Targeted Laser Ablation in the Embryo of Saccharina latissima

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^7,8. 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 embryo^9,10.

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 worldwide^12,13. 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 cycle^14,15,16.

S. latissima presents some interesting morphological features¹⁷. Its embryo develops as a monolayered planar sheet^15,18,19 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.

Protokół

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 remaining epiphytes by gently cleaning the cut pieces with the back of a scalpel and some absorbent paper.
4. Place the cleaned pieces in a glass dish filled with sterile natural seawater (see Table of Materials) for 45 min to release spores following previously published report²².
5. Remove the blade pieces and filter the seawater through a 40 µm cell strainer to remove debris or unwanted organisms.
6. Dilute the spores in the filtrate to a 20-40 spores/mL concentration in plastic Petri dishes²².
7. Place the spore solution in a culture cabinet (see Table of Materials) configured with the optimal culture conditions (13 °C, 24 µE.m^-2.s^-1, photoperiod 16:8 L:D).
8. Allow the spores to germinate and develop into gametophytes.
   NOTE: Spore germination is visible after 2 days in the cabinet, and the first cell division of the gametophytic cells usually occurs within the following 48 h.
9. Replace the growth medium after 5 days with micro-filtered natural seawater enriched with a 0.5x Provasoli solution (NSW1/2) (see Table of Materials).
   NOTE: To avoid repeating these steps, specific male and female gametophytes can be selected and vegetatively propagated for several months. The gametophytes remain vegetative when grown under red light (4 µE.m^-2.s^-1 with a wavelength of at least 580 nm)²³ in the same culture conditions described above (step 1.7).

2. Fragmentation and induction of oogenesis

1. Harvest gametophytes with a cell scraper.
2. Using a small plastic pestle, crush the collected gametophytes in a 1.5 mL tube into 4-5-celled pieces.
3. Fill the tube with 1 mL NSW1/2 (step 1.9).
4. Add 2.5 µL of the crushed gametophyte solution into 3 mL of natural seawater enriched with 1x Provasoli solution (NSW) and place them in a Petri dish.
   NOTE: A 25 mm, glass-bottomed Petri dish is recommended for easier handling.
5. Place the prepared dishes in a culture cabinet and induce gametogenesis at 13 °C under white light with an intensity of 24 µE.m^-2.s^-1 (dim light, photoperiod 16:8 L:D).
   NOTE: The first gametangia (female oogonia and male archegonia) can be observed after 5 days. The male is hyper-branched with small cells, and the female is composed of larger cells forming long filaments^15,22. The first eggs are observed ~10 days later, and the first division of zygotes usually occurs within the following 2 days.
6. Six days after observing the first eggs, transfer the dishes to brighter white light conditions: 50 µE.m^-2.s^-1, photoperiod 16:8 L:D, still at 13 °C.

3. Image acquisition for selecting embryos for ablation and monitoring subsequent growth

1. Image the entire Petri dish to (re)locate the embryos selected during the ablation step (no need to return the dish to the ocular microscope) and monitor the subsequent development of the selected embryos.
   NOTE: Use an inverted laser scanning confocal microscope (see Table of Materials) for imaging (Figure 1), and the laser ablation is described in step 5.
2. Place the Petri dish on the stage and orientate it with a visual mark (e.g., draw a line with a permanent marker).
3. Use the 10x/0.45 objective to focus on an embryo. Record the position of the four cardinal points of the Petri dish.
4. Start the tile scan. Acquire transmitted/fluorescent images of the whole Petri dish at low resolution: 256 x 256 pixels, a pixel dwell time of 1.54 µs with bidirectional scanning, and a digital zoom of 0.6x using a 561 nm laser at 1.2% transmission.
   NOTE: The scan time for a whole 2.5 cm Petri dish is ~6 min for 225 tiles (Figure 2). Here, the 561 nm laser was used for transmission and fluorescence imaging. The fluorescence signal was collected between 580–720 nm on the confocal photomultipliers (PMT) and the transmitted light was collected on the transmitted PMT. The 561 nm laser can also monitor chlorophyll at this step, but it is unnecessary because it only helps distinguish the signal noise and the organisms correctly.
5. Save the tile scan image and keep it open in the image acquisition software (see Table of Materials) window.
6. Change the objective, do not remove the Petri dish.
   NOTE: The 40x/1.2 water objective was moved to the side of the stage so that the immersion medium (water) could be added to the objective without moving the Petri dish from its initial position.
7. Navigate through the previously acquired tile scan image to select the appropriate embryo. Once an embryo has been identified on this image, move the stage to the exact position of the embryo and acquire transmitted/fluorescent images of that embryo at high resolution.
   NOTE: High-resolution settings: 512 x 512 pixels, 0.130 µm/pixel, 0.208 µs pixel dwell time with mono-directional scanning and 2x digital zoom using a 561 nm laser at 0.9% transmission.
8. Annotate the tile scan image for each embryo candidate for laser ablation (Figure 2B) and proceed to the laser ablation step.

4. Laser calibration

1. Calibrate the laser and synchronize the image acquisition software with the laser-driver software in the "Click & Fire mode" and a pulsed 355 nm laser.
   NOTE: This step is crucial to ensure perfect synchronization between the mouse cursor's position in the laser-driver software (see Table of Materials) with the position in the live image of the acquisition software.
2. Open the laser-driver software package and click on Live in the image acquisition software package.
3. Synchronize both software packages by clicking on Start acquisition in the laser-driver software package. The live image is now also recorded in the UV laser-driver software.
4. Define an area of interest (AOI) by clicking on Choose AOI button and clicking on the edges of the image (right, left, top and bottom) in the UV laser-driver software package.
   NOTE: After this calibration step, the settings for pixel size, image format, and zoom in the acquisition software package must remain constant.
5. Select an empty area on the dish and lower the level of the stage to 20 µm below the sample focal plane to focus on the glass bottom.
6. Set the ablation laser and imaging laser trajectories by clicking on Start calibration and choose Manual calibration.
7. Select a laser power high enough to see a black dot in the center of the live image corresponding to the hole in the glass coverslip (all the shutters must be open).
8. Click on this central black dot with the mouse cursor and click on 18 additional dots proposed by the software to complete the alignment procedure.
9. Check the calibration in the "Click & Fire mode" on the same coverslip.
   NOTE: Laser calibration depends on the imaging parameters. Once the laser has been calibrated, ensure that the imaging parameters (i.e., 512 x 512 pixels, 0.130 µm/pixel, 0.208 µs pixel dwell time with mono-directional scanning and 2x digital zoom) have not changed.

5. Laser ablation

1. Select an embryo of interest. Start a time-lapse recording in the image-acquisition software.
2. Acquire transmitted/fluorescence images with a 40x/1.2 W objective at high resolution (i.e., 512 x 512 pixels, 0.130 µm/pixel, 1.54 µs pixel dwell time with mono-directional scanning and 2x digital zoom using a 561 nm laser at 0.9% transmission). Acquire the time-lapse recording at maximum speed.
3. Zoom out of the area at the beginning of the time-lapse recording. Zoom in on the AOI.
4. Use the "Click & Fire" function of the laser-driver software to apply the damaging irradiation on the cell of interest in the embryo. Use the following parameters: 45% laser transmission (corresponding to a maximum of 40 µW) and 1 ms pulse time duration (step 4).
   NOTE: Video recording during the laser shooting is recommended.
5. Under 688 nm, monitor the ejection of autofluorescent chloroplasts from the cytoplasm.
6. If cell contents remain in the cell, use the "Click & Fire" function once more to increase the size of the breach in the cell. Repeat, keeping the number of shots to a minimum until most of the cell contents have been released.
7. Stop the time-lapse recording after the embryo has stabilized (i.e., no further intracellular movement can be detected (~1-5 min).
8. Update the annotation on the tile scan image, if necessary.

6. Monitoring the growth of irradiated embryos

NOTE: Monitoring is carried out over several days.

1. Determine the survival rate by monitoring the number of embryos that develop after laser ablation and compare them to those that die.
   NOTE: Some embryos die immediately after ablation for various reasons. A high and rapid mortality rate is usually a sign of inappropriate laser parameters or higher/longer exposure to stress during the experiment or subsequent transport.
2. Determine the growth delay by measuring the length of the laser-shot embryos every day and comparing it to intact embryos.
   NOTE: The growth rate of laser-shot organisms is generally slower than that of untreated organisms. However, some (inappropriate) laser settings can inhibit growth for more than a week, with growth resuming after that.
3. Find out the adjacent damage by monitoring the reaction of cells adjacent to the ablated cells. In some cases, post-burst depressurization may cause neighboring cells to burst.
4. Check for microbe contaminations. Monitor the growth of microalgae and bacteria in the medium. If an unusual level of microbes are present in the dish, then discard it and repeat the protocol from step 2 or step 3.
   NOTE: After laser ablation, damaged S. latissima embryos are already highly stressed, and additional external stress can cause increased mortality. Bacterial or viral outbreaks are possible because the treated embryos cannot grow in axenic conditions.
5. Check the global development of the shot organisms by studying the phenotype and understanding the role in the development of the targeted region.

Wyniki

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...

Dyskusje

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

Materiały

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