DEVELOPMENTAL DYNAMICS 00:000–000, 2014 DOI: 10.1002/DVDY.24109
TECHNIQUES
Combination of In Ovo Electroporation and Time-Lapse Imaging to Study Migrational Events in Chicken Embryos a
Maryna Masyuk,1 Gabriela Morosan-Puopolo,1 Beate Brand-Saberi,1 and Carsten Theiss1,2* 1
Institute of Anatomy, Department of Anatomy and Molecular Embryology, Ruhr-University Bochum, Bochum, Germany Institute of Anatomy, Department of Cytology, Ruhr-University Bochum, Bochum, Germany
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Background: During embryonic development cell migration plays a principal role in several processes. In past decades, many studies were performed to investigate migrational events, occurring during embryonic organogenesis, neurogenesis, gliogenesis or myogenesis, just to name a few. Although different common techniques are already used for this purpose, one of their major limitations is the static character. However, cell migration is a sophisticated and highly dynamic process, wherefore new appropriate technologies are required to investigate this event in all its complexity. Results and Conclusions: Here we report a novel approach for dynamic analysis of cell migration within embryonic tissue. We combine the modern transfection method of in ovo electroporation with the use of tissue slice culture and state-of-the-art imaging techniques, such as confocal laser scanning microscopy or spinning disc confocal microscopy, and thus, develop a method to study live the migration of myogenic precursors in chicken embryos. The conditions and parameters used in this study allow long-term imaging for up to 24 hr. Our protocol can be easily adapted for investigations of a variety of other migrational events and provides a novel conception for dynamic analysis C 2013 Wiley Periodicals, Inc. of migration during embryonic development. Developmental Dynamics 000:000–000, 2014. V Key words: confocal laser scanning microscopy; spinning disc confocal microscopy; tissue slice cultures; Rose chamber Key findings: New appropriate technologies are required to investigate cell migration in all its complexity. We combine in ovo electroporation of cultured chicken embryos with the use of tissue slice culture and state-of-the-art imaging techniques, such as confocal laser scanning microscopy or spinning disc confocal microscopy. The conditions and parameters used in this study allow long-term imaging for up to 24 hr. Submitted 30 July 2013; First Decision 16 December 2013; Accepted 21 December 2013
Introduction Cell migration plays a crucial role in an enormous number of physiological and pathological phenomena, such as hematopoiesis (Bleul et al., 1996), immunological processes (Carr et al., 1994; Springer, 1995), and tumor metastasis (Nicolson, 1993; Yeatman and Nicolson, 1993; Wang et al., 1998; M€ uller et al., 2001). Furthermore, migrational events are essential for several important steps during embryonic development. For instance, active and directed cell movements occur during gastrulation (Warga and Kimmel, 1990; Winklbauer et al., 1996; Nagel et al., 2004; reviewed by Keller, 2005), neural crest cell migration, which is prerequisite for the formation of such derivatives as neurons, glial cells, and melanocytes (Kalcheim and Le Douarin, 1986; Bronner-Fraser, 1993; Krull, 2001) and migration of primordial germ cells toward the gonads (Doitsidou et al., 2002; Knaut et al., 2003; Stebler et al., 2004). Moreover, it has been previously Additional Supporting Information may be found in the online version of this article. Drs. Brand-Saberi and Theiss contributed equally to this work. *Correspondence to: Carsten Theiss, Faculty of Medicine, Institute of Anatomy, Department of Cytology, Ruhr-University Bochum, 44780 Bochum, Germany. E-mail: [email protected]
shown that cell migration also plays a pivotal role during myogenesis. Thus, at the limb and cervical level the myogenic progenitor cells delaminate from the ventrolateral dermomyotome, a compartment of the somite, and actively migrate along defined migratory routes to their target locations to generate the hypaxial muscles of extremities, tongue and diaphragm (Chevallier et al., 1977; Christ et al., 1977; Ordahl and Le Douarin, 1992; Christ and Ordahl, 1995; Brand-Saberi et al., 1996a,b; Christ and Brand-Saberi, 2002). Recently, our and other groups have shown that cloacal muscles and muscles of the shoulder girdle develop by an “In-Out” mechanism, which consists of initial migration into the limb bud followed by a retrograde migration into the trunk region (Valasek et al., 2005, 2011; Rehimi et al., 2010). These groups of muscles are therefore referred to as secondary trunk muscles (reviewed by Yusuf and Brand-Saberi, 2012). To date, studies of the migrational patterns during myogenesis have relied on static morphological data, as approaches for the dynamic analysis of myogenic precursor cell migration within the embryonic tissue were lacking. However, migration of myogenic precursors is a motive process and new technologies are Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24109/abstract C 2013 Wiley Periodicals, Inc. V
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required to investigate this event in all its complexity. In this technical report, we describe the combination of in ovo electroporation and tissue slice-cultures with the state-of-the art imaging techniques of confocal laser scanning microscopy and spinning disc confocal microscopy and, thus, develop a novel approach to study live cell migration in embryonic tissues.
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Results and Discussion In recent years, significant progress has been made in developing new approaches for investigating embryonic myogenesis. Since decades, quail-chicken somitic grafting experiments serve as a tool to study the migration of myogenic precursors and consequently to trace the lineage of different muscle groups or single muscles (Beresford et al., 1978; Brand-Saberi et al., 1989; Zhi et al., 1996; Schweizer et al., 2004). The establishment of in ovo electroporation of avian somites delivered new options to label selectively the myogenic progenitor cells and, thus, to investigate embryonic myogenesis (Swartz et al., 2001a; Scaal et al., 2004). Furthermore, this method allowed directed overexpression or silencing of specific target genes and, therefore, provided a new possibility to study gene function in an efficient way (Swartz et al., 2001b; Dai et al., 2005). Nevertheless, although migration plays a crucial role during embryonic myogenesis, the most studies remained of static nature. Recently, Shiau et al. performed slice time-lapse imaging to visualize the behavior of ectodermal cells during sensory placode formation (Shiau et al., 2011). A similar technique was also adopted by McKinney and colleagues to study premigratory neural crest cells dynamics (McKinney et al., 2013). Here, we present a novel approach for dynamic analysis of myogenic migration within embryonic chicken tissue. We describe in detail the transfection method of in ovo electroporation in combination with the use of tissue slice culture and stateof-the-art imaging techniques like confocal laser scanning microscopy or spinning disc confocal microscopy. Thus, we show that with aid of our protocol of time-lapse imaging migrating myogenic precursors could be observed along their migration routes toward their target locations. More specifically, here, we visualize the migration of myogenic precursors from the somite into the developing limb bud as well as the retrograde migration of individual progenitor cells from the forelimb toward the trunk. Using our novel technique combination, we succeed to confirm the results from previous studies concerning the origin and developing mode of forelimb and shoulder musculature by demonstrating the migration of the myogenic precursors required for the formation of these muscle groups live (Chevallier et al., 1977; Christ et al., 1977; Brand-Saberi et al., 1996a,b; Christ and Brand-Saberi, 2002; Balased et al., 2011). This approach allows a dynamical analysis of the migrational events discussed and in addition live studies of other migrational events in future. We optimized the in ovo electroporation technique according to the used development stages and the transfection area and developed a procedure to prepare transverse slice cultures of the transfected region. Ideal conditions for the embryonic tissue culture were maintained by using a closed Rose-chamber filled with a nutrient medium and combination of different systems ensur ing incubation settings of 37 C and 5% CO2. Furthermore, to avoid damage of the tissue as well as of the fluorophore by phototoxicity, we give detailed instructions to reduce the excitation intensity and the number of time-intervals to a minimum.
As recommended in confocal laser scanning microscopy, an increase of the scanning time, detector gain and amplifier offset of the detection system is able to compensate these limitations on the excitation level. In spinning-disc confocal microscopy, the object is scanned by passing the excitation light through a disc with pinholes, with a microarray of lenses in front of each pinhole (van Munster et al., 2007). The main advantage of spinning disc confocal microscopy is the less photo bleaching (Wang et al., 2005). However, with conventional laser scanning microscopy as well as with aid of spinning disc confocal microscopy both settings enabled us to perform time-lapse imaging for up to 24 hr. During this long period the enhanced green fluorescent protein (EGFP) -labeled precursor cells remained viable and showed a bright FP-signal. Nevertheless, the monitoring period is limited, as the tissue starts to flatten out after 24 hr and the active cell migration is overlaid by this passive cell movement. Therefore, it is critical to think about the best Hamburger and Hamilton (HH) stage for electroporation and following time-lapse imaging. However, by using different stages the whole sequence of migrational events can be recorded. These advanced imaging techniques can be highly useful for investigations of embryonic myogenesis, but also to study other migrational events which occur during development. For instance, the migration of other dermomyotomal derivative progenitors, such as dermogenic precursors, or the migration of neural crest cells, which is prerequisite for neuro- and gliogenesis, and many others can be investigated with this protocol. With the aid of the described methods of electroporation and time-lapse imaging, we recently showed that the dermomyotome ventrolateral lip is essential for the hypaxial myotome formation (Pu et al., 2013). By manipulation of the gene expression by means of in ovo electroporation, the function of certain genes in regard to migrational events can be studied as well. Thus, this innovative method allows dynamic analysis of migration during embryonic development and can provide novel insights into these processes.
Experimental Procedures In Ovo Electroporation Preparation of the embryos Fertilized chicken eggs obtained from a local breeder were incu bated at 37.5 C and 80% relative humidity until the stages 14– 15 according to Hamburger and Hamilton (Hamburger and Hamilton, 1951) were reached. A total of 2–3 ml of albumin were withdrawn with a sterile syringe at the blunt end of the egg to lower the embryo. The upper side of the egg shell was reinforced by adhesive tape and an oval window approximately 2 cm in length was cut using scissors. For a better visualization of the embryo, black drawing ink diluted 1:10 with Locke’s solution containing penicillin G (Penicillin G sodium salt, PENNA, Sigma) was injected beneath the blastoderm using a sterile syringe with a fine needle. The next steps of the procedure were performed under a dissecting microscope (Fig. 1A). The vitelline membrane and the amnion overlaying the embryo were carefully removed with a tungsten needle. To prevent any damage of the blastoderm which can be attached to the vitelline membrane, it is useful to drip a small amount of Locke’s solution on the embryo to detach the vitelline membrane and thus facilitate its removal.
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Fig. 1. Experimental setup for in ovo electroporation of the somites. A–F: The ventrolateral dermomytomes of HH14–HH15 chicken embryos were electroporated using a Tol2-EGFP construct. A: The windowed egg was placed under the dissecting microscope. B,C: Using a borosilicate glass capillary, the Tol2-EGFP-construct mixed with Fast Green-solution was injected into the somites 16 to 21. C–E: During current passing negatively charged DNA moved toward the positive electrode (C,D) and, thus, the ventrolateral dermomyotome was transfected (E). F: After reincubation, at stage HH23, EGFP-labeled myogenic precursors are not only visible in the segmented somites, but numerous cells have already entered the developing limb bud.
Injection of the DNA solution
Electroporation
To ensure that the somites 16 until 21 are labeled, which give rise to myogenic precursor cells of the forelimb bud and the pectoral girdle, they were counted before injection. Here, it is crucial to know that in the stages HH14–HH15 the most cranial pair of the somites starts to disappear; therefore, the first fully formed somite pair was counted as somite pair number two. The Tol2-EGFP vector system (Koga et al., 1996; Kawakami et al., 2000; Kawakami, 2007; Sato et al., 2007) kindly provided as a gift from Koichi Kawakami (Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka 411–8540, Japan) was mixed in concentration of 9–10 mg/ml with transposase. To detect and check the injection, a few crystals of Fast Green FCF (F 7258, Sigma-Aldrich) were added to the plasmid solution as previously reported (Krull, 2004). A borosilicate glass capillary was assembled into a rubber aspirator tube and the Tol2-EGFP-Fast Green-solution was aspirated by mouth. The glass capillary filled with the DNA-solution was inserted into the most caudal somite on the right side of the embryo and pushed carefully in the cranial direction until the somite 16. While drawing back the capillary, the somites were filled with the DNA-solution by blowing it out with the aid of the rubber tube (Fig. 1B).
To ensure the conductivity during electroporation, Locke’s solution was applied on the newly injected area (Fig. 1C–E). The negative electrode (cathode) was placed left and the positive electrode (anode) right of the embryo, parallel to the craniocaudal axis, at a distance of approximately twice the width of the embryo. The uninsulated sides of the electrodes were positioned in line with the somites and five square pulses of 27.5 V, 20-ms pulse-width at 200-ms intervals between the pulses were applied with the aid of the TSS20 Ovodyne electroporator (Intracel, UK). Thus, the negatively charged DNA moved within the electric field toward the ventrolateral dermomyotome adjacent to the anode. During the pulse application, bubbles appear near the electrodes, indicating that the current passes through the embryo. The eggs were resealed with adhesive tape and reincubated for several time-intervals. The success of the procedure was evaluated by visualization of EGFP expression in transfected somitic cells with the aid of a fluorescent stereo microscope (M 165 FC, Leica, Germany) (Fig. 1F). The fluorescence can be observed already 4 hr after electroporation, whereby segmented somites are visible. For more detailed information on the procedure of in ovo electroporation and suggested solutions for possibly occurring problems please refer to the Table 1.
Control of efficiency at least 4 hr after electroporation
Electroporation
Injection
Step
Low or none number of cells are transfected
Embryos are bent, damaged or dead
During injection the DNA-solution spreads above or below the embryo During injection the DNA- solution diffuses out of the somites under the embryo, although the somites were initially entered No bubbles are visible during current pulse application
DNA-solution is flowing out of the capillary before injection or diffusing out of the somites after the injection occurs
There is a big resistance, while blowing DNA-solution out of the capillary into the somites
Problem
Volume of the injected DNA solution was too low
Plasmid DNA is damaged
Old electrodes DNA concentration is too low
Narrow electric field
Old electrodes Voltage is too low
Electrodes are too close to the embryo
Electrodes are dirty (for instance, driedup albumin sticks on the electrodes) Loose connection between the electrodes and the electroporator Voltage is too high
Somites were pierced on the ventral side
You did not hit the somites
Capillary opening is too large Plasmid DNA is too fluid
Adjust the electrode position. Higher distance between the electrodes enlarges the electric field and allows the transfection of a higher number of cells. Please note that an increased distance requires increased voltages Replace the electrodes, if they are older than 4 months Use higher DNA-concentrations. We recommend concentrations of 9–10mg/ml. Ensure that the DNA concentration is at least 2mg/ml Make sure that the DNA-constructs are not contaminated with bacterial DNA. Test the constructs for expression in cell lines Make sure that the entire somite is filled with the DNA-solution
Reduce the voltage. Perform pilot experiments trying different voltages in order to find an appropriate level. In general, keep the voltage as low as possible Increase the distance between the electrodes. Ensure that there is no contact between the electrodes and the embryonic tissue during current passage Replace the electrodes, if they are older than 4 months Increase the voltage
Clean the electrodes in Locke’s solution after every electroporation Make sure that the connection is not impaired
Dilute the DNA-solution with water or prepare a fresh solution Use a new glass capillary When you use as high concentrations as we used in our experiments, it is improbable that the plasmid solution is too fluid. If it is nevertheless the case, you can add carboxymethylcellulose, which will increase the viscosity Reposition the capillary and try again to penetrate the somites Reseal the egg and try again later when the damaged tissue healed
Plasmid DNA is too viscous
Solution Remove a larger part of the glass capillary using small scissors
Opening of the capillary tip is too small
Possible reason
TABLE 1. Troubleshooting During In Ovo Electroporation of Somites in Chicken Embryos
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Fig. 2. Preparation of slice cultures. A: The Tol2-EGFP electroplated chicken embryos were re-incubated for up to 72 hr and then excised from the yolk. B,C: Forelimb bud region of the chicken embryo (red arrow) was cut transversally into approximately 250-mm-thick slices with aid of a McIlwainTM tissue chopper. D,E: Using two small spatulas, the obtained sections were collected from the tissue chopper and placed into ice-cold HANKS solution. F,G: The elected slice (white arrow) was carefully transferred to a custom-made cover slip with a drop of HANKS solution by means of two spatulas. H: After withdrawing the HANKS solution with a pipette, the slice (white arrow) was fixed on the cover slip by adding a plasma clot coagulated with thrombin.
Slice Culture Methods Slice cultures of chicken limb regions were obtained from Tol2EGFP electroporated chicken embryos reincubated for up to 72 hr. The embryos were excised from the yolk with small scissors and collected in ice-cold HANKS solution (Fig. 2A). Thereafter, the embryos were trimmed of residual extraembryonic membranes and in the present study the forelimb bud region was separated from the rest of the body by means of binocular inspection. Limb bud regions of Tol2-EGFP electroporated chicken embryos were cut transversally into approximately 250mm-thick slices with aid of a McIlwain tissue chopper and collected in ice-cold HANKS (Fig. 2B–E). Slices were selected under
visual control with the binocular microscope (Fig. 2F) and carefully transferred to custom-made glasses (Ø 32 mm, no. 1, size 0.13–0.17 mm Kindler, Freiburg, Germany) using two small spatulas (Fig. 2G). Slices were attached to the cover glasses with a plasma clot (10 ml; P3266, Sigma, Germany) coagulated with thrombin (60U/10ml; 605157, Calbiochem, La Jolla, CA) and cov ered with nutrient medium preheated to 37 C (Fig. 2H). The nutrient minimal essential medium (MEM, M2279, Sigma-Aldrich, Germany) was supplemented with 10% fetal horse serum (HS, S9135, Biochrom, Germany), 1% chicken embryonic extract, 0.6% glucose, 1% penicillin (A321-42, Biochrom, Germany), and 1% L-glutamine (G7513, Sigma-Aldrich, Germany).
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Fig. 3. Rose chamber as a closed system for time-lapse imaging. A,B: The setup of a Rose chamber. The glass cover slip (2) with the specimen (red arrowhead) on its upper side is placed in a homemade Teflon chamber (1) and covered with a rubber gasket (3). A second glass cover slip (4) is placed above the gasket, thus creating an enclosed sealed chamber between the two cover slips. Finally, the construction is fixed with a V2A ring (5). C: Two needles are inserted into the chamber through two small apertures on the side of the Teflon chamber. During nutrient medium is slowly injected with a syringe through one needle, the enclosed air can escape out of the chamber through another needle. D: The Rose chamber with the specimen and nutrient medium inside. E,F: Engineering detail drawing of the Teflon chamber (1) and the V2A ring (5).
Time-Lapse Imaging and Image Analysis Confocal laser scanning microscopy For the quality and the reproducibility of live-cell imaging, a microscope system including the excitation lamp, the detection camera, as well as a heated microscope stage and a vibration free table is essential. For imaging the migration pattern of Tol2EGFP transfected myogenic precursor cells within the slice culture, we used a confocal laser scanning microscope (Zeiss LSM 510, Germany) equipped with a laser module containing an Ar laser (488 nm) and a HeNe laser (543 nm) in combination with Zeiss 10-Apochromate lens (Plan-Neofluar, NA 0.3). To main tain the incubation settings at 37 C and 5% CO2 on the microscope stage, a CTI-controller 3700 digital, O2-controller,
Tempcontrol 37-2 digital, and the Incubator Soxygen together with the heating insert P (Zeiss) was used. The coverslips with the slice cultures were placed in a Rose chamber and covered with 2 ml of nutrient medium, taking the advantage of a closed system and minimal distance between the lens and the specimen, and allows us to get best results in terms of brightness, contrast, acuity, and resolution (Figs. 3–5). According to the size of the specimen, which in turn depends on the developmental stage of the examined embryo, different magnifications can be selected. To reduce phototoxicity of the cells and photobleaching of the fluorescent protein (FP) -signal the excitation intensity at the LSM 510 was reduced to a minimum (488 nm, 2%; 543 nm, 11%) and images were taken every 10 min for a period of up to 24
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Fig. 4. Confocal laser scanning microscopy: Time-lapse imaging of Tol2-EGFP labeled myogenic precursor cells within a slice culture. Myogenic precursor cells were labeled with the Tol2-EGFP construct by electroporation of the ventrolateral dermomytomes of HH14 chicken embryos. After re-incubation up to stage HH18, time-lapse imaging on transverse slice cultures of the forelimb region was performed with the aid of confocal laser scanning microscopy. During recording, the EGFP-labeled myogenic progenitors (red arrows) actively migrated from the somite into the developing limb bud and hereby revealed numerous cell divisions and retained apparently normal motility. The EGFP-signal remained highly intense during the entire monitoring period. Time unit: hour. Scale bar ¼ 100 mm.
hr (zoom level 0.9-1; 1024 1024 pixels) (Fig. 4; Supp. Movie S1, which is available online).
Spinning disc confocal microscopy Slices of electroporated embryos were also recorded with aid of a CellVoyager CV1000 system (Visitron, Germany) (Fig. 5; Supp. Movie S2). This spinning disc confocal microscope contains a Nipkow spinning disc with approximately 20,000 pinholes and a second spinning disc containing the same number of microlenses. The main advantages are due to the multi-beam scanning, which result in a significantly reduced photo damage. In combination with the all-in-one unit of a CO2-incubator and controlled temperature inside the stage incubator a high-precision in the x-y-z stage for long periods of up to several days is possible. Imaging conditions were 37 C, 5% CO2, excitation with 488 nm combined with bright field and 10-Apochromate lens (Nikon), imaging positions with 9 fields, z-stack with 10 sections (3 micrometers apart), imaging interval with 10 min/stack. As a result of this time-lapse imaging, active movement of cells showing GFPexpression is detectable over a period of 24 hr without any damage of the tissue and the cells.
Efficiency of In Ovo Electroporation In numerous studies, in ovo electroporation was reported as an efficient gene transfection method for several embryonic tissues in chicken (Nakamura and Funahashi, 2001; Pekarik et al., 2003;
Katahira and Nakamura, 2003; Krull, 2004; Scaal et al., 2004). Here, we use in ovo electroporation of chicken somites as a tool to label the myogenic progenitor cells with EGFP and, thus, to visualize their migration by means of time-lapse imaging. Although the method of in ovo electroporation of avian somites and the associated problems and limitations have been well described in previous studies (Scaal et al., 2004; Dai et al., 2005), parameters such as distance between the electrodes and the voltage are highly dependent on the embryonic stage and the characteristics of the electrodes and have to be optimized for every experimental set-up. Therefore, we hereby provide a table containing problems, possible reasons and proposed solutions for electroporation of avian somites, especially at stages HH14– HH15, which we used in this study.
Combining In Ovo Electroporation, Slice Culturing, and Confocal Time-Lapse Microscopy Allows Live-Cell Imaging of Migrating Myogenic Precursors To examine the migration pattern of myogenic precursor cells at the forelimb level, we electroporated the ventrolateral dermomyotome of HH14–HH15 chicken embryos with the Tol2-EGFP vector system as described above and reincubated them for different periods. The main advantage of the electroporation of theTol2 construct in combination with transposase into chicken somites is to get stably integrated EGFP-genes, which allows us to perform time-lapse imaging of these cells until embryonic day
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Fig. 5. A–F: Spinning disc confocal microscopy: Time-lapse imaging of Tol2-EGFP labeled myogenic precursor cells within a slice culture. Myogenic precursor cells were labeled with the Tol2-EGFP construct by electroporation of the ventrolateral dermomytomes of HH14 chicken embryos. After re-incubation up to stage HH26, time-lapse imaging on transverse slice cultures of the forelimb region was performed with the aid of spinning disc confocal microscopy. During recording, numerous cell divisions and a normal active motility of the EGFP-labeled myogenic progenitor cells (red arrows) are visible. The EGFP-signal retained its intensity during the entire monitoring period. A0 –E0 : Approximately 3 magnification of the regions showing active cell migration from pictures A, C, and E. Time unit: hour. Scale bar ¼ 240 mm.
12 (Sato et al., 2007; Wang et al., 2011). Transverse slice cultures of the forelimb region of these embryos were captured with the aid of a confocal laser scanning microscope or a spinning disc confocal microscope for approximately 24 hr. The combination of these advanced transfection, tissue culturing and imaging techniques allowed us to observe the migrating myogenic precursor cells along their migration pathways and to study their behavior during this process. Here, we demonstrate the myogenic precursor cells delaminating from the ventrolateral dermomytome and actively migrating toward the dorsal and ventral developing limb bud, where they will later form forelimb muscles (Fig. 4). Furthermore, by reincubating the electroporated embryos until a later developmental stage (HJH26), we can observe the retrograde migration of individual cells from the forelimb bud toward the trunk—a process necessary for the formation of secondary trunk musculature such as muscles of the shoulder girdle (Fig. 5). During recording, the EGFP-labeled myogenic progenitor revealed numerous cell divisions and retained apparently normal active motility, indicating the cells were viable and intact. Furthermore, the cells showed an intense EGFP-signal during the entire monitoring period. To reduce photobleaching of the FP-signal and phototoxicity of the cells, the excitation intensity has to be reduced to a minimum and the number of time-intervals should be as low as possible. Additionally, the parameters for the scanning time, the detector gain and amplifier offset of the detection system should be increased to levels obtaining bright FP-signal even with low
excitation power. Another important parameter is the usage of the Tol2-EGFP construct, which allows a stable and sustained EGFPexpression until late developmental stages of chicken embryos in contrast to conventional electroporation techniques (Sato et al., 2007; reviewed by Kawakami, 2007). In all time-lapse imaging experiments, a critical point is to keep the region of interest in the focal plane for a long period. In many cases, thermal instability in the environment of the microscopic system is the reason for the focus shift. Therefore, we set a constant room temperature of 27 C at the LSM, whereas the used spinning disc confocal has the advantage of a closed system, in which all relevant microscopic apertures are temperatured at 37 C. In addition to this, we additionally use the z-stack mode in both systems, with a first and last image of the z-stack outside from the focal plane at the beginning of the time-series. Therefore, variability during the recording still gives best pictures in focus over time-periods of many hours.
Acknowledgments The authors thank Visitron for making available a Cell Voyager CV 1000 spinning-disc unit. The authors further acknowledge A. Lodwig for excellent technical assistance, A. Lenz and A. Conrad for secretarial work as well as Bj€ orn Thielker for the engineering detail drawing. This work was supported by MYORES project (511978) funded by the EU’s Sixth Framework Programme.
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Nagel M, Tahinci E, Symes K, Winklbauer R. 2004. Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling. Development 131:2727–2736. Nicolson GL. 1993. Paracrine and autocrine growth mechanisms in tumor metastasis to specific sites with particular emphasis on brain and lung metastasis. Cancer Metastasis Rev 12:325–343. Ordahl CP, Le Douarin NM. 1992. Two myogenic lineages within the developing somite. Development 114:339–353. Pekarik V, Bourikas D, Miglino N, Joset P, Preiswerk S, Stoeckli ET. 2003. Screening for gene function in chicken embryo using RNAi and electroporation. Nat Biotechnol 21:93–96. Pu Q, Abduelmula A, Masyuk M, Theiss C, Schwandulla D, Hans M, Patel K, Brand-Saberi B, Huang R. 2013. The dermomyotome ventrolateral lip is essential for the hypaxial myotome formation. BMC Dev Biol 13:37. Rehimi R, Khalida N, Yusuf F, Morosan-Puopolo G, Brand-Saberi B. 2010. A novel role of CXCR4 and SDF-1 during migration of cloacal muscle precursors. Dev Dyn 239:1622–1631. Sato Y, Kasai T, Nakagawa S, Tanabe K, Watanabe T, Kawakami K, Takahashi Y. 2007. Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol 305:616–624. Scaal M, Gros J, Lesbros C, Marcelle C. 2004. In ovo electroporation of avian somites. Dev Dyn 229:643–650. Schweizer H, Johnson RL, Brand-Saberi B. 2004. Characterization of migration behavior of myogenic precursor cells in the limb bud with respect to Lmx1b expression. Anat Embryol (Berl) 208:7–18. Shiau CE, Das RM, Storey KG. 2011.An effective assay for high cellular resolution time lapse imaging of sensory placode formation and morphogenesis. BMC Neurosci 12:37. Springer TA. 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57:827–872. Stebler J, Spieler D, Slanchev K, Molyneaux KA, Richter U, Cojocaru V, Tarabykin V, Wylie C, Kessel M, Raz E. 2004. Primordial germ cell migration in the chick and mouse embryo: the role of the chemokine SDF-1/CXCL12. Dev Biol 272:351–361. Swartz M, Eberhart J, Mastick GS, Krull CE. 2001a. Sparking new frontiers: using in vivo electroporation for genetic manipulations. Dev Biol 233:13–21. Swartz ME, Eberhart J, Pasquale EB, Krull CE. 2001b. EphA4/ephrin-A5 interactions in muscle precursor cell migration in the avian forelimb. Development 128:4669–4680. van Munster EB, Goedhart J, Kremers GJ, Manders EMM, Gadella Jr TWJ. 2007. Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy. Cytometry Part A 71A:207–214. Valasek P, Evans DJ, Maina F, Grim M, Patel K. 2005. A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells. Development 132:447–458. Valasek P, Theis S, DeLaurier A, Hinits Y, Luke GN, Otto AM, Minchin J, He L, Christ B, Brooks G, Sang H, Evans DJ, Logan M, Huang R, Patel K. 2011. Cellular and molecular investigations into the development of the pectoral girdle. Dev Biol 357:108–116. Wang JM, Deng X, Gong W, Su S. 1998. Chemokines and their role in tumor growth and metastasis. J Immunol Methods 220:1–17. Wang E, Babbey CM, Dunn KW. 2005. Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems. J Microsc 218: 148–159. Wang H, Bonnet A, Delfini MC, Kawakami K, Takahashi Y, Duprez D. 2011. Stable, conditional, and muscle-fiber-specific expression of electroporated transgenes in chick limb muscle cells. Dev Dyn 240:1223–1232. Warga RM, Kimmel CB. 1990. Cell movements during epiboly and gastrulation in zebrafish. Development 108:569–580. Winklbauer R, Nagel M, Selchow A, Wacker S. 1996. Mesoderm migration in the Xenopus gastrula. Int J Dev Biol 40:305–311. Yeatman TJ, Nicolson GL. 1993. Molecular basis of tumor progression: mechanisms of organ-specific tumor metastasis. Semin Surg Oncol 9:256–263. Yusuf F, Brand-Saberi B. 2012. Myogenesis and muscle regeneration. Histochem Cell Biol 138:187–199. Zhi Q, Huang R, Christ B, Brand-Saberi B. 1996. Participation of individual brachial somites in skeletal muscles of the avian distal wing. Anat Embryol (Berl) 194:327–339.
TECHNIQUES
Combination of In Ovo Electroporation and Time-Lapse Imaging to Study Migrational Events in Chicken Embryos a
Maryna Masyuk,1 Gabriela Morosan-Puopolo,1 Beate Brand-Saberi,1 and Carsten Theiss1,2* 1
Institute of Anatomy, Department of Anatomy and Molecular Embryology, Ruhr-University Bochum, Bochum, Germany Institute of Anatomy, Department of Cytology, Ruhr-University Bochum, Bochum, Germany
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Background: During embryonic development cell migration plays a principal role in several processes. In past decades, many studies were performed to investigate migrational events, occurring during embryonic organogenesis, neurogenesis, gliogenesis or myogenesis, just to name a few. Although different common techniques are already used for this purpose, one of their major limitations is the static character. However, cell migration is a sophisticated and highly dynamic process, wherefore new appropriate technologies are required to investigate this event in all its complexity. Results and Conclusions: Here we report a novel approach for dynamic analysis of cell migration within embryonic tissue. We combine the modern transfection method of in ovo electroporation with the use of tissue slice culture and state-of-the-art imaging techniques, such as confocal laser scanning microscopy or spinning disc confocal microscopy, and thus, develop a method to study live the migration of myogenic precursors in chicken embryos. The conditions and parameters used in this study allow long-term imaging for up to 24 hr. Our protocol can be easily adapted for investigations of a variety of other migrational events and provides a novel conception for dynamic analysis C 2013 Wiley Periodicals, Inc. of migration during embryonic development. Developmental Dynamics 000:000–000, 2014. V Key words: confocal laser scanning microscopy; spinning disc confocal microscopy; tissue slice cultures; Rose chamber Key findings: New appropriate technologies are required to investigate cell migration in all its complexity. We combine in ovo electroporation of cultured chicken embryos with the use of tissue slice culture and state-of-the-art imaging techniques, such as confocal laser scanning microscopy or spinning disc confocal microscopy. The conditions and parameters used in this study allow long-term imaging for up to 24 hr. Submitted 30 July 2013; First Decision 16 December 2013; Accepted 21 December 2013
Introduction Cell migration plays a crucial role in an enormous number of physiological and pathological phenomena, such as hematopoiesis (Bleul et al., 1996), immunological processes (Carr et al., 1994; Springer, 1995), and tumor metastasis (Nicolson, 1993; Yeatman and Nicolson, 1993; Wang et al., 1998; M€ uller et al., 2001). Furthermore, migrational events are essential for several important steps during embryonic development. For instance, active and directed cell movements occur during gastrulation (Warga and Kimmel, 1990; Winklbauer et al., 1996; Nagel et al., 2004; reviewed by Keller, 2005), neural crest cell migration, which is prerequisite for the formation of such derivatives as neurons, glial cells, and melanocytes (Kalcheim and Le Douarin, 1986; Bronner-Fraser, 1993; Krull, 2001) and migration of primordial germ cells toward the gonads (Doitsidou et al., 2002; Knaut et al., 2003; Stebler et al., 2004). Moreover, it has been previously Additional Supporting Information may be found in the online version of this article. Drs. Brand-Saberi and Theiss contributed equally to this work. *Correspondence to: Carsten Theiss, Faculty of Medicine, Institute of Anatomy, Department of Cytology, Ruhr-University Bochum, 44780 Bochum, Germany. E-mail: [email protected]
shown that cell migration also plays a pivotal role during myogenesis. Thus, at the limb and cervical level the myogenic progenitor cells delaminate from the ventrolateral dermomyotome, a compartment of the somite, and actively migrate along defined migratory routes to their target locations to generate the hypaxial muscles of extremities, tongue and diaphragm (Chevallier et al., 1977; Christ et al., 1977; Ordahl and Le Douarin, 1992; Christ and Ordahl, 1995; Brand-Saberi et al., 1996a,b; Christ and Brand-Saberi, 2002). Recently, our and other groups have shown that cloacal muscles and muscles of the shoulder girdle develop by an “In-Out” mechanism, which consists of initial migration into the limb bud followed by a retrograde migration into the trunk region (Valasek et al., 2005, 2011; Rehimi et al., 2010). These groups of muscles are therefore referred to as secondary trunk muscles (reviewed by Yusuf and Brand-Saberi, 2012). To date, studies of the migrational patterns during myogenesis have relied on static morphological data, as approaches for the dynamic analysis of myogenic precursor cell migration within the embryonic tissue were lacking. However, migration of myogenic precursors is a motive process and new technologies are Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24109/abstract C 2013 Wiley Periodicals, Inc. V
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required to investigate this event in all its complexity. In this technical report, we describe the combination of in ovo electroporation and tissue slice-cultures with the state-of-the art imaging techniques of confocal laser scanning microscopy and spinning disc confocal microscopy and, thus, develop a novel approach to study live cell migration in embryonic tissues.
DEVELOPMENTAL DYNAMICS
Results and Discussion In recent years, significant progress has been made in developing new approaches for investigating embryonic myogenesis. Since decades, quail-chicken somitic grafting experiments serve as a tool to study the migration of myogenic precursors and consequently to trace the lineage of different muscle groups or single muscles (Beresford et al., 1978; Brand-Saberi et al., 1989; Zhi et al., 1996; Schweizer et al., 2004). The establishment of in ovo electroporation of avian somites delivered new options to label selectively the myogenic progenitor cells and, thus, to investigate embryonic myogenesis (Swartz et al., 2001a; Scaal et al., 2004). Furthermore, this method allowed directed overexpression or silencing of specific target genes and, therefore, provided a new possibility to study gene function in an efficient way (Swartz et al., 2001b; Dai et al., 2005). Nevertheless, although migration plays a crucial role during embryonic myogenesis, the most studies remained of static nature. Recently, Shiau et al. performed slice time-lapse imaging to visualize the behavior of ectodermal cells during sensory placode formation (Shiau et al., 2011). A similar technique was also adopted by McKinney and colleagues to study premigratory neural crest cells dynamics (McKinney et al., 2013). Here, we present a novel approach for dynamic analysis of myogenic migration within embryonic chicken tissue. We describe in detail the transfection method of in ovo electroporation in combination with the use of tissue slice culture and stateof-the-art imaging techniques like confocal laser scanning microscopy or spinning disc confocal microscopy. Thus, we show that with aid of our protocol of time-lapse imaging migrating myogenic precursors could be observed along their migration routes toward their target locations. More specifically, here, we visualize the migration of myogenic precursors from the somite into the developing limb bud as well as the retrograde migration of individual progenitor cells from the forelimb toward the trunk. Using our novel technique combination, we succeed to confirm the results from previous studies concerning the origin and developing mode of forelimb and shoulder musculature by demonstrating the migration of the myogenic precursors required for the formation of these muscle groups live (Chevallier et al., 1977; Christ et al., 1977; Brand-Saberi et al., 1996a,b; Christ and Brand-Saberi, 2002; Balased et al., 2011). This approach allows a dynamical analysis of the migrational events discussed and in addition live studies of other migrational events in future. We optimized the in ovo electroporation technique according to the used development stages and the transfection area and developed a procedure to prepare transverse slice cultures of the transfected region. Ideal conditions for the embryonic tissue culture were maintained by using a closed Rose-chamber filled with a nutrient medium and combination of different systems ensur ing incubation settings of 37 C and 5% CO2. Furthermore, to avoid damage of the tissue as well as of the fluorophore by phototoxicity, we give detailed instructions to reduce the excitation intensity and the number of time-intervals to a minimum.
As recommended in confocal laser scanning microscopy, an increase of the scanning time, detector gain and amplifier offset of the detection system is able to compensate these limitations on the excitation level. In spinning-disc confocal microscopy, the object is scanned by passing the excitation light through a disc with pinholes, with a microarray of lenses in front of each pinhole (van Munster et al., 2007). The main advantage of spinning disc confocal microscopy is the less photo bleaching (Wang et al., 2005). However, with conventional laser scanning microscopy as well as with aid of spinning disc confocal microscopy both settings enabled us to perform time-lapse imaging for up to 24 hr. During this long period the enhanced green fluorescent protein (EGFP) -labeled precursor cells remained viable and showed a bright FP-signal. Nevertheless, the monitoring period is limited, as the tissue starts to flatten out after 24 hr and the active cell migration is overlaid by this passive cell movement. Therefore, it is critical to think about the best Hamburger and Hamilton (HH) stage for electroporation and following time-lapse imaging. However, by using different stages the whole sequence of migrational events can be recorded. These advanced imaging techniques can be highly useful for investigations of embryonic myogenesis, but also to study other migrational events which occur during development. For instance, the migration of other dermomyotomal derivative progenitors, such as dermogenic precursors, or the migration of neural crest cells, which is prerequisite for neuro- and gliogenesis, and many others can be investigated with this protocol. With the aid of the described methods of electroporation and time-lapse imaging, we recently showed that the dermomyotome ventrolateral lip is essential for the hypaxial myotome formation (Pu et al., 2013). By manipulation of the gene expression by means of in ovo electroporation, the function of certain genes in regard to migrational events can be studied as well. Thus, this innovative method allows dynamic analysis of migration during embryonic development and can provide novel insights into these processes.
Experimental Procedures In Ovo Electroporation Preparation of the embryos Fertilized chicken eggs obtained from a local breeder were incu bated at 37.5 C and 80% relative humidity until the stages 14– 15 according to Hamburger and Hamilton (Hamburger and Hamilton, 1951) were reached. A total of 2–3 ml of albumin were withdrawn with a sterile syringe at the blunt end of the egg to lower the embryo. The upper side of the egg shell was reinforced by adhesive tape and an oval window approximately 2 cm in length was cut using scissors. For a better visualization of the embryo, black drawing ink diluted 1:10 with Locke’s solution containing penicillin G (Penicillin G sodium salt, PENNA, Sigma) was injected beneath the blastoderm using a sterile syringe with a fine needle. The next steps of the procedure were performed under a dissecting microscope (Fig. 1A). The vitelline membrane and the amnion overlaying the embryo were carefully removed with a tungsten needle. To prevent any damage of the blastoderm which can be attached to the vitelline membrane, it is useful to drip a small amount of Locke’s solution on the embryo to detach the vitelline membrane and thus facilitate its removal.
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IN OVO ELECTROPORATION AND TIME-LAPSE IMAGING 3
Fig. 1. Experimental setup for in ovo electroporation of the somites. A–F: The ventrolateral dermomytomes of HH14–HH15 chicken embryos were electroporated using a Tol2-EGFP construct. A: The windowed egg was placed under the dissecting microscope. B,C: Using a borosilicate glass capillary, the Tol2-EGFP-construct mixed with Fast Green-solution was injected into the somites 16 to 21. C–E: During current passing negatively charged DNA moved toward the positive electrode (C,D) and, thus, the ventrolateral dermomyotome was transfected (E). F: After reincubation, at stage HH23, EGFP-labeled myogenic precursors are not only visible in the segmented somites, but numerous cells have already entered the developing limb bud.
Injection of the DNA solution
Electroporation
To ensure that the somites 16 until 21 are labeled, which give rise to myogenic precursor cells of the forelimb bud and the pectoral girdle, they were counted before injection. Here, it is crucial to know that in the stages HH14–HH15 the most cranial pair of the somites starts to disappear; therefore, the first fully formed somite pair was counted as somite pair number two. The Tol2-EGFP vector system (Koga et al., 1996; Kawakami et al., 2000; Kawakami, 2007; Sato et al., 2007) kindly provided as a gift from Koichi Kawakami (Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka 411–8540, Japan) was mixed in concentration of 9–10 mg/ml with transposase. To detect and check the injection, a few crystals of Fast Green FCF (F 7258, Sigma-Aldrich) were added to the plasmid solution as previously reported (Krull, 2004). A borosilicate glass capillary was assembled into a rubber aspirator tube and the Tol2-EGFP-Fast Green-solution was aspirated by mouth. The glass capillary filled with the DNA-solution was inserted into the most caudal somite on the right side of the embryo and pushed carefully in the cranial direction until the somite 16. While drawing back the capillary, the somites were filled with the DNA-solution by blowing it out with the aid of the rubber tube (Fig. 1B).
To ensure the conductivity during electroporation, Locke’s solution was applied on the newly injected area (Fig. 1C–E). The negative electrode (cathode) was placed left and the positive electrode (anode) right of the embryo, parallel to the craniocaudal axis, at a distance of approximately twice the width of the embryo. The uninsulated sides of the electrodes were positioned in line with the somites and five square pulses of 27.5 V, 20-ms pulse-width at 200-ms intervals between the pulses were applied with the aid of the TSS20 Ovodyne electroporator (Intracel, UK). Thus, the negatively charged DNA moved within the electric field toward the ventrolateral dermomyotome adjacent to the anode. During the pulse application, bubbles appear near the electrodes, indicating that the current passes through the embryo. The eggs were resealed with adhesive tape and reincubated for several time-intervals. The success of the procedure was evaluated by visualization of EGFP expression in transfected somitic cells with the aid of a fluorescent stereo microscope (M 165 FC, Leica, Germany) (Fig. 1F). The fluorescence can be observed already 4 hr after electroporation, whereby segmented somites are visible. For more detailed information on the procedure of in ovo electroporation and suggested solutions for possibly occurring problems please refer to the Table 1.
Control of efficiency at least 4 hr after electroporation
Electroporation
Injection
Step
Low or none number of cells are transfected
Embryos are bent, damaged or dead
During injection the DNA-solution spreads above or below the embryo During injection the DNA- solution diffuses out of the somites under the embryo, although the somites were initially entered No bubbles are visible during current pulse application
DNA-solution is flowing out of the capillary before injection or diffusing out of the somites after the injection occurs
There is a big resistance, while blowing DNA-solution out of the capillary into the somites
Problem
Volume of the injected DNA solution was too low
Plasmid DNA is damaged
Old electrodes DNA concentration is too low
Narrow electric field
Old electrodes Voltage is too low
Electrodes are too close to the embryo
Electrodes are dirty (for instance, driedup albumin sticks on the electrodes) Loose connection between the electrodes and the electroporator Voltage is too high
Somites were pierced on the ventral side
You did not hit the somites
Capillary opening is too large Plasmid DNA is too fluid
Adjust the electrode position. Higher distance between the electrodes enlarges the electric field and allows the transfection of a higher number of cells. Please note that an increased distance requires increased voltages Replace the electrodes, if they are older than 4 months Use higher DNA-concentrations. We recommend concentrations of 9–10mg/ml. Ensure that the DNA concentration is at least 2mg/ml Make sure that the DNA-constructs are not contaminated with bacterial DNA. Test the constructs for expression in cell lines Make sure that the entire somite is filled with the DNA-solution
Reduce the voltage. Perform pilot experiments trying different voltages in order to find an appropriate level. In general, keep the voltage as low as possible Increase the distance between the electrodes. Ensure that there is no contact between the electrodes and the embryonic tissue during current passage Replace the electrodes, if they are older than 4 months Increase the voltage
Clean the electrodes in Locke’s solution after every electroporation Make sure that the connection is not impaired
Dilute the DNA-solution with water or prepare a fresh solution Use a new glass capillary When you use as high concentrations as we used in our experiments, it is improbable that the plasmid solution is too fluid. If it is nevertheless the case, you can add carboxymethylcellulose, which will increase the viscosity Reposition the capillary and try again to penetrate the somites Reseal the egg and try again later when the damaged tissue healed
Plasmid DNA is too viscous
Solution Remove a larger part of the glass capillary using small scissors
Opening of the capillary tip is too small
Possible reason
TABLE 1. Troubleshooting During In Ovo Electroporation of Somites in Chicken Embryos
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IN OVO ELECTROPORATION AND TIME-LAPSE IMAGING 5
Fig. 2. Preparation of slice cultures. A: The Tol2-EGFP electroplated chicken embryos were re-incubated for up to 72 hr and then excised from the yolk. B,C: Forelimb bud region of the chicken embryo (red arrow) was cut transversally into approximately 250-mm-thick slices with aid of a McIlwainTM tissue chopper. D,E: Using two small spatulas, the obtained sections were collected from the tissue chopper and placed into ice-cold HANKS solution. F,G: The elected slice (white arrow) was carefully transferred to a custom-made cover slip with a drop of HANKS solution by means of two spatulas. H: After withdrawing the HANKS solution with a pipette, the slice (white arrow) was fixed on the cover slip by adding a plasma clot coagulated with thrombin.
Slice Culture Methods Slice cultures of chicken limb regions were obtained from Tol2EGFP electroporated chicken embryos reincubated for up to 72 hr. The embryos were excised from the yolk with small scissors and collected in ice-cold HANKS solution (Fig. 2A). Thereafter, the embryos were trimmed of residual extraembryonic membranes and in the present study the forelimb bud region was separated from the rest of the body by means of binocular inspection. Limb bud regions of Tol2-EGFP electroporated chicken embryos were cut transversally into approximately 250mm-thick slices with aid of a McIlwain tissue chopper and collected in ice-cold HANKS (Fig. 2B–E). Slices were selected under
visual control with the binocular microscope (Fig. 2F) and carefully transferred to custom-made glasses (Ø 32 mm, no. 1, size 0.13–0.17 mm Kindler, Freiburg, Germany) using two small spatulas (Fig. 2G). Slices were attached to the cover glasses with a plasma clot (10 ml; P3266, Sigma, Germany) coagulated with thrombin (60U/10ml; 605157, Calbiochem, La Jolla, CA) and cov ered with nutrient medium preheated to 37 C (Fig. 2H). The nutrient minimal essential medium (MEM, M2279, Sigma-Aldrich, Germany) was supplemented with 10% fetal horse serum (HS, S9135, Biochrom, Germany), 1% chicken embryonic extract, 0.6% glucose, 1% penicillin (A321-42, Biochrom, Germany), and 1% L-glutamine (G7513, Sigma-Aldrich, Germany).
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Fig. 3. Rose chamber as a closed system for time-lapse imaging. A,B: The setup of a Rose chamber. The glass cover slip (2) with the specimen (red arrowhead) on its upper side is placed in a homemade Teflon chamber (1) and covered with a rubber gasket (3). A second glass cover slip (4) is placed above the gasket, thus creating an enclosed sealed chamber between the two cover slips. Finally, the construction is fixed with a V2A ring (5). C: Two needles are inserted into the chamber through two small apertures on the side of the Teflon chamber. During nutrient medium is slowly injected with a syringe through one needle, the enclosed air can escape out of the chamber through another needle. D: The Rose chamber with the specimen and nutrient medium inside. E,F: Engineering detail drawing of the Teflon chamber (1) and the V2A ring (5).
Time-Lapse Imaging and Image Analysis Confocal laser scanning microscopy For the quality and the reproducibility of live-cell imaging, a microscope system including the excitation lamp, the detection camera, as well as a heated microscope stage and a vibration free table is essential. For imaging the migration pattern of Tol2EGFP transfected myogenic precursor cells within the slice culture, we used a confocal laser scanning microscope (Zeiss LSM 510, Germany) equipped with a laser module containing an Ar laser (488 nm) and a HeNe laser (543 nm) in combination with Zeiss 10-Apochromate lens (Plan-Neofluar, NA 0.3). To main tain the incubation settings at 37 C and 5% CO2 on the microscope stage, a CTI-controller 3700 digital, O2-controller,
Tempcontrol 37-2 digital, and the Incubator Soxygen together with the heating insert P (Zeiss) was used. The coverslips with the slice cultures were placed in a Rose chamber and covered with 2 ml of nutrient medium, taking the advantage of a closed system and minimal distance between the lens and the specimen, and allows us to get best results in terms of brightness, contrast, acuity, and resolution (Figs. 3–5). According to the size of the specimen, which in turn depends on the developmental stage of the examined embryo, different magnifications can be selected. To reduce phototoxicity of the cells and photobleaching of the fluorescent protein (FP) -signal the excitation intensity at the LSM 510 was reduced to a minimum (488 nm, 2%; 543 nm, 11%) and images were taken every 10 min for a period of up to 24
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IN OVO ELECTROPORATION AND TIME-LAPSE IMAGING 7
Fig. 4. Confocal laser scanning microscopy: Time-lapse imaging of Tol2-EGFP labeled myogenic precursor cells within a slice culture. Myogenic precursor cells were labeled with the Tol2-EGFP construct by electroporation of the ventrolateral dermomytomes of HH14 chicken embryos. After re-incubation up to stage HH18, time-lapse imaging on transverse slice cultures of the forelimb region was performed with the aid of confocal laser scanning microscopy. During recording, the EGFP-labeled myogenic progenitors (red arrows) actively migrated from the somite into the developing limb bud and hereby revealed numerous cell divisions and retained apparently normal motility. The EGFP-signal remained highly intense during the entire monitoring period. Time unit: hour. Scale bar ¼ 100 mm.
hr (zoom level 0.9-1; 1024 1024 pixels) (Fig. 4; Supp. Movie S1, which is available online).
Spinning disc confocal microscopy Slices of electroporated embryos were also recorded with aid of a CellVoyager CV1000 system (Visitron, Germany) (Fig. 5; Supp. Movie S2). This spinning disc confocal microscope contains a Nipkow spinning disc with approximately 20,000 pinholes and a second spinning disc containing the same number of microlenses. The main advantages are due to the multi-beam scanning, which result in a significantly reduced photo damage. In combination with the all-in-one unit of a CO2-incubator and controlled temperature inside the stage incubator a high-precision in the x-y-z stage for long periods of up to several days is possible. Imaging conditions were 37 C, 5% CO2, excitation with 488 nm combined with bright field and 10-Apochromate lens (Nikon), imaging positions with 9 fields, z-stack with 10 sections (3 micrometers apart), imaging interval with 10 min/stack. As a result of this time-lapse imaging, active movement of cells showing GFPexpression is detectable over a period of 24 hr without any damage of the tissue and the cells.
Efficiency of In Ovo Electroporation In numerous studies, in ovo electroporation was reported as an efficient gene transfection method for several embryonic tissues in chicken (Nakamura and Funahashi, 2001; Pekarik et al., 2003;
Katahira and Nakamura, 2003; Krull, 2004; Scaal et al., 2004). Here, we use in ovo electroporation of chicken somites as a tool to label the myogenic progenitor cells with EGFP and, thus, to visualize their migration by means of time-lapse imaging. Although the method of in ovo electroporation of avian somites and the associated problems and limitations have been well described in previous studies (Scaal et al., 2004; Dai et al., 2005), parameters such as distance between the electrodes and the voltage are highly dependent on the embryonic stage and the characteristics of the electrodes and have to be optimized for every experimental set-up. Therefore, we hereby provide a table containing problems, possible reasons and proposed solutions for electroporation of avian somites, especially at stages HH14– HH15, which we used in this study.
Combining In Ovo Electroporation, Slice Culturing, and Confocal Time-Lapse Microscopy Allows Live-Cell Imaging of Migrating Myogenic Precursors To examine the migration pattern of myogenic precursor cells at the forelimb level, we electroporated the ventrolateral dermomyotome of HH14–HH15 chicken embryos with the Tol2-EGFP vector system as described above and reincubated them for different periods. The main advantage of the electroporation of theTol2 construct in combination with transposase into chicken somites is to get stably integrated EGFP-genes, which allows us to perform time-lapse imaging of these cells until embryonic day
DEVELOPMENTAL DYNAMICS
8 MASYUK ET AL.
Fig. 5. A–F: Spinning disc confocal microscopy: Time-lapse imaging of Tol2-EGFP labeled myogenic precursor cells within a slice culture. Myogenic precursor cells were labeled with the Tol2-EGFP construct by electroporation of the ventrolateral dermomytomes of HH14 chicken embryos. After re-incubation up to stage HH26, time-lapse imaging on transverse slice cultures of the forelimb region was performed with the aid of spinning disc confocal microscopy. During recording, numerous cell divisions and a normal active motility of the EGFP-labeled myogenic progenitor cells (red arrows) are visible. The EGFP-signal retained its intensity during the entire monitoring period. A0 –E0 : Approximately 3 magnification of the regions showing active cell migration from pictures A, C, and E. Time unit: hour. Scale bar ¼ 240 mm.
12 (Sato et al., 2007; Wang et al., 2011). Transverse slice cultures of the forelimb region of these embryos were captured with the aid of a confocal laser scanning microscope or a spinning disc confocal microscope for approximately 24 hr. The combination of these advanced transfection, tissue culturing and imaging techniques allowed us to observe the migrating myogenic precursor cells along their migration pathways and to study their behavior during this process. Here, we demonstrate the myogenic precursor cells delaminating from the ventrolateral dermomytome and actively migrating toward the dorsal and ventral developing limb bud, where they will later form forelimb muscles (Fig. 4). Furthermore, by reincubating the electroporated embryos until a later developmental stage (HJH26), we can observe the retrograde migration of individual cells from the forelimb bud toward the trunk—a process necessary for the formation of secondary trunk musculature such as muscles of the shoulder girdle (Fig. 5). During recording, the EGFP-labeled myogenic progenitor revealed numerous cell divisions and retained apparently normal active motility, indicating the cells were viable and intact. Furthermore, the cells showed an intense EGFP-signal during the entire monitoring period. To reduce photobleaching of the FP-signal and phototoxicity of the cells, the excitation intensity has to be reduced to a minimum and the number of time-intervals should be as low as possible. Additionally, the parameters for the scanning time, the detector gain and amplifier offset of the detection system should be increased to levels obtaining bright FP-signal even with low
excitation power. Another important parameter is the usage of the Tol2-EGFP construct, which allows a stable and sustained EGFPexpression until late developmental stages of chicken embryos in contrast to conventional electroporation techniques (Sato et al., 2007; reviewed by Kawakami, 2007). In all time-lapse imaging experiments, a critical point is to keep the region of interest in the focal plane for a long period. In many cases, thermal instability in the environment of the microscopic system is the reason for the focus shift. Therefore, we set a constant room temperature of 27 C at the LSM, whereas the used spinning disc confocal has the advantage of a closed system, in which all relevant microscopic apertures are temperatured at 37 C. In addition to this, we additionally use the z-stack mode in both systems, with a first and last image of the z-stack outside from the focal plane at the beginning of the time-series. Therefore, variability during the recording still gives best pictures in focus over time-periods of many hours.
Acknowledgments The authors thank Visitron for making available a Cell Voyager CV 1000 spinning-disc unit. The authors further acknowledge A. Lodwig for excellent technical assistance, A. Lenz and A. Conrad for secretarial work as well as Bj€ orn Thielker for the engineering detail drawing. This work was supported by MYORES project (511978) funded by the EU’s Sixth Framework Programme.
IN OVO ELECTROPORATION AND TIME-LAPSE IMAGING 9
DEVELOPMENTAL DYNAMICS
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