C.Mouth reefs are exposed to a myriad of stressors, from pollution to climate change. Understanding how they respond to these threats at the molecular level is critical to predicting what will happen to them in the years to come and finding the best means to protect them, says Tali Mass, marine ecologist at the University of Haifa in Israel. Unfortunately, relatively little knowledge of the different cell types in your body has hampered this type of research.
To fill these gaps, she and her colleagues published a complete cell atlas from the smooth cauliflower coral (Stylophora pistillata) in the cell On the 3rd of May. Among the 40 cell types described were two types of immune cells, the first immunity-specialized cells reported for a coral.
“This is something we had to do forever in coral science,” says Nikki Traylor-Knowles, a coral immunologist at the University of Miami who was not involved in the study. Describing the paper as a “treasure trove”, she adds that it “will help so much of the molecular and functional work that has been stifled in the coral field for so long”.
The team chose the smooth cauliflower coral because it is well suited for this type of work. “It’s a very common species. It’s a hardy species. It grows quickly so you can grow [it] in the laboratory, ”explains Mass. Though the animals were collected from the wild for study, including the most recent, their ability to do well in captivity has made the species “almost like the lab rat in the coral community” and an excellent natural system for molecular work.
A wild colony of smooth cauliflower corals (Stylophora pistillata)
In order to achieve the enormous amount of sequencing required to create the atlas, Mass and her colleagues had to revise their proven RNA extraction protocols. Since the coral cells were slightly stressed when they were separated from their carbonate skeleton and isolated for individual sequencing, team members had to speed up their work. Ultimately, they cut cell prep time from a 48-hour protocol to a 15-minute protocol by combining chemical and engineering processes, explains Mass. The coral fragments had to be immersed in calcium-free filtered seawater using EDTA to reduce the presence of adhesion promoting metals as much as possible while the coral tissue was carefully scraped off the skeleton with a 10 microliter pipette tip, and the team ensured the viability of the Cells throughout use of calcein / Draq5 stain. “It took us about a year to develop this protocol,” she says.
The effort has paid off, as the new method enabled them to sequence the transcriptomes of more than 37,000 individual cells from three different life stages of the coral: free-swimming larvae, newly established and metamorphosed polyps, and mature adult colonies. The tens of thousands of transcripts from each cell were annotated by comparison with published gene sequences and then the cells were clustered based on gene expression patterns using MetaCell, resulting in the identification of 40 different coral cell types, including the first documentation of coral immune cells.
Whether the animals actually had specialized immune cells was “a topic of debate,” notes Traylor-Knowles, although her work has long supported their existence. “For me personally,” she says, “the discovery of not one but two different types of immune cells was” only so confirmatory “and will greatly aid her investigations into animal immunity.
Microscopic image of smooth cauliflower coral cells after separation
The process also identified key genes that can be useful as markers to visualize certain types of cells during histology or to separate them from other cells during flow cytometry, as would be required to identify a pure cell population for detailed in vitro testing. To produce work. For example, the newly discovered immune cells express both NFAT, a transcription factor originally discovered in activated T cells, as well as interferon regulatory factors and other immune genes, but which differ in the expression of other genes, including those involved in apoptotic pathways.
“Understanding how corals can defend and deal with it is urgently needed [with] The recent surge in numerous diseases affecting coral reefs around the world, ”says Mauricio Rodriguez-Lanetty, who studies coral physiology at Florida International University. He agrees that the immune cells are “a very valuable finding,” and relates The scientist that the ability to visualize and separate these immune cells enables coral immunity to be studied in much greater detail, and could even open up the possibility of “genetic manipulations to improve the response of the cells”.
What makes the study particularly thorough, says Traylor-Knowles, is that the team didn’t stop at transcriptomes. They “grounded it using other techniques” as well, including in situ Hybridization using the potential markers to highlight different cell types in actual coral tissues. They showed that cells that express the epidermal gene Peroxidasin were in fact, for example, on the outer surface of the animals, and those expressing a gene for gastrodermal collagen fringed the stomach cavity of the animals.
Debashish Bhattacharya, an evolutionary genomist at Rutgers University who studied symbioses in corals and other animals and was not involved in the study, says he looks forward to using the atlas to really dig into the animals’ so-called dark genes , which are highly expressed in some cells, but whose functions are currently unknown as this could provide new insights into the unique biology of corals.
For Rodriguez-Lanetty, the most exciting part of the job is the opportunity to study symbiosis. Dinoflagellate algae live in the tissues of the corals, supplying them with oxygen and helping to remove debris, while the corals provide the algae protection and some of the components they need for photosynthesis.
Close up of a coral polyp using fluorescence microscopy. The pigments of the dinoflagellates glow red, while some coral cells containing GFP glow green.
Until now it has been impossible to separate the cells involved in the symbiosis from other nearby cells in the tissue of the corals. Previous transcriptomic studies simply extracted RNA from ground pieces of coral, which meant the datasets had a lot of noise. “This study offers, for the first time, an approach that makes this possible [one] to specifically interrogate the cells that are important for the symbiosis and to be able to dissect and determine what these genes are and what [are] the cellular processes that take place to take up this symbiosis and to regulate that symbiosis, ”he says. “It’s very transformative.”
When categorizing different cell types, the von Mass group paid particular attention to signs of pluripotency. “A lot of people try to find out whether corals really have stem cells,” she says, so she goes to extra lengths to find them. But at least for the adult corals, their searches were empty. None of the 26,880 adult cells they sequenced carried the well-categorized markers of stem cells, although some of the cells from larvae did. “I mean, we were happy to find her [in the larvae]”She says, but future research must explain why they are lacking in adult corals.
Traylor-Knowles says she wonders if they are really absent from adults, or if their absence was an artifact of sampling. “That was an area as it was Why? . . . Why didn’t you find it? We know they are there! So that’s still the puzzle, ”she says.
She suggests that the study raises other questions. For example, the team was unable to assign each cell to a described category of cells from other animals. Some were ultimately classified as “unknown”. Because these cells aren’t well suited to other species, they could have functions unique to corals, she notes. The study “really opens a Pandora’s box. . . but in a good way. “Traylor-Knowles hopes that other researchers will follow the authors’ lead and create similar atlases for other coral species.
Bhattacharya agrees. “When people go out into nature and use modern tools, they will find a lot more information that is very, very exciting,” he says. And while it’s clearly important to study both models and natural systems, he says, “I’m just making the case that we should put more energy into the wild stuff.”
S. Levy et al., “A hard coral cell atlas sheds light on the molecular and cellular basis of coral symbiosis, calcification, and immunity.” cell, doi: 10.1016 / j.cell.2021.04.005, 2021.