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 insects^1,2, nematodes^3,4, vertebrates^5,6, and occasionally plants^7,

Protocol

1. Production of Saccharina latissima gametophytes

1. Collect mature sporophytes of S. latissima from the wild as previously described^20,21. Ensure that the selected sporophytes are devoid of epiphytes (small organisms visible on the blade's surface) or internal parasites (found in the bleached areas or spots on the blade).
2. Using a scalpel, cut the darkest part in the center of the blade (fertile spore-producing tissue²²) into 1-5 square pieces (1 cm²), avoiding any bleached spots, if present.
3. Remove any ....

Results

Gametophytes of S. latissima were grown, and gametogenesis was induced to produce zygotes and embryos. Twelve days after the induction of gametogenesis, the embryos underwent laser ablation. Here, the experiment aimed to assess the role of specific cells in the overall development of S. latissima embryos. The most apical cell, the most basal cell, and the median cells were targeted. After tile scanning, the entire Petri dish (Figure 2A), an embryo of interest, was identifie.......

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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