A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.
Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.
Congenital eye diseases can cause childhood blindness and are often due to abnormalities of the cranial neural crest. Neural crest cells are transient stem cells that arise from the neural tube and form numerous tissues throughout the body.1,2,3,4,5 Neural crest cells, derived from the prosencephalon and mesencephalon, give rise to the bone and cartilage of the midface and frontal regions, and the iris, cornea, trabecular meshwork, and sclera in the anterior segment of the eye.4,6,7,8 Neural crest cells from the rhombencephalon form the pharyngeal arches, jaw, and cardiac outflow tract.1,3,4,9,10 Studies have highlighted the contributions of the neural crest to ocular and periocular development, emphasizing the importance of these cells in vertebrate eye development. Indeed, disruption of neural crest cell migration and differentiation lead to craniofacial and ocular anomalies as observed in Axenfeld-Rieger Syndrome and Peters Plus Syndrome.11,12,13,14,15,16,17 Thus, a comprehensive understanding of the migration, proliferation and differentiation of these neural crest cells will provide insight into the complexities underlying congenital eye diseases.
The zebrafish is a powerful model organism for studying ocular development, as the structures of the zebrafish eye are similar to their mammalian counterparts, and many genes are evolutionarily conserved between zebrafish and mammals.18,19,20 In addition, zebrafish embryos are transparent and oviparous, facilitating the visualization of eye development in real-time.
Expanding on previously published work,6,7,20 the migratory pattern of neural crest cells was described using multi-photon fluorescence time-lapse imaging on transgenic zebrafish lines labeled with green fluorescent protein (GFP) under the transcriptional control of the SRY (sex-determining region Y)-box 10 (sox10) or Forkhead Box D3 (foxd3) gene regulatory regions.21,22,23,24. Multi-photon fluorescence time-lapse imaging is a powerful technique that combines the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with fluorophores.25,26,27 The use of the multi-photon laser has distinct advantages over standard confocal microscopy, including increased tissue penetration and decreased fluorophore bleaching.
Using this method, two distinct populations of neural crest cells varying in timing of migration and migratory pathways were discriminated, namely foxd3-positive neural crest cells in the periocular mesenchyme and developing eye and sox10-positive neural crest cells in the craniofacial mesenchyme. With this method, an approach to visualize the migration of ocular and craniofacial neural crest migration in zebrafish is introduced, making it easy to observe regulated neural crest migration in real time during development.
This protocol provides information for generating time-lapse videos during early eye development in Tg(sox10:EGFP) and Tg(foxd3:GFP) transgenic zebrafish, as an example. This protocol can be further applied for the high-resolution, three-dimensional, real-time visualization of the early development of any ocular and craniofacial structure derived from neural crest cells in zebrafish. Moreover, this method can further be applied for the visualization of the development of other tissues and organs in zebrafish and other animal models.
The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
2. Mounting of Embryo for Time-lapse Imaging
3. Microscope Set-up for Time-lapse Imaging
4. During Time-lapse Acquisition
5. Post-acquisition processing
Multi-photon fluorescence time-lapse imaging generated a series of videos that revealed the migration patterns of cranial neural crest cells that give rise to the craniofacial structures and anterior segment of the eye in the Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish lines. As an example, sox10-positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the craniofacial region (Video 1, Figure 2). The cells from the prosencephalon and mesencephalon migrate dorsal and ventral to the developing eye to populate the periocular mesenchyme. In addition, these sox10-positive cells form the frontonasal process. Neural crest cells from the rhombencephalon migrate ventrally and give rise to the pharyngeal arches. Time-lapse imaging of foxd3-positive neural crest cells between 30 and 60 hpf showed that these cells migrated between the optic cup and surface ectoderm and through the ocular fissure (Video 2, Figure 3). These foxd3-positive cells coalesced around the lens, forming the iris stroma.
Figure 1.Setup. A. Each component of the embryo mounting apparatus, including the open bath chamber, quick exchange platform and stage adapter. B. Assembly of the open bath chamber. C. Addition of 2% agarose solution (60-70 °C) to the open bath chamber. D. Placement of the embryo in the open bath chamber under a fluorescent microscope. E. Embryo (at 24 hpf) positioned laterally in the center of the polymerized agarose solution in the open bath chamber. F. Addition of media to the surface of the polymerized agarose solution to completely cover the embryo in the open bath chamber. G. Open bath chamber placed in the quick exchange platform and positioned in the stage adapter. H. Placement of the embryo mounting apparatus on the stage of the multi-photon microscope. I. Laser safety box. The sliding doors (1 and 2) for refilling the open chamber are indicated. Please click here to view a larger version of this figure.
Figure 2. Representative deconvolved and max-projected images from multi-photon time-lapse imaging of a Tg(sox10:EGFP) zebrafish embryo from 12 to 30 hpf. The sox10-positive cells migrated from the prosencephalon (P) and mesencephalon (M) to populate the periocular mesenchyme (the dotted circle denotes the eye) and frontonasal process (open arrow). Sox10-positive cells from the rhombencephalon (R) migrated ventrally to form the pharyngeal arches (closed arrow). Images were obtained every 20 min during the time frame and sewn together to create Video 1. Please click here to view a larger version of this figure.
Figure 3. Representative deconvolved and max-projected images from multi-photon time-lapse imaging of a Tg(foxd3:GFP) zebrafish embryo from 24 to 48 hpf. Foxd3-positive cells entered into the anterior chamber of the eye (asterisk denotes lens) between the surface ectoderm and optic cup, with more cells localized to the dorsal ("D")-posterior ("P") quadrant compared with the anterior ("A") and ventral ("V") quadrants. In addition, foxd3-positive cells migrated adjacent to and through the ocular fissure. Images were obtained every 20 min during the time frame and sewn together to create Video 2. Please click here to view a larger version of this figure.
Figure 4. Representative deconvolved and max-projected images from multi-photon time-lapse imaging of a Tg(foxd3:GFP) zebrafish embryo from 30 to 60 hpf. Foxd3-positive cells entered into the anterior chamber of the eye (outer dotted circle denotes peripheral edges of the eye, inner dotted circle denotes lens) between the surface ectoderm and optic cup, with more cells localized to the dorsal ("d")-posterior ("p") quadrant compared with the anterior ("a") and ventral ("v") quadrants. In addition, foxd3-positive cells migrated adjacent to and through the ocular fissure (white arrowhead denotes migrating neural crest, red dashed line demarcates ocular fissure). At 60 hpf, foxd3-positive cells completely encircled the lens (closed arrows), indicating the closure of the fissure. In addition, at 60 hpf, foxd3 was also expressed in photoreceptors (open arrowhead), which was not associated with the expression of this transcription factor in neural crest cells. Images were obtained every 20 min during the time frame and sewn together to create Video 3. Please click here to view a larger version of this figure.
Video 1. Time-lapse video of a Tg(sox10:EGFP) zebrafish embryo from 12 to 30 hpf. The asterisk denotes the eye. Please click here to download this video.
Video 2. Time-lapse video of a Tg(foxd3:GFP) zebrafish embryo from 24 to 48 hpf. The asterisk denotes the lens. The asterisk denotes the ocular fissure. d, dorsal; v, ventral; p, posterior; a, anterior. Please click here to download this video.
Video 3. Time-lapse video of a Tg(foxd3:GFP) zebrafish embryo from 30 to 60 hpf. The asterisk denotes the lens. The arrow denotes the ocular fissure. d, dorsal; v, ventral; p, posterior; a, anterior. Please click here to download this video.
Multi-photon time-lapse imaging enables the in vivo tracking of transient and migratory cell populations. This powerful technique can be used to study embryonic processes in real time, and in the present study, the results of this method enhanced the current knowledge of neural crest cell migration and development. Previous time-lapse imaging studies typically utilize confocal laser scanning microscopy.29,30,31,32 Here, we present the use of a multi-photon technique, which has many advantages over traditional confocal microscopy. The basis of multi-photon microscopy is that two photons of longer infrared wavelengths are used to excite the fluorophore. As a result, there is less scattering and thus decreased background, enabling deeper tissue penetration, achieving imaging depths exceeding 1 mm in biological tissue with more efficient detection than confocal microscopy. These properties also decrease phototoxicity, which is an important consideration with repeated and frequent image acquisition.25,26,27 From these properties, two-photon microscopy can image whole-organ preparations and tissue in small-animal models, (e.g. mice, zebrafish embryos at 12 hpf – as in the case of the present study). Further, these small animal models enable use of genetically encoded fluorescent proteins, which can be localized in the tissue of interest. Thus, the use of multi-photon microscopy can greatly enhance image acquisition for time-lapse experiments.
There are numerous steps in this protocol, which depend on the type of multi-photon laser and detection system being used. Most problems with this protocol arise with the laser settings and acquisition of images based on the detection system used. A working knowledge of the individual system and access to technical support are critical. In addition, previous experience with confocal laser scanning microscopy is helpful for troubleshooting. Moreover, the initial system set up may be time consuming. However, once the system set up is completed, only small adjustments are typically required when using the same transgenic lines.
In the present study, zebrafish embryos between 12 and 60 hpf were used for these experiments. During this age range, the embryos are small, easily embedded in agarose and readily anesthetized with tricaine. However, the embryos can become growth and developmentally delayed depending on the length of the experiment. Therefore, care must be taken to ensure that the embryo is appropriately staged at the end of the experiment.
Multi-photon time-lapse imaging yields high-resolution, three-dimensional, real-time visualization of cell migration, which not only enhances the capability to study in vivo zebrafish embryogenesis, but can also be applied to many other systems. Although the outlined protocol focuses on zebrafish neural crest cell migration, this technique can easily be adapted for other imaging purposes.
The authors have nothing to disclose.
The authors thank Thomas Schilling for kindly gifting the Tg(sox10:eGFP) fish and Mary Halloran for kindly gifting the Tg(foxd3:GFP) fish.
Breeding Tanks with Dividers | Aquaneering | ZHCT100 | Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert |
M205 FA Combi-Scope | Leica Microsystems CMS GmbH | Stereofluorescence Microscope – FusionOptics and TripleBeam | |
Sodium Chloride | Millipore (EMD) | 7760-5KG | Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3 |
Potassium Chloride | Millipore (EMD) | 1049380500 | Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8. |
Calcium Chloride Dihydrate | Fisher Scientific | C79-500 | Poly bottle; 500 g. CAS No. 10035-04-8 |
Magnesium Sulfate (Anhydrous) | Millipore (EMD) | MX0075-1 | Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2 |
Methylene Blue | Millipore (EMD) | 284-12 | Glass bottle; 25 g. Powder, Certified Biological Stain |
Sodium Bicarbonate | Millipore (EMD) | SX0320-1 | Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8 |
N-Phenylthiourea | Sigma | P7629-25G | >98%. CAS Number 103-85-5, EC Number 203-151-2 |
Dimethylsulfoxide | Sigma | D8418-500ML | Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3 |
Tricaine Methanesulfonate | Western Chemical Inc. | MS222 | Tricaine-S |
Low-Melt Agarose | ISC Bioexpress | E-3112-25 | GeneMate Sieve GQA Low Melt Agarose, 25 g |
Open Bath Chamber | Warner Instruments | RC-40HP | High Profile |
Glass Coverslips | Fisher Scientific | 12-545-102 | Circle cover glass. 25 mm diameter |
High Vacuum Grease | Fisher Scientific | 14-635-5C | 2.0-lb. tube. DOW CORNING CORPORATION 1658832 |
Quick Exchange Platform | Warner Instruments | QE-1 | 35 mm |
Stage Adapter | Warner Instruments | SA-20LZ-AL | 16.5 x 10 cm |
TC SP5 MP multi-photon microscope | Leica Microsystems CMS GmbH | ||
Mai Tai DeepSee Ti-Sapphire Laser | SpectraPhysics | ||
Laser Safety Box | Leica Microsystems CMS GmbH | ||
Leica Application Suite X (LAS X) Software | Leica Microsystems CMS GmbH | ||
Photoshop CS 6 Version 13.0 x64 Software | Adobe | ||
iMovie Version 10.1.4 Software | Apple |