Production and Bioassay of a Diffusible Factor That Induces Gametophyte-to-Sporophyte Developmental Reprogramming in the Brown Alga Ectocarpus.

The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.

. The Ectocarpus life cycle. The diploid sporophyte produces meio-spores via a single meiotic cell division (R! for reduction) followed by multiple mitotic divisions in each unilocular sporangium (US). The meio-spores are released and develop as gametophytes. Gametophytes are either male or female (dioicy) and produce either male or female gametes in plurilocular gametangia (PG). Pairs of male and female gametes fuse (F! for fusion) to form zygotes, which develop as diploid sporophytes to complete the sexual cycle (left). Unfused gametes can undergo parthenogenesis to produce partheno-sporophytes. Partheno-sporophytes produce spores in unilocular sporangia, which develop as gametophytes to complete the parthenogenetic cycle (right).
In addition, both diploid sporophytes and partheno-sporophytes produce mito-spores in plurilocular sporangia (PS), which germinate to produce a new sporophyte generation with the same ploidy as its parent (dotted line). Treatment with sporophyte-conditioned medium (+ SCM) induces a subset of meio-spores to switch from the gametophyte to the sporophyte developmental program (grey text). Adapted from Peters et al. (2008).
Alternation of generations in Ectocarpus has been shown to be controlled by two homeodomain transcription factors (HD TFs) of the three amino acid loop extension (TALE) class, OUROBOROS (ORO) and SAMSARA (SAM), which are necessary for the initiation of the sporophyte program (Coelho et al., 2011;Arun et al., 2019). In addition, the sporophyte has been shown to secrete a non-cell-autonomous, diffusible factor that can cause receptive cells to switch from the gametophyte to the sporophyte developmental program (Arun et al., 2013 and2019). This diffusible sporophyte-inducing factor is only effective on the single-cell stage of the gametophyte generation. Developing meio-spores became resistant to the factor at the same point in time as they synthesise a cell wall, about 24-48 h after release from unilocular sporangia (Arun et al., 2013). This observation suggests that the cell wall may play a 3 www.bio-protocol.org/e3753 role in locking the individual into the developmental program that has been initiated. oro and sam mutants do not respond to treatment with the diffusible factor, suggesting that ORO and SAM are part of the signalling network that detects the factor.
Preparations of the diffusible sporophyte-inducing factor are produced by filtering seawater medium in which Ectocarpus sporophytes have been cultivated, resulting in cell-free, sporophyte-conditioned medium (SCM). Procedures to produce and assay the diffusible factor were described briefly in Arun et al. (2013) and Arun et al. (2019). Activity of the diffusible factor can be assayed either by treating freshly released meio-spores or by treating gametophyte-derived protoplasts (Arun et al., 2013). The protocol below describes treatment of meio-spores, which are easier to obtain than gametophytederived protoplasts. For information about the method using gametophyte-derived protoplasts, please refer to Arun et al. (2013). The meio-spore-based procedure is described in detail and some specific tips to improve both SCM production and the bioassay for the diffusible factor are provided. This detailed protocol is currently being employed in experiments aimed at biochemically characterising the diffusible factor.   produced by fertile sporophytes. Depending on the strain of Ectocarpus, meio-spores can exhibit different levels of heteroblasty, i.e., spontaneous initiation of the sporophyte program instead of the gametophyte program (Müller, 1967). To assay the activity of the diffusible factor, it is important to use a strain that exhibits a low level of spontaneous heteroblasty, such as the strain Ec32.

Materials and Reagents
b. Induce the release of gametes from 45 to 60 mature gametophytes by grouping the material together in a small volume of PES (to simulate low tide conditions) and incubating in the dark at 13 °C for four hours (follow the procedure described in Steps A2a-A2c above). e. Alternatively, unilocular sporangia can be produced on cultured upright filaments, for example by transferring previously dissected upright filaments with unilocular sporangia that have released meio-spores back into culture. These filaments will adhere to the bottom of a 55-mm Petri dish and produce new upright filaments in a few days under standard culture conditions. The upright filaments will produce many unilocular sporangia after about one week. Cultivation of upright filaments results in the production of fewer (or no) plurilocular sporangia than cultivation of partheno-sporophytes (reducing the risk of contaminating meio-spore preparations with mito-spores). This method of producing unilocular sporangia is more rapid than cultivation of whole partheno-sporophytes.
2. Bioassay of the diffusible factor by treatment of meio-spores a. Under a binocular microscope, dissect a piece of sporophyte upright filament that bears one or more unilocular sporangia using a sterile glass Pasteur pipette that has been broken to create a sharp, cutting point ( Figure 3). 8 www.bio-protocol.org/e3753  be weakly attached to the coverslip at this stage and the added medium assures that they grow under optimal conditions. g. After an additional three or four days examine the Petri dish under an inverted microscope to score the numbers of gametophyte and sporophyte individuals (Figure 4). Sporophytes can be distinguished from gametophytes based on a symmetrical pattern of initial cell division and the presence of thick walled round cells rather than wavy rhizoid cells. Note that, if meio-spores are released within 48 h but then germinate slowly, the germination process can be accelerated by adding an additional 300 µl of the test medium to the 300 µl drop. This will allow the germlings to grow and attach to the coverslip before the plate is flooded with the 10 ml of PES. h. When working with active preparations of SCM, expect between 2% and 30% of the meiospores to be switched from gametophyte to sporophyte identity but note that the percentage of switching can be highly variable between assays. It is therefore preferable to carry out at least three assays for each test to obtain statistically robust estimations of diffusible factor activity.

Data analysis
Use the Wilcoxon rank sum test with Holm-Bonferroni P-value adjustment to determine whether test samples produce significantly more sporophyte individuals than the PES control treatments ( Figure   5).

Notes
One difficulty with this procedure is that the percentage of meio-spores that switch from the gametophyte to the sporophyte developmental program can be very variable (between 2% and 30%). This appears to due to two factors, variation in the quantity of diffusible factor in different batches of SCM and variation in the capacity of meio-spores to respond to the diffusible factor. The exact cause of this variation has not been determined but it is important that the operator be prepared to carry out additional replicate tests if the effect of the SCM is weak. 11 www.bio-protocol.org/e3753