CL
.
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Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods.
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???displayArticle.grants??? [+]
R01 NS083848 NINDS NIH HHS , R01 EY024844 NEI NIH HHS , EY023911 NEI NIH HHS , EY024844 NEI NIH HHS , EY12196 NEI NIH HHS , NS083848 NINDS NIH HHS , NS068012 NINDS NIH HHS , P30 EY012196 NEI NIH HHS , HD067140 NICHD NIH HHS , Howard Hughes Medical Institute , EY7145 NEI NIH HHS , AG041582 NIA NIH HHS , EY11379 NEI NIH HHS , HHMI_CEPKO_C Howard Hughes Medical Institute , R01 HD067140 NICHD NIH HHS , F31 NS068012 NINDS NIH HHS , R01 EY011379 NEI NIH HHS , T32 EY007145 NEI NIH HHS , F31 AG041582 NIA NIH HHS , F32 EY023911 NEI NIH HHS
Species referenced: Xenopus
Genes referenced: elavl3
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Figure for Editor: Recombinant VSV vectors can be used to transneuronally trace neurons in axolotls and Xenopus. (A) Schematic of injections into axolotl. (B-D) Recombinant VSV vectors can infect neurons when injected directly into the brain. Arrowheads in B-D indicate the brain of each animal. An animal injected with saline as a control is shown in (B). Note that the eyes are autofluorescent. (E-F) Sections of the medial pallidum of axolotl brains infected directly with either (E) rVSV(VSV-G) or (F) rVSV(RABV-G). Many infected cells co-stain with the anti-Tuj1 antibody, suggesting that both vectors can infect neurons. (G-H) A parasaggital image of an axolotl brain shows evidence of viral infection in the optic tectum (OT) (G). 100 nL of 1 x 1010 ffu/mL rVSV(VSV-G) were injected into the right eye, animals were sacrificed at 6 dpi (G) or 3 dpi (H). Infected cells and processes were observed in multiple areas in the brain, including within cells of the OT (white arrow in H) and in RGC axons (yellow arrowhead in G). (I) Schematic of injections into Xenopus embryos. Animals were injected with 100 nL of 1 x 1010 ffu/mL rVSV(VSV-G) into the right eye at 3 dpf, and allowed to incubate for 3 days. (J) As seen in zebrafish, virally labeled cell bodies could be observed in the contralateral OT. Scale bars: B-D = 3 mm; E, F, J = 50 μm; H= 7 μm. | |
Figure 1. rVSV(VSVâG) transmission shows an anterograde, and rVSV(RABVâG) shows a retrograde, polysynaptic pattern of transmission among neurons in mice. (AâH) rVSV(VSVâG) was injected into the dorsal striatum (DS) (AâD) or primary motor cortex (EâH), and animals were sacrificed 3 dpi. (A) Schematic of regions expected to be labeled by anterograde transsynaptic transmission (blue) of a virus from a DS injection (injection needle, green). (BâD) At 3 dpi, DS injections of rVSV(VSVâG) resulted in patterns of infection consistent with anterograde transsynaptic transmission. Infected cells were observed near the injection site in the DS (white arrowhead), as well as in the GPe (red arrowhead), SNr (pink arrowhead), and the thalamus. The cortex, which projects to the DS, was not labeled (brown arrowhead). Higherâmagnification images of neurons in the DS (C) and GPe (D) are shown. (E) Schematic of regions expected to be labeled by anterograde transsynaptic transmission (blue) of a virus from a primary motor cortex injection (injection needle, green). (FâH) Injection of rVSV(VSVâG) into the primary motor cortex labeled cells locally in the cortex (brown arrowhead), and the same regions as a direct DS injection, including the DS (white arrowhead), GPe (red arrowhead), STN (yellow arrowhead), SNr (pink arrowhead), and thalamus (purple arrowhead). Highâmagnification images of DS neurons (G), and neurons in the GPe (H) are provided. (I) Schematic of regions expected to be labeled by initial infection by retrograde uptake, and/or by retrograde transsynaptic transmission (orange), with the nucleus basalis (NB) predicted to be labeled only by retrograde transmission (magenta) of rVSV(RABVâG) from a DS injection (injection needle, green). (JâL) rVSV(RABVâG) was injected into the DS. At 3 dpi, this injection resulted in infected cells in retrograde targets, including local infection in the DS (white arrowhead), cortex (brown arrowhead), NB (red arrowhead), and the thalamus (purple arrowhead). High magnifications of a cortical neuron (K), and NB neurons (L) are shown. (M) Schematic of regions expected to be labeled by initial infection by retrograde uptake, and/or by transsynaptic transmission (orange) of a retrograde virus from a primary motor cortex injection (injection needle, green). (NâP) Injections of rVSV(RABVâG) into the primary motor cortex labeled neurons at the injection site in the cortex (brown arrowhead), as well as the NB (red arrowhead) and thalamus (purple arrowhead), but not the DS. Higher magnification of a cortical neuron (O) and thalamic neurons (P) are shown. Multiple types of neurons, including glutamatergic (e.g., cortical pyramidal neurons, panels K,O), GABAergic (DS medium spiny neurons, panels C,G), and cholinergic (NB neurons, panel L), were labeled. DSâ=âdorsal striatum, Thâ=âthalamus, STNâ=âsubthalamic nucleus, GPeâ=âglobus pallidus external segment, SNrâ=âsubstantia nigra pars reticulata, NBâ=ânucleus basalis. Scale barsâ=â1 mm in B,F,J,N; 50 μm in C,D,G,H,K,L,O,P. | |
Figure 2. A variety of cell lines from different organisms were infectable with VSV vectors. Cells were infected with either rVSV(VSVâG) or rVSV(RABVâG) and assayed for Venus or GFP fluorescence at 1 dpi. Chamber slides of (AâC) human 293T cells, (DâF) Vero monkey cells, (GâI) dog MDCK cells, (JâL) mouse NIH 3T3 cells, (MâO) hamster BSR cells, (PâR) chick DF1 cells, (SâU) salamander AL1 cells, or (VâX) Drosophila S2R cells were infected with 1 μL of 1âÃâ105 ffu/mL of either rVSV(VSVâG) (B,E,H,K,N,Q,T,W), or rVSV(RABVâG) (C,F,I,L,O,R,U,X). The virus used to infect is indicated above the panels. Negative controls (A,D,G,J,M,P,S,V) were not exposed to virus. Scale bars = 50 μm. | |
Figure 3. In ovo rVSV(VSVâG) infection of the chicken eye shows an anterograde transsynaptic pattern of spread. (AâD) Injection of rVSV(VSVâG) into the left eye of E14 chicken embryos resulted in viral gene expression in the retina (A,B). Transverse section through an infected retina at 48 hpi showing a column of labeled photoreceptor cells and associated GFPâpositive cells. Labeled cells were found in the outer nuclear layer (ONL) and inner nuclear layer (INL), but were absent from RGCs within these columns (C). This columnar transmission pattern was only detected near the injection site. In the posterior area of the retina in C, wholeâmount confocal images show a large number of labeled RGCs with axons directed towards the optic fissure (D). Inset in D shows high magnification of a labeled RGC. (EâE2) Schematic of expected anterograde transsynaptic transmission of rVSV(VSVâG) through the tectofugal (E,E2, red arrows) and thalamofugal (E,E2, blue arrows) visual pathways illustrated in parasagittal (E) and dorsal (E2) views. (FâK) The pattern of virus infection in whole brains at 24 hpi (FâF2) and 48 hpi (IâI2) after unilateral injection of rVSV(VSVâG) into the left eye. Red arrow (I2) indicates fluorescent protein expression in the OT. Sagittal sections at 24 hpi show the pattern of rVSV(VSVâG) transmission within the brain (G). Labeled RGC axons were present in the stratum opticum (SO) of the optic tectum (OT), and sparse labeled cell bodies were detected in the SGFS, IMC (G,G2) and the Rt (H). A few labeled cells were also detected within the GLd (H). At 48 hpi, fluorescent protein expression was greatly increased within primary, secondary, and higher order sites of the visual pathways (J). Higherâmagnification confocal image of the OT with DAPI staining to highlight tectal layers (J2) and detection of viral infection (J3) shows rVSV(VSVâG) transmission to retinorecipient layers of the SGFS and the SGC output layer of the OT (J3). As compared to expression at 24 hpi (H), fluorescent protein expression was greatly increased within the GLd and the Rt at 48 hpi (K). Aâ=âarcopallium, AIâ=âArcopallium intermedium, Eâ=âentopallium, GCLâ=âganglion cell layer, GLdâ=âdorsal lateral geniculate nucleus, GPâ=âglobus pallidus, Hâ=âhyperpallium, Hpâ=âhippocampus, IMCâ=ânucleus isthmi pars magnocellularis, INLâ=âinner nuclear layer (of retina), LStâ=âlateral striatum, Mâ=âmesopallium, Nâ=ânidopallium, OTâ=âoptic tectum, ONLâ=âouter nuclear layer (of retina), Rtâ=ânucleus rotundus, SGCâ=âstratum griseum central (of OT), SGFSâ=âstratum griseum et fibrosum superficiale (of OT), Stâ=âstriatum, SACâ=âstratum album central (of OT), SGPâ=âstratum griseum periventriculare (of OT), SOâ=âstratum opticum (of OT), Tnâ=ânucleus teaniae, VLâ=âventricular layer (of OT). Scale barsâ=â50 μm in C,D; 1 mm in G,J; 300 μm in G2,H,K; and 100 μm in J2,J3. | |
Figure 4. In ovo rVSV(RABVâG) infection of the chicken eye shows a retrograde pattern of spread into the centrifugal visual system. (AâA2) Illustrations of the centrifugal visual system (green arrows) shown in parasagittal (A) and dorsal (A2) views. Centrifugal neurons in the isthmoâoptic nucleus (ION) target amacrine cells in the retina (AâA2, large green arrow) and have collateral axons that transiently project to the OT during development (dashed arrow). Brain regions that provide visual input to the ION include afferents from the OT, mesencephalic reticular formation (MRF), pontine reticular formation (PRF), and the area ventralis of Tsai (AVT).(BâK) GFP expression reveals a retrograde pattern of rVSV(RABVâG) transsynaptic transmission within the brain following infection of the left retina at E14. GFP expression was not detected in the brain at 24 hpi or 48 hpi (BâB2). However, at 72 hpi widespread GFP expression was observed in the forebrain, right medial midbrain and cerebellum (CâC2). Sagittal sections through the right medial brain show rVSV(RABVâG) transmission in regions associated with the centrifugal visual pathway and accessory optic system including the ION (D,E), nBOR (D), brainstem (D, yellow arrowheads) and cerebellum (D,F). GFPâexpressing cells also were detected in several putative retrograde targets that project to the ION (and also receive inputs from nBOR) including the MRF, PRF, and AVT (G). Distal sections through the right (H,J) and left (I,K) brain show GFP expression in the contralateral OT, including in the SGC layer and layers 9â10 of the SGFS (J,K). Aâ=âarcopallium, AVTâ=âarea ventralis of Tsai, Cbâ=âcerebellum, Eâ=âentopallium, GLdâ=âdorsal lateral geniculate nucleus, GPâ=âglobus pallidus, Hâ=âhyperpallium, HAâ=âhyperpallium apicale, Hpâ=âhippocampus, IMCâ=ânucleus isthmi pars magnocellularis, IONâ=âisthmoâoptic nucleus, LHNâ=âlateral hypothalamic nucleus (visual suprachiasmatic nucleus), LStâ=âlateral striatum, Mâ=âmesopallium, MLdâ=âmesencephalicus lateralis pars dorsalis, MRFâ=âmesencephalic reticular formation, nBORâ=ânucleus of the basal optic root, Nâ=ânidopallium, NCâ=ânidopallium caudale, OTâ=âoptic tectum, PRFâ=âpontine reticular formation, Rtâ=ânucleus rotundus, SGCâ=âstratum griseum central (of OT), SGFSâ=âstratum griseum et fibrosum superficiale (of OT), Stâ=âstriatum. Scale barsâ=â1 mm in D,G,H,I; 100 µm in E,F,J,K. | |
Figure 5. Infection with rVSV(RABVâG), but not rVSV(VSVâG), in the chicken optic tectum results in retrograde transmission from the brain to the retina. (AâD2) Injection of rVSV(VSVâG) into the right optic tectum (OT) (arrow) resulted in an anterograde transsynaptic pattern of spread in the brain. Wholeâmount dorsal views of brightfield (A) and GFP expression (A2) in the brain at 48 hpi. Sagittal section showing GFPâpositive cells (B) in the Rt, GLd (inset from a semiâadjacent section), and telencephalon (nidopallium, mesopallium, and hyperpallium) consistent with anterograde labeling from the OT with rVSV(VSVâG). Brightfield (C,D) and fluorescent protein expression (C2,D2) in posterior (C) and anterior (D) views of retinae after rVSV(VSVâG) infection of the OT. GFP expression was not detected in the retina at 48 or 72 hpi (nâ=â7) (CâD2). In 2/7 embryos, GFPâpositive cells were detected in the ciliary body surrounding the lens (D,D2, yellow arrow) but were absent from the retina. (EâI) Injection of rVSV(RABVâG) into the OT resulted in retrograde transmission and GFP expression in the retina. Dorsal views of brightfield and fluorescent (GFP) images (EâE2) of the brain at 72 hpi. Sagittal section showing GFP expression in the brain (F) including in the ION (inset), nBOR, cerebellum, Rt, and telencephalon. At 72 hpi, OT injections with rVSV(RABVâG) resulted in in clusters of GFPâpositive cells throughout the retina (nâ=â3) (GâG2). Confocal zâstack projection showing GFP expression in a RGC (H, red arrow) and an adjacent amacrine cell (H, yellow arrow) in a flatâmount preparation of the left retina. Transverse section through a retina shows GFP expression in several retinal cell types including RGCs (I, red arrow), Müller glia (I, purple arrow), and amacrine cells in the lower half of the INL (I, yellow arrow). GFPâexpressing cells in the INL were only found in locations proximal to GFPâpositive RGCs. Cbâ=âcerebellum, GCL=ganglion cell layer (of retina), GLdâ=âdorsal lateral geniculate nucleus, HAâ=âhyperpallium apicale, Hpâ=âhippocampus, INLâ=âinner nuclear layer (of retina), LStâ=âlateral striatum, Mâ=âmesopallium, nBORâ=ânucleus of the basal optic root, Nâ=ânidopallium, NCâ=ânidopallium caudale, ONLâ=âouter nuclear layer (of retina), Rtâ=ânucleus rotundus. Scale barsâ=â1 mm in B,F; 50 μm in H,I. | |
Figure 6. Transsynaptic labeling of the visual pathway in larval zebrafish infected with rVSV(VSVâG). (A) Diagram of unilateral eye injection. RGC axons (red arrows) project contralaterally to the optic tectum, pretectum, and thalamus. The locations of rVSV(RABVâG)âinfected cells are indicated by green shaded areas. Putative axonal projections from the OT to the pallium, habenula, and cerebellum are labeled as dashed arrows. (BâD) rVSV(VSVâG) infection of the OT. Dorsal (B) and lateral (B2) views of a 24 hpi zebrafish stained for Venus (green), HuC/D (red, panâneuronal marker), and HNK1 (blue, neuropil). Labeled RGC termini and tectal cell bodies can be seen in the right (contralateral) tectum. Boxed area in B is shown at higher magnification in C. Spectrum of colors represents depth from the dorsal surface of the tectum (redâyellow) to the ventral surface (blueâpink) for Venusâlabeled cells. (D) High magnification of contralateral Venusâlabeled tectal cells at 48 hpi, in location similar to C (boxed area). The majority of the labeled tectal neurons had a single process extending medially, consistent with the previously described morphology of retinorecipient neurons (Robles et al., 2011). (E,F) Dorsal views of confocal maximal projections show rVSV(VSVâG) labeling at 24 hpi (E) and 72 hpi (F). Areas delineated by dashed lines are the OT, habenula, and olfactory bulb. Transverse optical sections from each stage are shown in panels below (E2âE4 for 24 hpi and F2âF4 for 72 hpi). (GâI) Dorsal view of the zebrafish brain (3 dpf), stained with HuC (G). Areas delineated by dashed lines are the habenula and cerebellum, which are shown at higher magnification in H and I, respectively. 24 hpi labeling was restricted to the optic tectum, pretectum, and thalamus. At 72 hpi, labeling broadened and included cells in the pallium (F3), habenula (H), and the cerebellum (I). Labeled cells are indicated by white arrows or arrowheads and axons are labeled with yellow arrowheads). Venusâexpressing cells were not present in the olfactory bulb (E4, F4). CB: cerebellum, HABâ=âhabenula, Meâ=âmedulla, OBâ=âolfactory bulb, OTâ=âoptic tectum, Pâ=âpallium, PTâ=âpretectum, Thâ=âthalamus. Scale barsâ=â100 μm in BâB2; 20 μm in C,D; 50 μm EâF4; 20 μm in H,I. | |
Figure 7. Labeling of centrifugal circuits in zebrafish with rVSV(RABVâG). (A,B) Dorsal view of confocal maximal projections showing rVSV(RABVâG) labeling (green, GFP) and HU (magenta, panâneuronal marker) at 24 hpi (A) and 48 hpi (B). GFP channel from (A,B), with signal enhanced to show cells in the brain, is shown in (A2,B2). Images in (BâB2) were captured after dissection of the brain from surrounding tissues. GFPâlabeled axonal projections from the contralateral to the ipsilateral habenula (white arrowhead) and projections from the OT to the cerebellum (yellow arrowheads) are indicated in (BâB2). (C) Illustration showing injection of rVSV(RABVâG) into the eye of 3 dpf larval zebrafish. Centrifugal axons in the terminal nerve (tn) originate from the contralateral olfactory bulb (OB) and ventral pallium (P), and project to target cells in the retina (dark green arrow). The locations of rVSV(RABVâG)âinfected cells after unilateral eye injection is indicated by green shaded areas. Projections from other centrifugal neurons previously reported among teleost fish are indicated by dashed arrows from the thalamus (Th), OT, and pretectum (PT). (DâJ) Transverse optical sections from 24 hpi or 48 hpi are shown. At 24 hpi, labeling is restricted to the RGC axons in the neuropil of OT, and cell bodies in the OB (D), OT (E, 48 hpi shown), pallium (F), thalamus (G), preoptic area (H), habenula (I), medulla (J, 48 hpi shown), and pretectum (not shown). At 48 hpi, additional labeling was seen, including GFPâpositive efferent axons (yellow arrowhead) projecting into the contralateral and ipsilateral cerebellum (J). (KâL2) Confocal maximal projections of GFP expression and HU after injection of 1âÃâ108 ffu/mL (K,L), or 1âÃâ109 ffu/mL (K2,L2) rVSVÎG(RABVâG), at 24â48 hpi. Yellow arrowheads in (KâL2) indicate RGC axons in the neuropil of the OT. In zebrafish injected with the higher dose of rVSVÎG(RABVâG), labeling revealed primary infection in centrifugal neurons. (MâU) Representative transverse optical sections from 24â72 hpi show the results of centrifugal labeling, as sparse GFPâexpressing cells in the OB (M,N), OT (O,P), pretectum (Q), pallium (R), thalamus (S), habenula (T), and medulla (U). GFPâlabeled cells were not detected in the preoptic area (not shown) and labeled axons were not present in the cerebellum (U) following infections with rVSVÎG (RABVâG). CBâ=âcerebellum, HABâ=âhabenula, Meâ=âmedulla, OBâ=âolfactory bulb, OTâ=âoptic tectum, P= pallium, PTâ=âpretectum, POâ=âpreoptic area, tnâ=âterminal nerve, Scale bars:â=â50 μm. | |
Figure 8. rVSVs can infect squirrel monkeys and macaques. (A) Schematic drawing of a squirrel monkey brain, indicating location of the injection sites in primary visual cortex of monkey #1 (dark green), in the motor cortex (light green), and in the somatosensory cortex (red) of monkey #2. Arrows indicate projections originating at the site of the motor cortex injection, small red and green dots represent retrogradely labeled neurons from the sites of somatosensory and visual cortex injections, respectively. The area demarcated by the red oval corresponds to the nucleus basalis of Meynert (NBM) (B) A GFPâexpressing neuron in the visual cortex after injection of rVSV(RABVâG) encoding GFP into the primary visual cortex in monkey #1. This pyramidal neuron is located â¼3 mm dorsal to the injection site and is presumably labeled by retrograde uptake of the primary inoculum. (C,D) Neuronal labeling by presumed retrograde uptake after injection of an mCherryâencoding rVSV(RABVâG) into the primary somatosensory cortex in monkey #2. (C) Labeled pyramidal neuron in cerebral cortex â¼3 mm dorsal to the injection site. (D) Presumptive cholinergic neurons in basal forebrain nucleus of Meynert. (E,F) Results of the injection of rVSV(VSVâG)âexpressing Venus into the primary motor cortex of squirrel monkey #2. (E) Labeled pyramidal neurons and interneurons at the injection site. (F) Venus was seen in the axons originating from the infected neurons at the injection site, projecting away from cortex into the white matter going towards internal capsule. (GâK) Labeling of cortical neurons after rVSV injections into the primary visual area in a macaque monkey. (G) Schematic drawing of a macaque brain showing injection sites of rVSV(VSVâG) (dark green) and rVSV(RABVâG) (red) in the primary visual cortex. Small red and green dots represent location of rVSV(VSVâG) (green) or rVSV(RABVâG) (red) labeled neurons from the sites of somatosensory and visual cortex injections (H,I) Labeled neurons near the site of injection of rVSV(VSVâG) encoding Venus into V1. (J,K) Labeled neurons near the site of injection of rVSV(RABVâG) encoding mCherry into V1. FCâ=âcentral fissure; FSâ=âSylvian fissure; NBMâ=ânucleus basalis of Meynert; STSâ=âsuperior temporal sulcus, V1â=âprimary visual cortex. Scale barsâ=â50 μm in B,C,E,GâJ; 100 μm in D,F. | |
Figure 9. rVSV(VSVâG) can infect seahorse retinae and allows mapping of retinorecipient areas in the brain. (A) Skeletal preparation of a seahorse (Hippocampus erectus) and schematic drawing of a seahorse skull with the brain. Dashed lines indicate approximate brain regions for sections in CâE. (B) Injection of rVSV(VSVâG) into the retina resulted in infection of various retinal cell types (3 dpi). (CâE) Brain sections depicting labeled RGC arbors throughout the seahorse brain, following injection of rVSV(VSVâG) into the retina. C2, D2, E2 are higherâmagnification images of RGC axons in CâE. A small number of infected cell bodies were detected in the brain. Redâ=âGFP, blueâ=âDAPI. PRâ=âphotoreceptors, Mâ=âMüller glia, RGCâ=âretinal ganglion cells. Scale barsâ=â50 μm. | |
Figure 10. Recombinant VSV vectors can infect invertebrate organisms. (A,B) Image of the box jellyfish, Tripedalia cystophora (A). The visual system is comprised of four sensory structures, or rhopalia (white arrow in (A). Image in (B) shows higher magnification of rhopalium in (A). There are six eyes located on each rhopalium, two of which are cameraâtype lens eyes (blue arrow, B). The vitreous space of the lower lens eye was injected with rVSV(VSVâG) (injection site indicated by needle in B) and the infection was monitored for a total of 7 dpi. (C,D) Photoreceptors in all 10 injected animals expressed Venus by 2 dpi. (C) Lateral view of entire rhopalium, with orientation as in (B), shows Venus expression in the photoreceptors of the lower lens eye (C, boxed area). Greenâ=âVenus, blueâ=âDAPI. (D) Higher magnification of boxed region in C showing rVSV(VSVâG) infection in retinal photoreceptors of the everted lens eye (dashed lines). Images taken at 2 dpi. (EâG) Recombinant VSV vectors could also infect Drosophila melanogaster. (E) Uninfected flies were nonfluorescent, while those infected with rVSVâVenus(VSVâG) (F) or rVSVâGFP(RABVâG) (G) showed fluorescent protein expression. Images taken 1 dpi. Scale barsâ=â5 mm in A; 100 μm in B,C; 50 μm in D; 0.5 mm in EâG. |
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