Mixed clonal-aggregative multicellularity entrained by extreme salinity fluctuations in a close relative of animals

  1. Núria Ros-Rocher
  2. Josean Reyes-Rivera
  3. Yeganeh Foroughijabbari
  4. Chantal Combredet
  5. Ben T. Larson
  6. Maxwell C. Coyle
  7. Erik A. T. Houtepen
  8. Mark J. A. Vermeij
  9. Nicole King
  10. Thibaut Brunet

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Abstract

Multicellularity evolved multiple times independently during eukaryotic diversification. Two distinct mechanisms underpin multicellularity: clonal development (serial cell division of a single precursor cell) and aggregation (in which independent cells assemble into a multicellular entity). Clonal and aggregative development are traditionally considered to be mutually exclusive and to result from independent acquisitions of multicellularity. Here, we show that the choanoflagellate Choanoeca flexa, a close relative of animals that forms contractile monolayers of cells (or "sheet colonies"), develops by an unconventional intermediate mechanism that we name "clonal-aggregative multicellularity". We find that C. flexa sheets can form through purely clonal processes, purely aggregative processes, or a combination of both, depending on experimental conditions. To assess the ecological relevance of these findings, we characterize the natural context of multicellular development in the native environment of C. flexa on the island of Curaçao. We show that the C. flexa life cycle is environmentally regulated by extreme salinity fluctuations in splash pools undergoing cycles of evaporation and refilling. Upon desiccation, C. flexa colonies dissociate into drought-resistant quiescent cells, which resume activity and reform multicellular sheets after rehydration. We hypothesize that clonal-aggregative development reflects selection for fast transitions into and out of multicellularity in the ephemeral context of coastal splash pools. Our findings underscore the potential of the exploration of biodiversity to reveal new fundamental biological phenomena and expand the known option space for both multicellular development and for the origin of animal multicellularity.

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  2. Arcadia Science

    Thanks a lot for your question! C. flexa cultures (in their standard growth conditions) usually have abundant multicellular sheets but can also have single swimmers (flagellate cells). We used cultures that mostly had sheets (and few single cells), and both gradual evaporation and addition of salts triggered a transition into cyst-like cells. A key point is that sheets first dissociate into single flagellate cells, and then transition into cyst-like cells. However, we have not tried to induce “encystation” in cultures with purely single swimmer cells, yet we could assume that they likely have the ability to transition into cyst-like cells given the dissociation response of sheets under gradual evaporation/addition of salts conditions. Rapid evaporation of sheets, on the other hand, impaired sheet dissociation and the transition to cyst-like cells; so it seems that this step-wise transition is essential for cell survival.

  3. Arcadia Science

    That’s a very good question! We additionally measured other abiotic parameters, including pH, but we did not explore the composition of splash pools (and that is something that we are indeed looking forward to further exploring in the future, both at the chemical and also at the species community level). pH did not vary much (most splash pools had pH around 8), and thus we did not find any correlation regarding the observation of sheets and pH. Thanks for your question!

  4. Arcadia Science

    Choanos are fascinating! And your questions are on point:

    • We consider cells to have “integrated” in a colony (or “sheet”) when they have established stable connections with the other cells of the sheet, as assessed by: 1) alignment of apicobasal polarity; 2) connection by inter-microvillar contacts; and 3) stable long-term adhesion to other cells.

    • We’ve seen quite some movement during colony formation: cells or small groups of cells joining a colony and cells or small groups of cells leaving a colony. However, we have not observed any translocation event (a cell switching its position within a colony) other than cells making space for daughter cells after division. At least at the time scale we imaged (a few minutes up to 36 hours in timelapse microscopy), most cells remained in the same position. Yet, we have not quantified/tracked individual cells moving around or switching positions inside a colony in larger time scales (more than 2 days), so we cannot be 100% sure. We will soon investigate the adhesion molecules responsible for colony formation, so that’s something worth looking at.

    • We are not aware of peripheral cells having any visible adaptation that would help “capture” newly aggregating cells (but you never know), but that is something very interesting to further explore, both at the molecular and the phenotypic level.

    • Regarding the difference with clonal species, species with clonal multicellularity form colonies of very different geometry, such as chains, rosettes and trees. The sheet geometry might be especially amenable to aggregation, as new cells can easily join the periphery of the colony and establish collar-collar contacts. By contrast, aggregating into a rosette would involve “squeezing your way in” (not inconceivable but that would be a more complex process). Similarly, in chain colonies, cells are linked by cytoplasmic bridges that are remnants of incomplete cell division, and the chain morphology can therefore only develop clonally. However, correlations between geometry and developmental mode are based on a relatively small number of well-characterized species and might be nuanced or completed with future discoveries.

    Hope that helps, happy to continue the discussion!

    Thanks a lot for your comments and your interest in our research!

  6. Arcadia Science

    sheets dissociate and differentiate into solitary, non-motile, and non-proliferative cyst-like cells

    for cells that aren't a part of sheets, do you see similar differentiation patterns into cysts? Any difference in outcomes/ability to do so? Curious if the multi-cellular state is somehow helpful to actively mediate this transition.

  7. Arcadia Science

    We never observed sheets in splash pool water with a salinity above 94 ppt (2.35-fold seawater salinity

    Are there any other notable differences between splash pools, like pH or metals/mineral content?

  8. Arcadia Science

    integrating into the sheet

    I don't know much about choano colony dynamics, so I have a bunch of questions!. How do you generally assess integration? Also, do you see much cell movement within a community structure, i.e. outside cells or newly integrated ones that eventually move to the center? Or would you expect that peripheral cells tend to stay peripheral and have some unique features that enable aggregation? And finally, is this there any known difference in these spatial features relative to species known to only clonally divide?

  9. Version published to 10.1101/2024.03.25.586565v1 on bioRxiv