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DEVELOPMENTAL DYNAMICS 240:820–827, 2011

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TECHNIQUES

Labelling Cell Structures and Tracking Cell Lineage in Zebrafish Using SNAP-Tag

Developmental Dynamics

Claúdia Campos,1† Mako Kamiya,2 Sambashiva Banala,2 Kai Johnsson,2* and Marcos Gonza´lez-Gaitań1*

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP-tag technology and O6-benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long-term imaging and allows, unlike most of the autofluorescent proteins, the use of acid fixatives such as trichloroacetic acid. Furthermore, the use of O6-benzylguanine derivatives bearing caged fluorescein allows cell lineage tracing through photo-deprotection of the fluorophore and its detection either through fluorescence microscopy or through immunohistochemistry after fixation using anti-fluorescein antibodies. Developmental Dynamics 240:820–827, 2011. V 2011 Wiley-Liss, Inc. C

Key words: SNAP-tag; zebrafish; imaging; lineage tracing Accepted 28 December 2010

INTRODUCTION Fluorescent proteins (FPs) enable the visualization of proteins of interest in live cultured cells and in animal models (Giepmans et al., 2006; Detrich and Kevin, 2008) and have become indispensable tools in cell and developmental biology. However, with respect to applications in Zebrafish, FPs also have certain shortcomings. Most importantly, the dramatic decrease in fluorescence intensity by photobleaching during long imaging intervals and the fading of fluorescence upon immersion in harsh acid fixatives often used for Zebrafish limit their usage (AuCoin et al., 2006). In the latter case, the tagged protein can be

visualized through an antibody detection step. Yet, such detection is incompatible with the use of photoactivatable FPs for tracking cell lineage. Furthermore, an antibody detection step limits the amount of antigens available for detecting other targets by immunostaining. In principle, the specific labelling of protein with highly photostable fluorophores that are also insensitive to harsh fixatives could overcome these problems. It has previously been shown that Halotag fusion proteins can be specifically labelled with tetramethylrhodamine (TMR) in live embryos (Li et al., 2008). However, the employed labelling protocol, incubating live embryos expressing a HaloTag fusion protein

with a TMR derivative, restricts the labelling to membrane-permeable fluorophores and thereby significantly limits the utility of the approach. In this study, we introduce a technique for the specific labelling of proteins with synthetic fluorophores in live Zebrafish embryos based on SNAP-tag. SNAP-tag is a 20-kDa mutant of the human O6-alkylguanineDNA alkyltransferase (hAGT) protein that specifically reacts with O6-benzylganine (BG) derivatives carrying a variety of different synthetic fluorophores (Fig. 1A, B) (Keppler et al., 2003). The monomeric tag can be attached to a wide variety of proteins and labelled in all cellular compartments of both in live and fixed cells

Additional Supporting Information may be found in the online version of this article. 1 Departement de Biochimie, Universite´ de Gene`ve, Gene`ve, Switzerland 2 ´ cole Polytechnique Fe´de´rale de Lausanne, Lausanne, Switzerland Institute of Chemical Sciences and Engineering, E †Claúdia Campos’s present address is Plant Development, Instituto Gulbenkian Cieˆncia, 2780-156 Oeiras, Portugal Grant sponsor: University of Geneva; Grant sponsor: ERC (SARA); Grant sponsor: SystemsX (lipidsX); Grant sponsor: Swiss National Science Foundation (SNF); Grant sponsor: NCCR (Chemical Biology); Grant sponsor: Fondation Claude et Giuliana; Grant sponsor: FCT; Grant number: SFRH/BD/15210/2004; Grant sponsor: Oncasym. *Correspondence to: Marcos Gonza´lez-Gaitań, Biochemistry Department, University of Geneva, Sciences II, Quai Ernest Ansermet, 1211 Geneve4 Switzerland. E-mail: [email protected] or to Kai Johnsson, Institute of Chemical Sciences ´ cole Polytecnhique Fe´de´rale de Lausanne, CH 1015, Lausanne, Switzerland. E-mail: kai.johnsson@epfl.ch and Engineering, E DOI 10.1002/dvdy.22574 Published online 28 February 2011 in Wiley Online Library (wileyonlinelibrary.com).

C 2011 Wiley-Liss, Inc. V


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(Johnsson and Johnsson, 2007; Gautier et al., 2008; Johnsson, 2009). In addition, a large variety of different fluorescent substrates allows detection of SNAP-tag fusion protein at any desired wavelength (Juillerat et al., 2003; Johnsson and Johnsson, 2007; Johnsson, 2009). Moreover, the photostability of several BG substrates is in the range of eGFP and mRFP, with relative photostabilities ranging from 50 to 90% (Keppler et al., 2006; Johnsson and Johnsson, 2007; Johnsson, 2009). SNAP-tag has been used for localized calcium sensing and for the generation of semisynthetic fluorescent sensor proteins for metabolites (Brun et al., 2009; Kamiya and Johnsson, 2010). In spite of its attractive properties, the application of SNAP-tag so far has been limited to in vitro and/or in cell culture systems (Lemercier et al., 2007; Bannwarth et al., 2009; Keppler and Ellenberg, 2009). Here we demonstrate the use of SNAP-tag to perform live imaging and cell tracking in the Zebrafish embryo. To take advantage of the large variety of substrates available, we developed an efficient labelling protocol that involves the co-injection of capped mRNA and the SNAP-tag substrate of interest. We demonstrate that fluorescencelabelled SNAP-tag fusion proteins are suitable for long-term time-lapse imaging and are not affected by acidic fixatives, allowing visualization without antibody labelling. Furthermore, caged BG substrates can be used to track cell lineage. The specificity of the labeling and the possibility to utilize fluorophores with properties that cannot be found in any FP should make SNAP-tag a powerful tool for studying Zebrafish.

RESULTS AND DISCUSSION SNAP-tag Is Not Toxic for the Zebrafish Embryo and Allows Specific Protein Labelling SNAP tag has been successfully used in cell culture to label nuclear structures (Keppler et al., 2004; Jansen et al., 2007), membranes (Gautier et al., 2008), microtubules (Keppler and Ellenberg, 2009), and to visualize G-protein-coupled receptor oligomerization (Maurel et al., 2008) or to mea-

Fig. 1. General procedure and substrates used for fluorescence labeling of SNAP fusion proteins. A: Covalent labeling of SNAP fusion proteins with BG derivatives. B: Structures of BG derivatives used for fluorescence labeling of SNAP fusion proteins; the maxima in the fluorescence excitation (ex.) and emission (em.) spectra of the substrates after reaction with SNAP-tag are listed.

sure Ca2þ concentrations in cells (Bannwarth et al., 2009; Kamiya and Johnsson, 2010). To test the compatibility of the SNAP-tag with imaging in Zebrafish, we injected Zebrafish embryos with capped RNA of different SNAP-tagged proteins (Fig. 2). We first tagged a nuclear protein, Histone 2A (H2A). Injection of H2ASNAP did not cause significant lethality compared to wild-type controls and the surviving H2A-SNAP animals were morphologically similar to wild-type embryos both at early and late stages of development (Fig. 2A; see Supp. Fig. S1, which is available online). Upon injection of the substrate alone, no nuclear staining is observed (Fig. 2B–D). The substrate is fluorescent by itself, but in the absence of the labelling SNAP-tag, the fluorescent signal is diluted in the embryo upon injection. Indeed, combined injection of H2A-SNAP and BGDAF, TMR-Star or BG-Cy5 led to a specific nuclear signal in different wavelengths (Fig. 2E–G), indicating that the fluorescence observed after co-injection of H2A-SNAP and the BG-substrates was specific and dependent on H2A-SNAP expression. In their SNAP-bound form, the tested BG substrates display high quantum yields in relation to eGFP or mRFP, which also explains the increase in brightness when H2A-SNAP is added during injection (Keppler et al.,

2006). Injection of TMR-Star alone leads to a weak unspecific signal (Fig. 2C). This could be due to the known fact that rhodamine derivatives bind unspecifically to cellular components (Chen, 1989; Koide et al., 2007). SNAP-tagging also allowed prolonged and continuous imaging, with a detectable signal after more than 8 hr of imaging (Supp. Movie S1). During the course of this long-term time-lapse, we could observe cell movements (Supp. Movie S1) and mitosis occurring at later times points (Supp. Fig. S2A and B). In addition, SNAP-tag fluorescence decreased by only 30% when a 10% 546-nm laser power was applied (Supp. Fig. S2C). To further test if the SNAP-tag could be used to label other cellular structures, we generated Lgl2-SNAP, which labels the basolateral membrane of epithelial cells (Sonawane et al., 2005) and SNAP-Rab5, which labels the early endosomes (Ulrich et al., 2005). Indeed, co-injection of either Lgl2-SNAP or SNAP-Rab5 with BG substrates allowed specific visualization of the baso-lateral cell surface (Fig. 3A,B) and the early endosomes (Fig. 3C) in 36hpf embryos, respectively. The observed SNAP-Rab5 vesicles are likely to be labelled by SNAP-Rab5 synthesized de novo, since the half life of Rab5 is in the range of only a few hours (Rink et al., 2005). Furthermore, the subcellular localization of


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Fig. 2. H2A-SNAP labeling in live zebrafish embryos. A: Quantification and comparison of lethality and abnormal development between wild type and SNAP injected embryos at 15–17 somites and at 36 hr post-fertilization (hpf). B–D: Wild type embryos injected with (B) BG-DAF, (C) TMR Star, or (D) BG-Cy5 substrates. E–G: Confocal pictures of embryos injected with H2ASNAP and (E) BG-DAF, (F) TMR Star, and (G) BG-Cy5. F: Embryo was labeled in a mosaic fashion, therefore nuclei on the right side of the midline are more strongly labeled than nuclei on the left side of this structure. B–D: Projection of 45 planes encompassing the spinal cord (dotted lines delimit the spinal cord). E–G: Projection of 5 planes in the spinal cord. B–G: 36-hpf embryos; anterior to the left. Scale bars ¼ 20 mm.

Fig. 3. SNAP-tag allows labelling of different cell structures in the zebrafish embryo. A,B: Mosaic labelling of the basolateral membrane with Lgl2-SNAP and TMR Star (laser power to image Lgl2SNAP: 20%). B: Zoom of the boxed area in A. C: Mosaic labelling of early endosomal membranes with SNAP-Rab5 and BG-DAF; arrows point at the endosomal population labelled with SNAPRab5 (laser power to image SNAP-Rab5: 3%). D: Mosaic labelling of early endosomal membranes with Rab5-CFP. A–D: 36-hpf embryos; projection of 5 planes of the embryo spinal cord; dotted lines delimit the embryo’s spinal cord; anterior to the left; scale bars ¼ 10 mm.

Fig. 4. Imaging of SNAP labelled structures is compatible with the use of other FPs. A: Twelve-somite-old embryo co-injected with SNAP-Rab5, BG-Cy3, and Gap43-GFP; single plane confocal micrograph of an embryonic spinal cord (laser power to image SNAP-Rab5: 3%). B: Twenty-two-somite-old transgenic Hu:GFP embryo injected with H2A-SNAP and BG-Cy5 (laser power to image H2A-SNAP: 2%). Projection of 5 planes of the embryonic spinal cord. A,B: Anterior to the left. Scale bars in A,B ¼ 10 mm.

Fig. 5. Live imaging of cellular events using the SNAP-tag. A: Tracking of Rab5 endosomal movement in a 12-somite-old embryo labelled with SNAP-Rab5, BG-Cy3, and Gap43-GFP allows analysis of the distribution of these structures across mitosis; yellow arrows point at detected endosomal vesicles (laser power to image SNAP-Rab5: 3%). B: Projection of 5 confocal micrographs of a mitotic cell in the zebrafish spinal cord at 22 somites, labelled with H2A-SNAP and BG-Cy5; yellow arrows indicate the selected nucleus to follow during mitosis (laser power to image H2A-SNAP: 10%). A,B: Anterior to the left; time in min:sec. Scale bars ¼ 10 mm.



the fluorescent signal deriving from the SNAP-Rab5 fusion is similar to the localization in mosaic embryos, which were injected with Rab5-CFP construct (Fig. 3D). Therefore, the SNAP-tag can be used in Zebrafish embryos to tag different proteins of interest in any desired wavelength, without the need for recloning. We also injected BG-DAF and BGCy3 substrates in the cardiac atrium of 36hpf embryos expressing H2A-SNAP mRNA (injection at 1-cell stage). The substrates could be observed in the already formed blood vessels of the labeled embryos (data not shown). However, they could not diffuse across the endothelium. Therefore, this injection method proved to efficiently label blood vessels upon late injections of the substrate, but cannot be used to label other structures. SNAP-tag fusions were also injected and visualized in embryos expressing other fluorescent proteins (Fig. 4): SNAP-Rab5 mRNA was coinjected with Gap43-GFP (Fig. 4A) and H2A-SNAP was injected in the HuC:GFP transgenic line (Fig. 4B). Through the visualization of SNAPRab5, the distribution of these early endosomes could be followed during mitosis (Fig. 5A). Mitosis was also imaged in the live embryo, with the H2A-SNAP fusion (Fig.5B) allowing nuclei tracing. Hence, the SNAP tag is compatible with imaging of other structures with other FPs by choosing BG-substrates with appropriate wavelengths and adequate quantum yields. By enabling imaging of different cellular structures in the live embryo, we showed that the SNAP-tag technology is a versatile method for imaging in the developing Zebrafish embryo.

SNAP Labelling Is Resistant to Acid-Based Fixatives GFP fluorescence, as well as other FPs, is quenched upon immersion of labelled cells or tissues in fixatives with acid pH, such as methanol (Baumstark-Khan et al., 1999) or trichloroacetic acid (Fig. 6A). Later detection of structures labelled by these proteins requires detection of the FP with specific antibodies. BG substrates are not pH sensitive in the physiological pH range in vitro (Keppler et al., 2006). However, to test if the fluorescence deriving from the

SNAP labelling was resistant to fixation with acid solutions in Zebrafish, we fixed embryos injected with H2ASNAP or Lgl2-SNAP and BG-DAF in a solution of 10% trichloroacetic acid (TCA), or 4% PFA/PBS, followed by 100% methanol incubation overnight at 20 C. Before fixation, the fluorescence emitted by H2A:GFP and H2ASNAP embryos was similar (Fig. 6A, B live). However, analysis of the 36hpf fixed embryos showed that while most H2A:GFP fluorescence was bleached (Fig. 6A), H2A-SNAP fluorescence was still detected (Fig. 6B). Lg12-SNAP could also be detected after incubation in methanol and HuC/ D antigen detection, showing its resistance to the methanol fixation and posterior HuC/D detection (Fig. 6C). While performing multiple antigen labelling, the choice of antibodies can be limited, requiring different combinations of immunostainings to study co-localization. In addition, immunostaining generally requires the use of GFP as this is so far the FP for which the most efficient antibodies have been developed (Chapouton et al., 2006). As the SNAPtag signal can be detected at different wavelengths before and after fixation without the need to resort to antibodies, the tag represents a versatile tool to image cell structures and events.

Cell Lineage Analysis With SNAP-Tag While performing cell lineage analysis, it is important to track cell movements and changes in cell shape. Photoactivatable fluorophores are particularly useful for investigating cell lineage as they can be injected early in development when the cells are large, and then later activated in the growing embryo when the cells of interest may be small and buried deep within the tissue (Vincent and O’Farrell, 1992). Caged fluorescein dextrans have proven useful for analyzing regional cell movement and tissue patterning in zebrafish embryos (Kozlowski et al., 1997), however, this reagent is no longer commercially available. A labeling technique using the SNAP-tag may be a suitable alternative to replace the caged fluorescein dextrans to perform cell tracking in the Zebrafish embryo. In addition, SNAP-tag can be fused to proteins with specific sub-cellular

localization (Fig. 7A). For this purpose, we synthesized a BG-derivative of caged fluorescein, which shows no fluorescence but can be activated by UV illumination (Fig. 7B). Notably, the synthesis of this molecule consists of a simple one-step coupling reaction (Supp. Fig. S3). We injected zebrafish embryos with H2A-SNAP and a mixture of synthesized caged substrate, BG-CMNB-caged carboxyfluorescein, and BG-Cy5 substrates. BG-Cy5 was co-injected in order to visualize and select the nuclei for uncaging. We selected a mitotic nucleus in the neuroepithelium of a 20hpf zebrafish embryo and exposed it to pulses of UV illumination, resulting in fluorescence visualization in the irradiated nuclei (Fig. 7C– E). The activated signal was still visible after cytokinesis (Fig. 7F, arrows), and we could visualize a third nucleus, in which the substrate was also uncaged to a lower extent (Fig. 7F, arrowhead). The embryo was allowed to develop for 24 hr and fixed for subsequent immunodetection of fluorescein and HuC/D (Fig. 7G,H). In summary, using this procedure we are able to label individual nuclei and track them in the zebrafish neuroepithelium. In this particular case, it allowed us to conclude that none of the uncaged nuclei divided (Fig. 7G) and that all three cells express HuC/D (a postmitotic marker) and, therefore, differentiated (Fig. 7H). Hence, by means of the SNAP-tag technology and caged fluorophores, cell behaviour could be assessed. We foresee the application of this approach to other tissues and developmental scenarios for cell lineage analysis.

SNAP-Tag in Zebrafish: Conclusions Our results showed that SNAP-tagging can be used to label several cellular structures in the zebrafish embryo, such as the nuclei, membranes, or early endosomal populations at different developmental times with a variety of different fluorophores. Without the need for recloning, SNAP-tag can be imaged at varying wavelengths by simply varying the applied BG substrate and it can also be used to perform cell lineage analysis by using caged substrates. To generate a transgenic zebrafish line,

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the researcher is obliged to choose a fixed fluorescent protein to tag its protein of interest, which can lead to limitations when performing live imaging. However, when using a SNAPtag as an alternative to a fluorescent protein, it is sufficient to inject the transgenic embryos with the desired fluorescent BG substrate, to image it in any desired wavelength. The flexibility of SNAP-tag together with the advantageous properties of synthetic fluorophores and other chemical probes should make the approach an important tool for investigating key questions in developmental biology.

EXPERIMENTAL PROCEDURES Fish Maintenance and Stocks Fig. 6.


Embryos were obtained from wildtype (AB) and transgenic H2A:GFP

Fig. 7.


(Pauls et al., 2001) or HuC:GFP zebrafish (Park et al., 2000) and maintained by standard procedures in the Gonzalez Zebrafish Facility at the University of Geneva. Zebrafish were maintained at standard conditions. Embryos were staged by hpf at 28.5 C (Kimmel et al., 1995) or according to somite number.


DNA Constructs For the generation of H2A-SNAP, the SNAP tag was cloned into the XhoI/ XbaI sites of a pCS2þ vector containing the open reading frame of h2afvl (NM_001043323.1). The coding region of zebrafish lgl2 (NM_212582.1) was amplified from cDNA and cloned into the ClaI/XhoI restriction sites of the expression vector pCS2þ with a Cterminal SNAP tag. To obtain the fusion SNAP-Rab5, with SNAP on the N-terminus of Rab5a, SNAP was inserted into the BamHI/BglII sites of the pCS2þ CFP-Rab5a plasmid, as previously described (Scholpp and Brand, 2004).

BG Substrates SNAP-tag substrates (BG-DAF, TMRStar, BG-Cy3, BG-Cy5) were obtained from Covalys Biosciences (Witterswil, Switzerland) and New England Biolabs (Ipswich, MA). For the synthesis of the BG-CMNB-caged carboxyfluorescein, 5-carboxyfluorescein-bis-(5carboxymethoxy-2-nitrobenzyl) ether b-alanine-carboxamide, succinimidyl

ester (CMNB-caged carboxyfluorescein, SE) was purchased from Invitrogen (Carlsbad, CA). BG-PEG-NH2 was synthesized according to published procedures (Banala et al., 2008). To a solution of CMNB-caged carboxyfluorescein, SE (0.5 mg, 0.52 mmol) in anhydrous DMF (0.5 ml), BG-PEG-NH2 (2 mg, 4.0 mmol), and triethylamine (1 ml) were added. The mixture as stirred overnight at room temperature and a few drops of water were added. The product was purified by semi-preparative reversed-phase HPLC to obtain BG-CMNB-caged carboxyfluorescein (0.25 mg, yield. 36%). HRMS(ESI) (m/z): [Mþ2H]2þ calculated for C65H64O23 676.2068; found 676.2090. High-resolution mass spectra (HRMS, ESI-TOF) were recorded on a Q-TOF Ultima spectrometer (Micromass). HPLC purification was performed on a Waters (Milford, MA) 1525 binary HPLC pump using Nova-Pak C18-column (6 mM; 7.8 300 mm) at a flow rate of 4 mL/min. Product was detected at 280 and 350 nm using a Waters 2487 dual wavelength absorption detector.

RNA and Injections DNA constructs were transcribed using the SP6 MessageMachine kit (Ambion, Austin, TX). In vitro synthesized capped mRNA was dissolved in water. Typically, 50 pg of H2A-SNAP and 200 pg of Lgl2-SNAP and SNAPRab5 RNA were dissolved in 0.1M

Fig. 6. SNAP labelling resists acid fixatives and can be further detected without antibody detection. A: Histone:GFP transgenic embryo imaged before (GFP live) and after (GFP fixed) fixation in TCA: (DAPI indicates that the presence of nuclei). B: Mosaic embryo expressing H2A-SNAP and labelled with BG-DAF, imaged before (SNAP live) and after (SNAP fixed) fixation in TCA (DAPI staining indicates co-localization between the SNAP signal and the nuclei). C: Mosaic expression of Lgl2-SNAP (labelled with BG-DAF) in an embryo fixed in PFA/MeOH, further stained for HuC/D expression. A–C: Projection of 5 planes in the spinal cord of 36hpf embryos, anterior to the left. GFP- and SNAP-labeled structures were imaged with a laser power of 0,6%. Scale bars ¼ 20 mm. Fig. 7. Cell lineage analysis by combining SNAP-tag and a caged BG substrate. A: Mechanism for substrate uncaging. B: Structure of BG-CMNB-caged carboxyfluorescein; the maxima in the fluorescence excitation (ex.) and emission (em.) spectra of the substrates after reaction with SNAP are listed. C–F: Projection of confocal micrographs of an embryonic spinal cord co-injected with H2A-SNAP, BG-CMNB-caged carboxyfluorescein, and BG-Cy3. C: Before irradiation, labelling with BG-Cy3 allowed selection of a mitotic nucleus (arrow). D: Irradiation of selected nucleus with UV laser and visualization of BG-CMNB-uncaged carboxyfluorescein fluorescence. E: Zoom of box present in D. F: Daughters of mitotic cell observed in D (arrows point at daughters from cell labeled in D, arrowhead points at a cell in which uncaging also occurred at lower efficiency). G: Twenty-four hours after uncaging of the BG-CMNB-caged carboxyfluorescein, two nuclei with a stronger labeling were detected with the anti-fluorescein antibody (1 and 3), along with a third nucleus with weaker labeling (2), which was marked by an arrowhead in F. H: Sequential Z-stacks showing the three detected nuclei (1–3) in green; co-staining the labeled embryo with an antiHuC/D antibody (white) shows that all three nuclei belong to HuC/D-expressing cells. Scale bars ¼ 10 mm. Laser power to image H2A-SNAP and uncaged fluorophone: 0,6%.

KCl and 0.1% phenol red. BG substrates were dissolved in RNAse free DMSO at a concentration of 200 mM and one-cell-stage embryos were injected with 1 nl of this solution, followed by a second injection with the desired capped mRNA, at one-cell stage or two–four-cell stages in the case of desired mosaic expression, or at 36hpf in the cardiac atrium, to assess labeling at later stages of development. To test the optimal amount of BG fluorophore, we co-injected embryos with both H2A-SNAP mRNA and increasing concentrations of each substrate. The amount of used substrate was optimized in order to minimize the amount to obtain the best signal-to-noise ratio and to keep normal embryo development. As the procedure for BG substrate synthesis is not mRNAse free, the second injection step insures that the capped mRNA is not degraded. BG-CMNB-caged carboxyfluorescein was co-injected with BG-Cy3 to allow monitoring of the injections.

Time-Lapse Imaging Prior to live imaging, injected embryos were selected under a fluorescent lamp, mounted in 1% low melting agarose in a self-assembled imaging chamber previously described (Concha and Adams, 1998) and anesthetized with 0.03% Tricaine (Sigma, St. Louis, MO). Images were obtained with an Olympus Fluoview FV1000 confocal laser-scanning microscope, equipped with a PlanApoN 60 oil objective and Fluoview software for imaging. Lasers were adjusted to an intensity allowing a good signal quality in the middle portion of thestacks, using high gain, ensuring that photobleaching was reduced. For 3D images, stacks with a z-spacing of 1 mm were acquired. For long-term time-lapse in HuC:GFP-injected embryos, imaging conditions were performed as above and stacks of 12 images with a z-spacing of 2 mm and a time interval of 5 min were acquired over 8 hr:15 min. The laser intensities were adjusted to minimize bleaching and the room temperature was kept at 28 C. To uncage the BG-CMNB-caged carboxyfluorescein, 20hpf embryos were mounted for live imaging as above. A scanning area of 240240 pixels was selected in the spinal cord around the

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cell to uncage; a region of interest (ROI) with the shape matching the nucleus to uncage was created. This ROI was exposed to a single 405-nm laser pulse with 20% intensity (20 frames/ UV pulse), after which fluorescent signal could be observed. To create a reference, cells in the adjacent somite to the uncaged area in the spinal cord were also subjected to UV illumination. This allowed to create a bright uncaged area in the somite, which could be used after fixation to relocate the area of the uncaged cell in the spinal cord. Embryos were removed from the agarose, allowed to recover for 24 hr at 28.5 C and fixed for subsequent antibody staining.


Immunohistochemistry Immunohistochemistry was performed as previously described (HorneBadovinac et al., 2001) and the following antibodies were used: mouse anti-HuC/D (1:50, Molecular Probes, Eugene, OR) and rabbit anti-fluorescein AlexaFluor 488 conjugate (1:500, Molecular Probes). For fluorescent detection of anti-HuC/D, we used a goat anti-mouse Cy5 conjugated antibody (1:500, Molecular Probes). H2A:GFP and embryos injected with SNAP fusions were fixed in 4% PFA/ PBS at 4 C overnight, or in 10% trichloroacetic acid/water for 10 min at 4 C. Following PFA fixation, embryos were transferred to methanol at 20 C overnight, permeabilized in acetone at 20 C for 5 min, and rehydrated in PBS. After fixation, embryos were rinsed and permeabilized with 0.3% Triton X-100 in PBS for 210 min followed by blocking for 30 min. Prior to mounting, stained tissue was transferred through a series of graded glycerol of 25, 50, and 75%. Embryos were deyolked, flatmounted in glycerol between two coverslips with silicone bridges, and imaged with an Olympus FV1000 confocal microscope.

Image Analysis and Processing Image stacks were opened with the FV10-ASW 1,6 viewer and images saved as tiff files, or opened with the Imaris software to assemble 4D stacks for live cell tracking or uncaging. Using

the ImageJ Bioformats plug-in allowed quantification of fluorescence intensity when necessary.

ACKNOWLEDGMENTS M.G.G. and C.C. were supported by the University of Geneva as well as by ERC advanced investigator (SARA), SystemsX (lipidsX), Swiss National Science Foundation (SNF), NCCR (Chemical Biology), and R’equip (SNF) grants. K.J. was supported by the Swiss National Science Foundation, the Swiss SystemsX.ch initiative, and the Fondation Claude et Giuliana. C.C. was funded by FCT (SFRH/BD/ 15210/2004) and Oncasym. C.C. thanks the PGDB PhD programme and A.C. Borges and M. Franco from IGC for providing advice and technical help for revision experiments. M.K. was supported by a JSPS stipend. K.J. and M.G.G. are members of the NCCR Chemical Biology.

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