Chromosome Visualization in a Temperate Anemone, Anthopleura Elegantissima

Home , Aggregating anemone, Exaiptasia

Chromosome visualization in a temperate anemone, Anthopleura elegantissima

by Lucy Shaffer

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in BioResource Research (Honors Scholar)

Presented May 25, 2018 Commencement June 2018

AN ABSTRACT OF THE THESIS OF

Lucy Shaffer for the degree of Honors Baccalaureate of Science in BioResource Research presented on May 25, 2018. Title: Chromosome visualization in a temperate anemone Anthopleura elegantissima.

Abstract approved: ______Eli Meyer

Anthopleura elegantissima (Cnidaria: Anthozoa) is an emerging model organism for studying the endosymbiotic relationship between dinoflagellate (genus Symbiodinium) and the cnidarian host. To be developed as a model organism, more genetic resources, like integrated genome and linkage map, are needed. An important validation for developing genomic resources is chromosome number. To count chromosomes, we need to be able to visualize them. In this study, I developed protocols for single cell dissociation, tissue imprinting, fixation, and DAPI staining for Anthopleura elegantissima. I visualized chromosomes for this anemone and propose that these methods can be used to karyotype Anthopleura elegantissima, to further develop this model.

Key Words: cell biology, cell visualization, chromosome visualization, anemone, Anthopleura elegantissima

Corresponding e-mail address: [email protected]

by Lucy Shaffer

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in BioResource Research (Honors Scholar)

Presented May 25, 2018 Commencement June 2018 Honors Baccalaureate of Science in BioResource Research project of Lucy Shaffer presented on May 25, 2018.

APPROVED:

______Eli Meyer, Mentor, representing Department of Integrative Biology

______Stephen Atkinson, Secondary Mentor, representing Department of Microbiology

______Holland Elder, Committee Member, representing Department of Integrative Biology

______Katherine Field, Committee Member, Director, BioResource Research

______Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request.

______Lucy Shaffer, Author

INTRODUCTION

Coral reefs are estimated to harbor around one third of all described marine species (Wilkinson 2004). However, the rapid decline in coral abundance and loss of reef habitats are one of the most pressing environmental issues of our time. The coral reef depends upon a functional symbiosis between cnidarian hosts and unicellular, photosynthetic, dinoflagellate algae of the genus Symbiodinium (Baumgarten et al. 2015). The endosymbiotic relationship between the coral host and dinoflagellates is necessary for the survival of most reef building corals in the nutrient-poor seas of the tropics (Muscatine and Porter 1977). The endosymbiont provides 95% of the coral’s energy budget in the form of photosynthetically-fixed carbon - 3- (Trench 1993; Yellowlees et al. 2008). The host supplies nitrates (NO3 ) and phosphates (PO4 ) to sustain the endosymbiont’s productivity (Rädecker et al. 2018). The host and the endosymbiont live interdependently as they exchange nutrients. However, this relationship is disrupted by heat stress. Sea temperatures above the average maximum for a region cause corals to eject their symbionts in a process called bleaching (Hughes et al. 2017). Without their symbionts, the corals risk starvation and death. If temperatures do not return to the mean temperature for the region, there is often coral mortality (Hoegh-Guldberg et al. 2017). Despite the importance of coral dinoflagellate symbiosis, the specific molecular and cellular biological mechanisms of onset and the breakdown of this symbiosis are not well known. This is in part because of the difficulties of maintaining corals in the laboratory and experimental set-ups. Nearly half of all cnidarians contain symbiotic algae, including sea anemones, therefore other classes of Cnidaria can be used as an algal-symbiosis model (Douglas et al. 2009). Aiptasia pallida, a tropical sea anemone species, has been used as a model system to make advances in the field of cnidarian dinoflagellate symbiosis (Baumgarten et al. 2015). However, A. pallida. associates with multiple Symbiodinium species (LaJeunesse et al. 2004). This introduces additional complications to studying symbiosis in this system. Anthopleura elegantissima, commonly known as the aggregating anemone or clonal anemone, is a good candidate for a model of cnidarian dinoflagellates symbiosis, because it is symbiotic with a species of the dinoflagellate, Symbiodinium muscatinei, and with a single-celled alga, Elliptochloris marina (Lajeunesse and Trench 2000). The anemone is easy to maintain in a laboratory setting, and is the most abundant intertidal sea anemone on the Pacific Coast of North America (Quesada et al. 2016). Anthopleura elegantissima also exists naturally in three distinctly colored symbiotic states: zooxanthellate (brown,hosting the dinoflagellate S. muscatinei), zoochlorellate (green, hosting the chlorophyte Elliptochloris marina), and asymbiotic (white, lacking symbionts) (Lajeunesse and Trench 2000; Lewis and Muller-Parker 2004; Letsch et al. 2009). Having symbiotic relationships with fewer species and distinct symbiotic states will simplify experimental designs which can determine the mechanisms for of onset and breakdown of symbiosis.

1 Despite its potential as a model for symbiosis, there is a need for genomic resources for A. elegantissima, which are required to design complicated cell biology experiments targeting specific genes that play a role in mechanisms of symbiosis. A transcriptome assembly of functional genes has been published (Kitchen et al. 2015). An integrated genome and linkage map is in development by the Meyer lab at Oregon State University. The draft genome for the A. elegantissima is 310 Mb, with a linkage map that indicates that there are 16 to 18 linkage groups, which suggests that there are 32 to 36 chromosomes in the diploid organism (i.e 2n=32-36). We expected that A. elegantissima would have 32 to 36 chromosomes, both from the number of linkage groups, and from the karyotypes of other anemones and corals (Order: Anthozoa) which have been karyotyped have similar numbers of chromosomes. Karyotyped anemones include Haliplanella luciae with 2n=36 (Fukui, 1993) and Nematostella vectensis with 2n=30 (Guo et al. 2018). Corals that have been karyotyped include Goniopora lobata with 2n=14 (Heyward 1985), Favia pallida (2n=28), Galaxea fascicularis (2n=26), Acropora millepora (2n=28), Acropora spathulata (2n=28), Acropora papillare (2n=26) and Acropora nasuta (2n=40) (Flot et al. 2006). While a great diversity of chromosome numbers is represented here, two anemones and three coral species have similar numbers of chromosomes to the number of chromosomes for A. elegantissima indicated by the linkage map. My project was to support the conclusions of the linkage map, with a karyotype of the anemone to confirm the number of chromosomes present. Previous anemone and coral karyotyping studies used fertilized eggs or embryos because they are naturally in high mitotic states (Fukui 1993; Choe et al. 2000). Gamete collection is time consuming and limited due to A. elegantissima displaying annual cycle of sexual reproduction, only spawning in the fall. (Ford 1964). Mitotic karyotype from live polyp of hydra has been done before, which suggests that chromosome visualization can be done in adult sea anemone because both are in the classes of Cnidaria, and are similar in body structure including tissue layers (Anokhin and Nokkala 2004). However, karyotyping from adult anemone tissue have not been done before, therefore specific conditions under which the chromosomes can best be visualized had to be developed. This study followed the general karyotyping procedures for specimen preparation, chromosome preparation and chromosome staining (Anokhin et al. 2010). Specimen preparation included a single-cell dissociation protocol, tissue imprinting and DNA staining. Through these techniques, we visualized cell nuclei and chromosomes in A. elegantissima. These techniques could be applied to karyotype not only A. elegantissima but other species of sea anemones.

MATERIALS AND METHODS

Field sampling Anthopleura elegantissima were collected from Seal Rock Oregon from the rocky intertidal coast under Oregon Department of Fish and Wildlife (ODFW) scientific permit #18876. They were transported to Oregon State University in Corvallis, OR, USA and maintained in recirculating aquaria at 12˚C.

Single cell dissociation protocol To obtain single cells from live Anthopleura elegantissima specimens, a modified coral cell dissociation procedure was used (Rosental et al. 2017). Anemones treated with colchicine were kept in a 0.25% colchicine solution with sterilized seawater for 6 or 24 hours to arrest cells in metaphase (Guo et al. 2018). The gastrovascular cavity was obtained using a razor blade and scissors by cutting the specimen vertically. One half of the gastrovascular tissue was scraped with a razor blade. The other half of the specimen was kept on the ice for later use in tissue imprinting. On ice, scraped tissue was minced using two razor blades for two minutes, to mechanically separate cells. Mechanical separation continued with addition of 10mL cell medium containing 3.3mL of 10x Phosphate Buffered Saline (PBS), 1mL of 100mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.5mL of Fetal Bovine Serum (FBS) and 4.2mL of deionized water. The tissue mixture was back-pipetted for a minimum of 10 minutes until the tissues were visibly dissociated, then filtered through 40µm mesh, then centrifuged at 500g and 10 °C for 5 minutes.

Fixation of single cells The cell pellet obtained from centrifuging each sample to a collection tube with 0.5 mL of cell medium. The pellet was flicked several times to create a homogeneous solution. The fixing solution was 3:1 methanol: acetic acid (-2 °C) and was added to the cells drop by drop while mixing to keep the solution homogenous. The cell - fixative mixture was kept at -2°C for 5 minutes, then centrifuged at 500g for 5 minutes to pellet the cells. The supernatant was discarded, then another 1mL of fixative solution was added, and the cells were mixed to create a homogenous sample. These fixed cells were kept in the refrigerator for future use. To mount cells for visualization, about 50µm of sample was dropped on a clean slide and dried at room temperature or in a ventilated hood.

Hypotonic treatment An alternate treatment is to take the cell pellet from the centrifuge and subject it to hypotonic treatment to cause cells and nuclei to swell, which allows chromosomes to separate for better viewing. Hypotonic solutions were made by diluting PBS concentrations by 10%, 25% and 35%. Each hypotonic solution was added to the cell pellet and mixed to create a homogenous sample. The cells were held in the hypotonic solution from 10 - 20 minutes.

Tissue imprint Remaining samples that had been vertically cut from each specimen were used to make additional slides. Mucus on the surface of gastrovascular tissue was removed using scissors and by blotting the tissue onto a paper towel. When the inner layer was visible, it was dabbed on a clean slide at least ten times. The slide was dried on ice in the fume hood, then a few drops of

3 fixing solution were added twice. This fixation kept fragile single cells from breaking down, and letting them stick and dry onto a clean glass slide.

DAPI(4′-6-diamidino-2-phenylindole) dye staining Fixed slides were incubated at 50°C for 3 minutes. VECTASHIELD® mounting medium for fluorescence with DAPI (Vector Laboratories, California) was provided by Dr. Stephen Akinson from the Bartholomew Lab, Oregon State University. DAPI is a DNA-specific probe which forms a fluorescent complex by attaching to A-T rich regions and causes only DNA to emit a strong blue fluorescence (Kapuscinski 2009). It was used to specifically stain the nuclei and chromosomes. Each fixed sample was stained using DAPI by placing few drops of DAPI, then laying the cover slip on top. Stained slide was viewed under a Leica DMR fluorescence microscope (Leica Microsystems Wetzler GmbH Ernst-Leitz-Strasse, Germany) with excitation/emission of 358⁄461nm. Images were collected using SPOT Advanced™ software.

RESULTS

In this study, different preparation conditions for single cell dissociation, fixation, and hypotonic treatment were tested to optimize the protocols. From the optimized protocols, I successfully separated single cells from Anthopleura elegantissima and made tissue imprints, then stained the cells with DAPI, and visualized their DNA under the fluorescent microscope.

Table 1 Anthopleura elegantissima. Cell counts at various stages of cell preparation. Single cell dissociation protocol was followed by various numbers of washings at different concentrations of fixative solution. Protocol stage Cells per mL Single cell separation 2,790,000 cells per mL Fixation 1 wash methanol:acetic acid (3:1) 800,000 cells per mL Fixation 1 wash methanol:acetic acid (2:1) 500,000 cells per mL Fixation 3 washes methanol:acetic acid (3:1) 220,000 cells per mL

I quantified the number of intact anemone cells from my single cell separations. 10 uL of re-suspended cell solution was used to count cells at 400x magnification on a hemocytometer. Mean cell counts from each quadrant were multiplied by 104 to get numbers of cells/milliliter. The single cell dissociation protocol yielded up to 2.8 x 106 cells per mL (Table 1), which is similar to the concentration of cells (107 cells/mL) that Rosental and colleagues were able to attain using a similar protocol (Rosental et al. 2017). I also determined the most effective fixative solution and the number of fixative washes by counting only intact cells. Table 1 suggests that three washings with the fixation solution dramatically reduced the number of cells: 2,800,000 to 220,000. After one wash in the fixative (3:1), the cell mixture did not spread on the glass slide after being mounted. The droplet held its

4 spherical shape, which means that there was still too much water left in the mixture. To keep as many cells as possible and properly fix the cells, a series of two fixation washes with 3:1 methanol:acetic acid were chosen. After three washing steps with fixative solution, some cell nuclei were found outside of their cell membranes, which was unexpected. I suspected that cellular stress from multiple centrifugation and harsh fixation solution caused cell membrane to dissociate from the nucleus.

Single cell Anthopleura elegantissima

Nematocyst

Symbiodinium muscatinei

Figure 1 Anthopleura elegantissima cell solution in isotonic cell medium after cell dissociation protocol at 400x magnification. Arrows point to different cell types. The red circle indicates a broken cell with nucleus slightly outside of the cell membrane.

I successfully obtained large numbers of individual cells with my cell dissociation method and I was able to visualize different types of cells. Figure 1 shows a typical single cell anemone slide in isotonic cell medium at magnification of 400x. The slide image was taken from a tentacle sample after single-cell dissociation without colchicine treatment. Three types of cells can be seen: a single anemone cell, a S. muscatinei cell, and a nematocyst. The broken cell and escaping nucleus are outlined in red. Even after one centrifugation, cellular stress may have broken some cell membranes, allowing nuclei to escape. The isolated nucleus problem was minimized by limiting centrifugation and fixative washes.

5 A B

Isotonic 10% hypotonic, 10min

C D E

10% hypotonic, 20min 25% hypotonic, 20min 35% hypotonic, 20min

Figure 2 A comparison among hypotonic cell treatments with isotonic and hypotonic cell medium. The amount of hypotonicity and times are indicated on each image. (A) Single cell dissociated tentacle sample in isotonic cell medium at 400x magnification. (B) Single cell dissociated tentacle sample in 10% hypotonic cell medium, 10 minutes at 630x magnification. (C) Single cell dissociated tentacle sample in 10% hypotonic cell medium, 20 minutes at 630x magnification. (D) Single cell dissociated tentacle sample in 25% hypotonic cell medium, 20 minutes at 630x magnification. (E) Single cell dissociated tentacle sample in 35% hypotonic cell medium, 20 minutes at 630x magnification.

I compared single cells in isotonic cell medium with various hypotonic treatments to determine an effective cell swelling concentration and length of time. Even in the lowest hypotonic solution gradient (B), the abundance of cells decreased and that trend continued for each subsequent concentration. Although some single cells swelled up and remained intact in (B) and (C), as indicated by red circles, the number of cells lost was deemed too great. Most cells in (D) and (E) were fragmented. Overall, overwhelming loss of cells in all hypotonic treatments lead to excluding this entire step.

B A

Figure 3 Comparison between gastrovascular tissue of Anthopleura elegantissima nuclei samples treated with colchicine at 6 AM (left) and 2 PM (right). Microscope magnification 1000x. (A) Anthopleura elegantissima nuclei in the cytokinesis phase of cell division. (B) Symbiodinium muscatinei’s nuclei

I determined that freshly fixed cells were better for visualization and that the hour of the day that cells are treated with colchicine may matter. While searching for the chromosomes of A. elegantissima, Symbiodinium muscatinei’s nuclei (B) could easily be distinguished by its natural auto-fluorescence, which appeared red (Pasaribu et al. 2015). Also, the nucleus size of S. muscatinei was larger than that of A. elegantissima, making it easy to tell them apart. Large numbers of doublet cells were observed in this sample that had been prepared at 6 AM and viewed the same day. The doublets in the left image indicate that the specimen was going through active cell division. The image on the right shows A. elegantissima cell nuclei from cells treated in the same way as the photo on the left except the tissue sample was treated with colchicine at 2 PM. Very little cell division was observed in the slide of cells treated at 2 PM, and this observation was consistent throughout the course of this study.

Figure 4 Condensed chromosomes of Anthopleura elegantissima at 1000x

I found condensed chromosomes in a sample that was colchicine treated at 6 AM (Figure 4). This image shows a tissue imprint of gastrovascular cavity cells in which condensed chromosomes were visualized. The chromosomes were fluorescing more brightly than normal nuclei, which is expected since condensed DNA will have more DAPI per unit area, thus increasing the fluorescence. Also, the chromosomes have finger-like morphology that results from DNA tightly condensing for division.

DISCUSSION

The mechanism of onset and breakdown of cnidarian algal symbiosis is an important field of study, which is hindered by difficulties inherent to studying corals. Anthopleura elegantissima is being developed as a model organism for studying endosymbiotic mechanisms. Finding its chromosome number is a basic piece of information needed for validation of the genomic resources that are being developed for this organism. Despite the challenges I encountered during this project, I succeeded in optimizing cell separation and fixation techniques to allow chromosome and nuclei visualization in this species. The sample in which the chromosome was visualized in Figure 4 was prepared and viewed on the same day. Previous samples that were not viewed an hour after fixation, but went through the same tissue imprinting and fixation protocol did not show any condensed chromosomes. Previous samples were left at room temperatures for 2-3 days before staining and viewing on the fluorescent microscope. It is known that methanol:acetic acid fixative is highly hygroscopic, meaning the fixative tends to absorb moisture from the air and methanol denatures proteins by replacing water in the tissue (Huang and Yeung 2015). Corvallis experiences high fluctuation in humidity throughout the day. For example, over the course of 3 days, one sample experienced humidity fluctuations ranging from 52% to 93% humidity. Humidity may have affected condensed chromosomes. If chromosome spreading takes place after the humidity induced rehydration, metaphase chromosomes may breakup and spread too much to be seen (Deng et al. 2002). The abundance of A. elegantissima’s nuclei pairs in Figure 3 suggests that the tissue sample was going through active mitosis. This contrasts with other tissue samples taken at different times: the cells from the gastrovascular tissue sample taken in the afternoon went through the same tissue imprinting and fixation techniques, but did not consistently exhibit cells in mitosis. With previous gastrovascular tissue imprints, there were only a few nuclei pairs. The cells in Figure 3A were treated with colchicine at 6 AM. Previous samples were treated at 2 PM. No one has calculated the mitotic index of an anemone, however, a species of Symbiodinium has a mitotic index which is higher from 6:00 AM to 10:00 AM (Wilkerson et al. 1983). As I noticed

8 more division in the sample treated within these hours, it is possible that the anemone host’s cell division is associated with the circadian rhythm of the symbiont, and hence have a higher rate of cell division in the morning hours. In conclusion, tissue imprinting, two washes with fixative solution, and DAPI staining protocol within 24 hours, using adult gastrovascular cells resulted in successful visualization of condensed chromosomes of A. elegantissima. Although a definite chromosome count could not be done to karyotype A. elegantissima, the methodology established in this paper can be used to in future studies to karyotype A. elegantissima and other sea anemones from adult tissues, instead of larvae. Additionally, mitotic index of A. elegantissima can be done to verify the observation about the potential association between the host’s and the symbiont’s circadian rhythm.

Acknowledgements I wish to especially thank Dr. Eli Meyer and Ph.D. student Holland Elder for their instructions, guidance, encouragements and generosity in letting me work on my thesis in their lab, and being great mentors. Dr. Stephen Atkinson provided me with much appreciated mentorship, advice and microscopy equipment and materials for this work. I would also like to thank the Weis Lab at Oregon State University for providing and taking care of the anemones. This study was supported by the E.R Jackman Internship Support Program from Oregon State University College of Agricultural Science and an Experiential Learning Award from Oregon State University Honors College.

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