Visualizing neurons one-by-one in vivo: Optical dissection and reconstruction of neural networks with reversible fluorescent proteins

Authors

  • Shinsuke Aramaki,

    1. Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN, Kobe, Japan
    2. Faculty of Medicine, Kobe University, Kobe, Japan
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  • Kohei Hatta

    Corresponding author
    1. Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN, Kobe, Japan
    • Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, 2-2-3 Minatojima Minami, Chuo-ku, Kobe 650-0047, Japan
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Abstract

A great many axons and dendrites intermingle to fasciculate, creating synapses as well as glomeruli. During live imaging in particular, it is often impossible to distinguish between individual neurons when they are contiguous spatially and labeled in the same fluorescent color. In an attempt to solve this problem, we have taken advantage of Dronpa, a green fluorescent protein whose fluorescence can be erased with strong blue light, and reversibly highlighted with violet or ultraviolet light. We first visualized a neural network with fluorescent Dronpa using the Gal4-UAS system. During the time-lapse imaging of axonal navigation, we erased the Dronpa fluorescence entirely; re-highlighted it in a single neuron anterogradely from the soma or retrogradely from the axon; then repeated this procedure for other single neurons. After collecting images of several individual neurons, we then recombined them in multiple pseudo-colors to reconstruct the network. We have also successfully re-highlighted Dronpa using two-photon excitation microscopy to label individual cells located inside of tissues and were able to demonstrate visualization of a Mauthner neuron extending an axon. These “optical dissection” techniques have the potential to be automated in the future and may provide an effective means to identify gene function in morphogenesis and network formation at the single cell level. Developmental Dynamics 235:2192–2199, 2006. © 2006 Wiley-Liss, Inc.

INTRODUCTION

Discriminating between individual neurons is essential in any attempt to analyze a neural network, as many axons and dendrites intermingle to fasciculate, making synapses as well as glomeruli. During live imaging in particular, it is often impossible to distinguish between individual neurons when they are labeled in the same fluorescent color and are connected or partially overlapped. Using the green-to-red photoconvertible fluorescent protein Kaede (Ando et al., 2002), it is possible to distinguish individual neurons from each other in the living zebrafish (K. Hatta and H. Tsujii, unpublished observation). The neurons expressing Kaede were initially fluorescent green, but by irradiating a single neuron with (ultra-) violet light, we were able to turn the targeted neuron to fluorescent red in color. This change enabled us to distinguish the morphology of a single neuron from other neurons in a complex network. Only a single neuron was distinguished, however, because this conversion is irreversible.

In this study, we have been able to take advantage of Dronpa (Ando et al., 2004), a green fluorescent protein whose fluorescence can be erased with strong blue light, and reversibly highlighted with ultraviolet (UV) or violet light (Fig. 1A). Dronpa is a genetically engineered version of green fluorescent protein cloned from Pectiniidae (Ando et al., 2004). Its fluorescence can be switched on and off by using two different wavelengths of light. Strong excitation at around 490 nm bleaches Dronpa more efficiently than other fluorescent proteins, and the bleached protein is able to completely regain its green fluorescence with minimal irradiation at around 400 nm. The off state is thermally stable (Ando et al., 2004). Here, we describe the first successful application of the reversible fluorescent protein in vivo as well as novel techniques for analyzing the anatomy of neural networks during imaging: visualization of multiple individual neurons one-by-one in a tangled network, optical antero- and retrograde labeling, time-lapse imaging, and two-photon microscopy to specifically label single neurons buried inside of tissues.

Figure 1.

Repeated elimination and reactivation of Dronpa fluorescence, a reversible green fluorescent protein (GFP), with blue and violet lights, in zebrafish in vivo. A: Schematic diagram explaining the reversible nature of Dronpa fluorescent proteins. After synthesis, Dronpa exhibits green fluorescence. This fluorescence is eliminated after exposure to a strong blue light but is recovered after a brief exposure to a (ultra-) violet light. This process can be repeated. B–G: Highlighting rhombomeres one after another in the same embryo at 19 hours postfertilization (hpf). Anterior is to the top. Dorsal view of the hindbrain of an embryo that expresses Dronpa and membrane-bound monomeric red fluorescent protein (mRFP) after co-injection of the corresponding synthesized RNAs at the single cell stage. Dronpa is a fluorescent green color, and mRFP is a fluorescent red color. All images have been pseudo-colored magenta. B: The embryo is initially doubly fluorescent in green and red. C: Scanning with a strong 488-nm laser eliminated entirely the green fluorescence. D–G: Rhombomere 4 was scanned with the 405-nm laser. This specifically re-highlighted the area in green. By repeating this process, we could highlight rhombomeres 4, 5, 6, and 7 one at a time. Note that the red fluorescence of mRFP is not affected during these procedures. H–K: Imprinting an identical pattern but in reversed color combinations on the same embryo. The embryo was at the stage of 50% epiboly to early gastrula (5–6 hpf). Each of two alternative methods based on either elimination with blue light or reactivation with (ultra-) violet light. I: Twelve radial lines were first imprinted on the embryo expressing Dronpa and mRFP by eliminating the green fluorescence by scanning with a strong 488-nm laser in this pattern. J,K: The embryo was then scanned with the strong 488-nm laser entirely (J), then, with the 405-nm laser in the same pattern of radiation (K). The arrow marked B indicates exposure to the blue light, and V indicates exposure to the (ultra-) violet. See also Supplementary Movies S1a and S1b. Scale bars = 100 μm in B, 200 μm in I.

RESULTS AND DISCUSSION

Repeated Elimination and Reactivation of Green Fluorescence of Dronpa In Vivo

We first tested whether Dronpa can be expressed in zebrafish embryos and show the same photochromic behavior as reported in HeLa cells (Ando et al., 2004). We injected synthesized Dronpa mRNA into zebrafish embryos together with mRNA of membrane localized monomeric RFP (mRFP) in an attempt to visualize the cell membrane in red and cytoplasm in green, and detect the shape of the cells expressing the protein even in the absence of Dronpa's green fluorescence. The embryos showed strong green fluorescence by 50% epiboly or 5 hours postfertilization (hpf) and maintained this fluorescence for at least a few days. A 488-nm argon laser, as used for GFP, was used for observation. For photobleaching, we repeatedly scanned the specimen with 488-nm argon laser at the maximum strength, 10–20 times stronger than that used for observation, by conventional confocal microscopy until most of the green fluorescence became nonvisible. For photoactivation, a very brief scanning with 405-nm diode laser or 364-nm UV laser was sufficient. Figure 1B–G shows such an example. In this case, the entire hindbrain in the 19 hpf embryo was photobleached (Fig. 1C), and rhombomere 4 (r4) was specifically scanned and highlighted with the 405-nm laser (Fig. 1D). The entire hindbrain was then photobleached as before, and r5 was specifically scanned and highlighted with the 405-nm laser (Fig. 1E). This process could be repeated several times without showing any significant decay in the brightness of activated Dronpa, thereby allowing us to label rhombomeres 4, 5, 6, 7, and other structures sequentially, one at a time (Supplementary Movie S1a, which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat).

It was also easy to imprint various patterns on an embryo expressing Dronpa. In the case of Figure 1H–K, we imprinted a radiation pattern on the gastrula in two different ways. The first method was to scan repeatedly with a strong 488-nm laser in a predetermined pattern. This resulted in a non–green-fluorescent pattern becoming visible against the green fluorescence background (Fig. 1I). As mRFP was also coexpressed, the nonfluorescent area appeared as red. In the second method, the entire embryo was photobleached with a strong 488-nm laser (Fig. 1J), then scanned briefly with a 405-nm laser in a predetermined pattern. This strategy resulted in a green fluorescent pattern appearing against a nongreen background (Fig. 1K). These results showed that Dronpa could be expressed efficiently by using the conventional mRNA injection into zebrafish embryos, and its photochromic behavior could be easily achieved with confocal microscopes, in vivo.

Optical Dissection of Neural Networks Expressing Dronpa to the Single Cell Level

We next attempted to use Dronpa to analyze the morphologies of several individual neurons that were overlapped or intermingled with each other (Fig. 2). Here, we were able to take advantage of a genetic mosaic system to express DRONPA in vivo (K. Hatta et al., unpublished observations). We injected UAS-dronpa construct (pUD) into the DeltaD-Gal4 transgenic fish (Tg(deltaD:Gal4; Scheer et al., 2001), in which Dronpa was preferentially expressed in neurons. After selecting areas that contain rather complex wiring patterns (Fig. 2B,F), we optically dissected neural networks to the single cell level using the procedure schematically illustrated in Figure 2A. After complete irradiation with a strong 488-nm laser, a small dot-like fluorescence sometimes remained, especially in the soma of neurons, in which Dronpa was strongly expressed. Even further irradiation had little effect on any attempt in eliminating this (Fig. 2C,D). Although the exact nature of this dot is unknown (it might be a denatured form of Dronpa), it could potentially be used as a marker for locating the soma after the complete elimination of green fluorescence. A brief scanning with 405- or 364-nm laser on the area of the soma was sufficient to re-highlight the green fluorescence. After the diffusion of fluorescent proteins along neurites, the whole morphology of each neuron became apparent (Fig. 2C,D,G; see also Supplementary Movies S2 and S3). By merging these optically isolated neuronal images, we successfully reconstructed the neural networks in which single neurons are distinguished by different pseudo-colors (Fig. 2E,H).

Figure 2.

Visualization of individual neurons one-by-one: optical dissection and reconstruction of neural networks using Dronpa. A: Schematic representation of the technique used for optical dissection of individual neurons from a network. The entire visual field, including multiple neurons expressing fluorescent Dronpa, was irradiated with blue light (B) to eliminate the fluorescence. The position of the single soma is locally irradiated with (ultra-) violet light (V). The fluorescence in the soma is specifically reactivated and diffused into the neurites. By repeating the same procedure three times on a different neuronal soma each time, we were able to visualize and trace individual neurons. The network can then be reconstructed with each neuron labeled in a different pseudo-color. B–E: As an example, for mosaic expression of Dronpa in the neurons, pUD was injected into the Tg(DeltaD:Gal4) fish at the one- to four-cell stage. Dorsolateral view of the hindbrain. Anterior is to the right. B: There were several neurons expressing Dronpa extending axons that are intermingled with each other. The entire brain was first scanned with a strong 488-nm laser to eliminate most of the fluorescence. Note that rudimental fluorescence remained in some neuronal somas. A single soma of the neuron was then irradiated with the 405-nm laser. C: Anterograde diffusion of the fluorescent Dronpa specifically visualized the neurites of this neuron. D: This procedure was repeated, but a different neuronal soma was irradiated. E: By adding different pseudo-colors (green and magenta) to these two neurons using LSM510 (Zeiss), we reconstructed the network, revealing that these two neurons were ascending commissure neurons that cross at the midline of each other. F–H: Another example. The caudal hindbrain of an embryo at 26 hours postfertilization (hpf) prepared similarly as above. Dorsal view. Anterior is to the top. Several neurons were intermingled, and the morphology of each neuron was obscure. G: After repeating complete elimination and local reactivation of the fluorescence four times (B,Vx4), the shape of each neuron became evident. Neurons 1 and 2 were T-shaped commissure neurons. Neuron 3 was an ascending commissure neuron. Neuron 4 was starting to extend its neurite anteriorly. H: We could interpret the network constructed by these four neurons. The broken line represents the ventral midline, where the floor plate resides. See also Supplementary Movies S2a and S2b. Scale bars = 20 μm in B, 50 μm in F.

Retrograde Labeling of Neurons from Axons to the Soma With Dronpa

Retrograde labeling is useful in identifying the position of soma from the visible axons, and this becomes possible when the axon is sufficiently illuminated with Dronpa. The entire area of interest was first irradiated with a strong blue light to eliminate the fluorescence of Dronpa (Fig. 3B). The axon of interest was then irradiated with a 405-nm laser (Fig. 3C). It proved necessary to wait for the diffusion of the fluorescent Dronpa throughout the cell, including the soma. The time necessary for diffusion is most likely dependent on the diameter, shape, and length of the axon. In the case of the motoneuron in Figure 3, it took approximately 3 min for Dronpa to diffuse from the axon to the soma (Fig. 3C,D). A single irradiation was sufficient to obtain the selective highlighting of a single neuron in the cluster of three somas. The resulting brightness may also depend on the ratio between the volume of the axon in the area of reactivation and that of the rest of the cell. If the ratio is small, a single irradiation may be insufficient to adequately label the soma. We believe that it may be necessary to repeat the irradiation process at the same position after the diffusion has taken place to a certain extent or completed. For example, after 13 repetitions, we were able to obtain a better contrast for this neuron (Fig. 3E; see also Supplementary Movie S3).

Figure 3.

Retrograde labeling from axon to soma using Dronpa. A: Mixtures of motoneurons and interneurons in the spinal cord expressing fluorescent Dronpa at 27 hours postfertilization (hpf), as prepared in Figure 2. Anterior is to the right. Dorsal is to the top. B: The image after scanning the entire visual field several times with the strong 488-nm laser. Fluorescence was mostly eliminated. C: The image immediately after scanning with the 405-nm laser (violet waved arrow) of a small portion of the spinal nerve innervating the body muscle. D: The image 3 min after irradiation. Retrograde diffusion of the re-highlighted fluorescent Dronpa revealed the shape of the axon and the position of the soma (arrow) previously hidden by a few interneurons partially overlapped in view A. E: The image after repeated irradiation and diffusion for 13 times (49 min after the initial irradiation). The shape of the neuron is more clearly distinguishable. F: Outline of the neuron (in green), interpreted and drawn on the same photo as A. SC, spinal cord. See also Supplementary Movie S3. Scale bar = 50 μm.

Time-Lapse Imaging With Dronpa

The wavelengths required for eliminating fluorescence and for observation are the same. The relatively fast bleaching rate of Dronpa at 488 nm may be a drawback due to the limited number of images that can be acquired after photoactivation. To obtain multiple bright images, the excitation light must be reasonably attenuated (Ando et al., 2004). In in vivo, in particular, it is often necessary to take several z-slices to obtain three-dimensional information at each time point. As we used a relatively strong laser for observation to obtain sufficient resolution, the fluorescence became considerably weaker after taking a set of z-slices at one time point. This weakening makes it difficult to perform four-dimensional (time-lapse) analysis. We, therefore, decided to irradiate the neuron each time, or after every few times, following a set of scanning for observation, to illuminate it to a sufficient brightness. This technique is especially effective for imaging of neurons that are just starting to extend their neurites. Figure 4 illustrates such an example. In this case, a single neuron strongly expressing Dronpa was targeted (Fig. 4A). The fluorescence of the neuron remained strong by additional repeated irradiation with violet light after every three observations (Fig. 4B–E). It was possible to observe the neuron extending the axon ventrally, crossing under the floor plate at the midline, to reach the lateral wall of the opposite side, then, to bifurcate both anteriorly and posteriorly. During this period, the scan was repeated for 74 times at 15-min intervals without significant decay in brightness (see Supplementary Movie S4).

Figure 4.

Time-lapse imaging of neural development using Dronpa. A: A single neuron expressing Dronpa starting to extend axon at 27 hours postfertilization (hpf). Anterior is to the left. B–E: Time-lapse series of axonal navigation of the T-shaped commissural spinal neuron imaged at 27 hpf (B), 31 hpf (C), 39 hpf (D), and 42 hpf (E). The axon extended ventrally (B), crossed the midline (C), reached the contralateral side, and bifurcated both anteriorly and posteriorly (D). The neuron was irradiated with the 405-nm laser, immediately after every three time points for observation. This strategy resulted in recovery of the weakening fluorescence caused by observation using the laser with the same wavelength for the elimination. F: Depth pseudo-color code representation of the neuron at 42 hpf, showing the three-dimensional structure of the neuron. The scale for the depth is shown at the bottom. See also Supplementary Movie S4. Scale bar = 50 μm.

Activation of Dronpa in Cells Buried Inside of Tissues Using Two-Photon Confocal Microscopy: Visualization of the Early Axonal Trajectory of a Single Mauthner Neuron

As shown above, it was possible to re-highlight a single cell if its target soma is separated from other cells expressing Dronpa, with conventional confocal microscopy. It was not possible, however, to re-highlight exclusively a single cell or a small group of cells buried in the tissue and covered by layers of cells expressing Dronpa due to the difficulty in avoiding unintended re-highlighting of the cells in the light path out of the focal plane (Fig. 5A). If it was possible, however, to use two-photon confocal microscopy for activating Dronpa, in which a laser whose wavelength is approximately two times longer than usual is used, we could highlight cells buried inside of tissues without activating neighboring cells in the light path (Fig. 5A). In an attempt to test this possibility, we chose an area in which Dronpa was expressed in layered cells in a double-hetero transgenic of Tg(deltaD:gal4) and Tg(UAS:dronpa). In this embryo at 21–22 hpf, many neurons and some neuroepithelial cells at the site of neurogenesis were found to be fluorescent (Fig. 5B). After eliminating fluorescence of the visual field by scanning with a strong blue laser of a conventional confocal microscope, we were able to re-highlight fluorescence of the cell of interest by scanning with a 405-nm laser, but this also re-highlighted the cells located dorsally or ventrally along the path of the laser (Fig. 5C). In contrast to these findings, we were able to successfully re-highlight fluorescence in a single or a small group of cells buried inside of the tissue by repetitive scanning at 780 nm using two-photon excitation microscopy equipped with Titan-Sapphire laser, without highlighting the dorsally or ventrally located cells (Fig. 5D). In the case shown here, we were able to label almost exclusively a single neuron belonging to the Mauthner cells, paired interneurons present in the rhombomere 4 near the anterior edge of the otic vesicle. In the specimen observed before eliminating the fluorescence, we could see numerous axons crossing the midline in the hindbrain (Fig. 5E). After bleaching and the two-photon reactivation, we were able to visualize exclusively the earliest axonal trajectory of the Mauthner neuron among others (Fig. 5F). It crossed the midline, gradually turned posteriorly, and started to extend caudally along the contralateral longitudinal pathway at stage 19–21 hpf. As shown here, using two-photon microscopy, we could improve the specificity of the reactivation of Dronpa along the z (depth) -dimension in general. However, it should be noted that, when the cells expressing Dronpa are densely packed in the tissue, it is often challenging to label strongly a single cell without highlighting the cells directly neighboring to it to some degree.

Figure 5.

Labeling of a single neuron located inside of the brain using Dronpa by two-photon confocal microscopy: Visualization of a Mauthner cell. A: Schematic representation of the comparison between the results of photoreactivation of Dronpa in a layered tissue by conventional or two-photon confocal microscopy. The former results in the label as a column, whereas the latter results in the label specifically in the focused cells. All cells initially expressing Dronpa are fluorescent green in color. After scanning with strong blue laser (B), the fluorescence is eliminated. Scanning on the cell of interest with (ultra-) violet laser reactivates the fluorescence, not only in the cell of interest but also in the neighboring cells along the path of the laser. Conversely, by using two-photon confocal microscopy, the cells in focus can be specifically labeled, as red laser cannot reactivate the fluorescence of Dronpa and the two-photon event that is required to reactivate the fluorescence is limited to the focal plane. B: Confocal observation of the hindbrain near the otic vesicle (OT, outlined with broken lines), expressing Dronpa with the 488-nm laser. The embryo is a double-hetero transgenic of Tg(dld:Gal4) and Tg(UAS:dronpa) at around stage 21 hours postfertilization (hpf). Dorsal view (a projection of 14 optical slices taken at the interval of 5.4μm) is at the bottom, and an optically reconstructed transverse section is on the top. d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral. The midline of hindbrain is indicated by long dashed dotted line. C: The same specimen after an attempt at re-highlighting a neuron inside of the brain by conventional confocal microscopy. The specimen was first scanned repeatedly with the strong 488-nm laser to eliminate entire fluorescence in the area, then scanned twice at the center of a chosen neuron with 405 nm by conventional confocal microscope. These scans were made from the dorsal side. The cell was highlighted, but the fluorescence of the dorsally and ventrally neighboring cells along the laser path (arrowheads) were also activated to obtain sufficient brightness at the target cell. D: The same specimen after the successful re-highlighting of a single neuron inside of the brain by two-photon microscopy. It was located at the middle of rhombomere 4 near the anterior edge of the otic vesicle (outlined). After elimination of the fluorescence as described above, specimen was scanned repeatedly (1,000 times) at the center of the chosen neuron with 780 nm by two-photon confocal microscopy from the dorsal side. The cell was highlighted without activating the fluorescence of the dorsally and ventrally neighboring cells along the laser path. E: An optical slice of B at the floor of the hindbrain, with enhanced brightness and contrast, demonstrating numerous axons and growth cones crossing the midline. The axon from the left Mauthner neuron is indicated by arrowhead. F: An optical slice of C at the floor of the hindbrain, demonstrating a single axon crossing the midline (M-axon, arrowheads), gradually turning posteriorly and joining the contralateral medial longitudinal track, confirming that the single cell we labeled was indeed the left Mauthner neuron (M-cell in D). Scale bars = 20 μm.

PERSPECTIVES

In this study, we showed that it is possible to use reversible fluorescent proteins to optically dissect neuronal networks in vivo. This novel procedure of visualizing individual neurons involves the repetitive process of complete elimination, local reactivation, and imaging of the fluorescence of Dronpa with two wavelengths, and reconstruction of the network in pseudo-colors. It is consequently an operation that is well suited for automation. In the future, it may be possible to automatically visualize all of the neurons in a neural network one-by-one by reconstructing them in multiple pseudo-colors. Dronpa is also useful in studying morphogenesis of multicellular structures. The reversible nature of Dronpa makes the cycle of elimination, imprinting patterns, and subsequent observation of pattern deformation feasible many times on the same embryo, thereby enabling the analysis of cellular movements on a global scale and the sum of the entire process in simulation. As shown in Figure 1 and Supplementary Movies S1a and S1b, it is easy to highlight various structures such as rhombomeres and organs, if they can be defined by light. This highlighting helps in dissecting particular structures or organs manually or in sorting by a cell sorter after dissociation of the tissue for the use of transplantation, culture, and gene-expression profiling. Using the technique described here means that it is not necessary to touch the cells when labeling them. This noninvasive feature is of potential use in visualizing the neuron of interest before electrophysiological recording in vivo or in slice preparation. Our recent results have indicated that a similar technique is also feasible in mammals (K. Hatta and T. Omura, unpublished observation), so that detecting cell movement, axonal guidance, and mapping of the brain will become easier to perform than before and will facilitate single cell labeling and electrophysiological studies.

Co-injection or cotransfection of a Dronpa construct with mRNA or DNA encoding a dominant-negative or constitutively active form of developmentally important molecules or with siRNAs would be an effective means for identifying their roles in cellular morphogenesis of interest. Dronpa is monomeric and was fused to proteins such as ERK to track their movement between the cytoplasm and nucleus (Ando et al., 2004). By expressing such fused proteins in vivo, we expect to analyze their transport and polarized localization in individual neurons or other cell types during embryogenesis.

We also reported the generation of a permanent transgenic zebrafish line carrying UAS:dronpa (Tg(UAS:dronpa)) in this article. Dronpa could be expressed in cell types of interest by crossing this line with appropriate Gal4 transgenic lines (Fig. 5A and unpublished observation). These fish may further facilitate a systematic analysis of neuronal networks.

EXPERIMENTAL PROCEDURES

Fish

Wild-type zebrafish used in this article were hybrids of India and Oregon AB lines. Tg(DeltaD:Gal4) lines (Scheer et al., 2001) were obtained from the Zebrafish International Research Center (ZIRC; Eugene, OR). Each embryo was morphologically staged according to Kimmel et al. (1995) and expressed by hours postfertilization (hpf) at 28.5°C.

Injection of DNA and RNA

The UAS-Dronpa (pUD) DNA was constructed by isolating the EcoRI/PvuII-blunt Dronpa fragment from pDG1-S1 (MBL, Nagoya, Japan) and cloning it into the XhoI-blunt/EcoRI fragment of the pUS. pUS was constructed by isolating the EcoRI-blunt/EcoRI-blunt UAS-E1B fragment of pB-UAS-E1B (Koster and Fraser, 2001) and cloning into the HindIII-blunt/SalI-blunt vector fragment of pCS2+ (constructed by M. Takeuchi and K. Hatta). pB-UAS-E1B was a gift from Drs. Koster and Fraser.

For injection, plasmid DNAs were prepared using the Endofree plasmid kit (Qiagen, Tokyo, Japan). Fertilized eggs were dechorionated at the one-cell stage either manually with forceps or by digestion with 0.5 mg/ml pronase (Sigma-Aldrich, St. Louis, MO) for several minutes at room temperature followed by washing three times with fish tank water. For the circular plasmids, pUD was injected into the cytoplasm of single- to four-cell stage embryos at a concentration of 10 pg/embryo (0.5 nl of 20 ng/μl) in 100 mM KCl containing 0.02% phenol red (Sigma-Aldrich) using air pressure (IM-30, Narishige, Japan). In some cases, nondechorionated fertilized eggs were injected with plasmids. Injected embryos were raised in E2 embryo media (EM; 15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2.2H2O, 1 mM MgSO4.7H2O, 0.15 mM KH2PO4, 0.05 mM Na2HPO4.2H2O, and 0.7 mM NaHCO3, pH 7.0–7.5) containing 0.5 ppm methylene blue on 1% agarose-coated culture dishes. To inhibit pigmentation, embryos were transferred to a solution of EM containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) before 24 hpf.

Capped mRNA was prepared from pUD and pCS2+mRFP by in vitro transcription using the mMESSAGE mMACHINE kit (Ambion, Austin, TX) with the SP6 primer. pCS2+mRFP was a gift from Dr. Tsien. The mRNA was injected into the cytoplasm of dechorionated single-cell stage embryos at a concentration of 250 pg/embryo (0.5 nl of 500 ng/μl) in H2O containing phenol red.

Generation of Tg(UAS:dronpa)

A transgenic line carrying UAS:dronpa (Tg(UAS:dronpa)) was generated using Tol2 transposon-mediated gene transfer (Kawakami et al., 2000, 2004). The pTol2UD was constructed by isolating the EcoRV/ApaI UAS:dronpa fragment from pUD (see above) and cloning it into the NruI/ApaI vector fragment of the pT2KSAG. pTol2UD (25 ng/μl) and transposase mRNA (25 ng/μl) synthesized from pCS-TP were co-injected into fertilized eggs as described above. pT2KSAG and pCS-TP were gifts from K. Kawakami.

Imaging

To permit imaging, embryos were anesthetized with 0.2 mg/ml Tricaine (3-amino benzoic acid ethylester, Sigma-Aldrich) and mounted on a Lab-Tek Chambered Coverglass (Nunc, Naperville, IL) in a drop of 1% low melting point agarose (Sigma-Aldrich) in fish Ringer solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.2) or EM, or set free in small holes made in a layer of 1% low melting point agarose filled with EM containing 1-phenyl-2-thiourea and methylene blue. Images of z-slices of multiple time points were obtained by laser confocal microscopy, processed to projections or depth coded projections, and pseudo-colored using LSM 510 (Zeiss, Jena, Germany). Elimination of Dronpa's green fluorescence was achieved by repeated scanning with the 488-nm argon laser at the maximum strength. Reactivation of the fluorescence of Dronpa was achieved by a brief scanning of a selected region of interest, using the bleach function of LSM510, with the 405-nm diode laser or the 364-nm UV laser by conventional confocal microscopy, or the 780-nm Titan-Sapphire laser by two-photon confocal microscopy. The fluorescence of Dronpa was observed as for GFP, i.e., with the 488-nm argon laser but at an attenuated strength (1–10% of the maximum depending on the specimen) to minimize photobleaching. The objective lenses used for the elimination, reactivation, and observation of Dronpa by confocal microscopy was Plan-Neofluar 20x/0.5 in general. For the specimen in Figure 1H and Supplementary Movie 1b, Plan-Neofluar 10x/0.3 was used. For the specimen in Figure 2B, 10x/0.3 was only used for the elimination and observation. For observation of the fluorescence of Dronpa with conventional fluorescent microscopes (DMRA2, Leica, Solms, Germany) or fluorescent dissection microscopes (MZFL3, Leica), we used a system involving a mercury lamp passed through a GFP filter set (GFP2 or GFP3). For observation of mRFP, we used either a 543-nm helium-neon laser for confocal microscopy or the light from the mercury lamp passed though a rhodamine filter set (G) for fluorescent microscopes.

Acknowledgements

We thank S. Aizawa for support; R. Köster, S. Fraser, K. Kawakami, and R. Tsien for plasmids; A. Miyawaki, S. Hayashi, and M. Hibi for encouragement; and M. Royle for comments. K.H. received a Grant-in-aid for Scientific Research from The Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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