Targeted Laser Ablation in the Embryo of Saccharina latissima
Summary
The destruction of specific cells in the embryo is a powerful tool for studying cellular interactions involved in cell fate. The present protocol describes techniques for the laser ablation of targeted cells in the early embryo of the brown alga Saccharina latissima.
Abstract
In Saccharina latissima, the embryo develops as a monolayered cell sheet called the lamina or the blade. Each embryo cell is easy to observe, readily distinguishable from its neighbors, and can be individually targeted. For decades, laser ablation has been used to study embryo development. Here, a protocol for cell-specific laser ablation was developed for early embryos of the brown alga S. latissima. The presented work includes: (1) the preparation of Saccharina embryos, with a description of the critical parameters, including culture conditions, (2) the laser ablation settings, and (3) the monitoring of the subsequent growth of the irradiated embryo using time-lapse microscopy. In addition, details are provided on the optimal conditions for transporting the embryos from the imaging platform back to the lab, which can profoundly affect subsequent embryo development. Algae belonging to the order Laminariales display embryogenesis patterns similar to Saccharina; this protocol can thus be easily transferred to other species in this taxon.
Introduction
Laser ablation has been used for decades to study embryo development. Irradiating embryo cells with a laser beam makes it possible to monitor the regenerative potential and the modification of the cell lineage during embryogenesis and investigate the impact of targeted ablation on cell division and cell fate. The model organisms used in laser ablation methods are typically animals, such as insects1,2, nematodes3,4, vertebrates5,6, and occasionally plants7,8. In addition, a laser micro-ablation approach was used on the brown alga Fucus in 1994 and 1998 to demonstrate the role of the cell wall in the photopolarization of the early embryo9,10.
Brown algae belong to the group Stramenopiles, diverged at the root of the eukaryotic tree 1.6 billion years ago. As a result, they are phylogenetically independent of other multicellular organisms, such as animals and plants11. Saccharina latissima belongs to the order Laminariales, more commonly known as kelps, and they are among the largest organisms on earth, reaching sizes of over 30 m. Saccharina sp. is a large seaweed used for many applications such as food and feed, and its polysaccharides are extracted for use in the agricultural, pharmacological and cosmetic industries worldwide12,13. Its cultivation, mainly in Asia and more recently in Europe, requires the preparation of embryos in hatcheries before releasing juveniles in the open sea. Like all kelps, it has a biphasic life cycle composed of a microscopic gametophytic phase, during which a haploid gametophyte grows and produces gametes for fertilization, and a diploid macroscopic sporophytic phase, where a large planar blade develops from its holdfast attached to the seafloor or rocks. The sporophyte releases haploid spores at maturity, thereby completing the life cycle14,15,16.
S. latissima presents some interesting morphological features17. Its embryo develops as a monolayered planar sheet15,18,19 before acquiring a multilayered structure coinciding with the emergence of different tissue types. In addition, Laminariales is one of the only taxa of brown algae whose embryos remain attached to their maternal gametophytic tissue (Desmarestiales and Sporochnales do too15). This feature offers the opportunity to study the role of maternal tissue in this developmental process and compare maternal control mechanisms in brown algae with those in animals and plants.
This article presents the first complete protocol for laser ablation in an early kelp embryo. This protocol involving UV ns-pulsed technique results in the specific destruction of individual embryo cells to study their respective roles during embryogenesis. The procedure offers a reliable approach for investigating cell interactions and cell fate during embryogenesis in Laminariales.
Protocol
Representative Results
Discussion
Local cellular laser ablation allows for temporal and spatial ablation with a high level of precision. However, its efficiency can be hampered by the non-accessibility of target cells; for example, all the cells are of a three-dimensional embryo. This protocol was developed on the embryo of the alga Saccharina latissima, which develops a monolayered lamina in which all cells can be easily distinguished and destroyed individually with a laser beam.
Laser power and wavelength</s…
Divulgations
The authors have nothing to disclose.
Acknowledgements
S.B.'s PhD grant is funded by Region Bretagne (ARED grant Number COH20020) and Sorbonne Université. I.T.is PhD grant is funded by Region Bretagne (ARED grant Number COH18020) and the Norvegian NMBU University. This project has received financial support from the CNRS through the MITI interdisciplinary programs. MRic is member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04).
Materials
| 25 mm glass bottom petri dish | NEST | 801001 | |
| Autoclaved sea water | – | Collected offshore near the Astan buoy (48°44.934 N 003°57.702 W) close to Roscoff, France, at a depth of 20 m. | |
| Cell scraper | MED 2 | 83.3951 | |
| Cell strainer 40 µm | Corning / Falcon | 352340 | |
| Culture cabinets | Snijders Scientific Plant Growth Cabinet ECD01 | Any other brand is suitable provided that the light intensity, the photoperiod and the temperature can be controlled. | |
| LSM 880 Zeiss confocal microscope | Carl Zeiss microscopy, Jena, Germany | Ablation and imaging were performed using a 40x/1.2 water objective | |
| Pellet pestles | Sigma Aldrich | Z359947 | Blue polypropylene (autoclavable) |
| Provasoli supplement | – | Recipe is available here: http://www.sb-roscoff.fr/sites/www.sb-roscoff.fr/files/documents/station-biologique-roscoff-preparation-du-provasoli-2040.pdf | |
| Pulsed 355 laser (UGA-42 Caliburn 355/25) | Rapp OptoElectronic, Wedel, Germany | ||
| Scalpel | Paramount | PDSS 11 | |
| SysCon software | Rapp OptoElectronic, Wedel, Germany | Laser-driver software | |
| ZEN software | Carl Zeiss microscopy, Jena, Germany | Imaging software, used together with the SysCon software; Black 2.3 version |
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