Nanos2+ cells give rise to germline and somatic lineages in the sea anemone Nematostella vectensis

Andreas Denner, Julia Steger, Alexander Ries, Elizaveta Morozova-Link, Josefine Ritter, Franziska Haas, Alison G. Cole, Ulrich Technau

Preprint posted on 17 December 2023 https://www.biorxiv.org/content/10.1101/2023.12.07.570436v2

The search for i-cells: @TechnauLab characterizes a putative stem cell population in the sea anemone Nematostella vectensis

Selected by Isabella Cisneros

Categories: cell biology, developmental biology, molecular biology

Background:

Cnidarians are well known for their fantastic regenerative abilities, which can range from replacing an entire section of their body to reforming from dissociated single cells [1]. However, most of the studies characterizing regeneration have only looked at the hydrozoan models Hydra vulgaris and Hydractinia symbiolongicarpus. This includes studies that have aimed to characterize a population of stem cells called i-cells. Interstitial cells, or i-cells, are a stem cell population that has been found to drive the regenerative response in Hydra and Hydractinia. In the former, these cells are multipotent and give rise to both gametes and some somatic cell types, not including epithelial cells [2, 3]. In the latter, these cells are considered pluripotent adult stem cells as they give rise to both germ cells and all somatic lineages [4]. While great strides have been made towards understanding cnidarian regeneration and the stem cells that drive it in hydrozoans, it remains to be seen whether cnidarians from other classes also share this cell population.

The search for an i-cell population has been ongoing in a popular anthozoan cnidarian–the sea anemone Nematostella vectensis. Despite having a well characterized regenerative response, the nature of the stem cells driving this process remains unclear. While a previous study did characterize two populations of stem cells in Nematostella–a slow-cycling quiescent population and a fast-cycling proliferative population–whether these cells bear resemblance to hydrozoan i-cells remains to be seen. In this study, the authors set out to characterize this elusive stem cell population in Nematostella by using a combination of sequencing and molecular techniques. In doing so, they continue to build on efforts to isolate and characterize i-cells in Nematostella, an endeavor that could have wide-ranging effects on evolutionary studies of regeneration both within cnidarians and beyond.

Main Findings:

Transgenic reporters for piwi1 and nanos2 reveal similar–but not identical–expression patterns in somatic cell types and gametes

Given that it is difficult to identify i-cells by morphology alone in Nematostella, the authors began their study by assessing the expression of marker genes associated with stemness, specifically that of piwi and nanos. Both genes have been previously characterized as members of the germline multipotency program [5]; piwi interacts with piRNAs to protect the genome, while nanos encodes zinc-finger RNA-binding proteins that help to inhibit apoptosis and differentiation processes [6, 7]. The authors visualized the expression of the markers piwi1 and nanos2 in Nematostella using a transgenic reporter line expressing mOrange.

They found that the piwi1 reporter recapitulated mRNA expression seen in in situ experiments, noting how expression shifted from a ubiquitous pattern during gastrulation to a more restricted pattern of expression in endomesodermal tissues during metamorphosis. In adults, this expression extended to the epithelia surrounding the gonads and reticulate tract. The authors also dissociated adult animals and found piwi1 expression in putative germline stem cells, oocytes, spermatogonia, and epithelia. Similarly, the nanos2 reporter also recapitulated expression from in situexperiments, with ubiquitous expression during gastrulation becoming restricted to the primary mesenteries during the planula stage. In juvenile animals, nanos2 expression was associated with multiple cell types and organs; additionally, dissociated animals showed expression in all neuroglandular cell types, albeit a more residual expression likely retained from precursor cells. Because the reporters showed similar expression patterns, the authors chose to focus on the gonadal region to tease apart more subtle differences. Here, they noticed nanos2 expression in trophocytes–accessory cells located close to oocytes–while piwi1 expression was localized directly within the oocytes.

Throughout these experiments, the authors observed that nanos2-positive cells were more commonly found in the descendants of somatic lineages, whereas piwi1 was much stronger in the germline. This, in addition to the intensity of nanos2 expression and its more restricted epithelial expression pattern, led the authors to focus on the nanos2-positive population for the rest of the study.

EdU pulse chase experiments reveal fast-cycling and slow-cycling populations within nanos2-positive cells

Having singled out the nanos2-positive population, the authors next wanted to understand if this cell population was actively cycling. They labeled transgenic animals with EdU for 24 hours and observed that, while some nanos2 cells were doubly labeled, others were not. This led the authors to suspect that the nanos2-positive population includes both fast-cycling proliferative and slow-cycling quiescent cells. The authors subsequently decided to do a 5-hour EdU pulse followed by cell dissociation and cell counting as a means of identifying the fraction of fast-cycling cells. Cells positive for nanos2 were found to comprise a quarter of all cells, with almost half of that population being proliferative. Interestingly, some cells negative for nanos2 were also found to be proliferative, suggesting the possibility of another cycling cell population in Nematostella. The authors also did an even more extensive EdU pulse chase experiment to identify quiescent or slow-cycling stem cells, with cryosections revealing both EdU and nanos2 positive cells present in samples ranging from 1 to 3 months post-pulse. Given that other characterized i-cell populations have both fast-cycling and slow-cycling subpopulations, this result demonstrates important similarities between nanos2-positive cells and i-cells.

Functional analysis of nanos2-positive cells using CRISPR knock-outs reveals that nanos2 is necessary for the formation of the germline

Next, the authors wanted to take a closer look at the role of nanos2 in cellular differentiation, which led them to generate a CRISPR/Cas9 knock-out mutant. While the F1 offspring showed no increased mortality or developmental defects, they observed that the homozygous mutants did not spawn gametes. Closer observation of the gonads revealed that they did not have any oocytes, sperm or Vasa2-positive cells, indicating sterility. The authors also wanted to understand how the homozygous mutation affected cell type composition, which they assessed using single-cell RNA sequencing. Interestingly, when compared to the wild-type tissue data, they noted no significant differences in overall cell type composition. When they looked closer at the putative stem cell and primary germ cell clusters, however, they found that nanos2 mutant cells were not contributing to the primordial germ cells and that gametes could not be detected in the mutants. This led the authors to conclude that nanos2 is required for both germline formation and gamete differentiation in Nematostella.

Taking all their data together, the authors concluded that piwi1 and nanos2 positive cells act as a population of putative stem cells that give rise to both germ cells and neuronal progenitors, which may be indicative of the presence of i-cells in Nematostella. While future studies will be necessary to confirm a multipotent or pluripotent cell population, this study confirms previous proposals regarding the function of piwi1+ cell populations and the role of nanos2 as an ancestral key determinant of germline establishment—evolutionary significant findings in and of themselves.

Why I Chose This Preprint:

I was thrilled to see this preprint when it came out in December! The question of whether there are i-cells in Nematostella has remained largely open until now, and I was excited to see a group tackle this question head on. While we are still learning more about i-cells in the hydrozoan models Hydra and Hydractinia, it is important not to lose sight of the evolutionary picture. Characterization of this cell population in other cnidarian classes provides us with more information on just how shared this regenerative toolkit is, both within Cnidaria and outside of it. This study marks a crucial step towards understanding where i-cells exist in an evolutionary context, which could have broad implications for regenerative and stem cell biology studies moving forward.

Questions For The Authors:

Do you have plans to assess other markers associated with i-cells in Hydra and/or Hydractinia or with pluripotency more generally?
Would a rescue experiment be feasible in Nematostella as another means of assessing the functional nature of the nanos2-positive cell population?
While further study will be needed to completely tease out the characteristics of the nanos2-positive population, which hydrozoan i-cell population do you think it will bear more resemblance to?

References:

[1] Holstein, T.W., Hobmayer, E., Technau, U. Cnidarians: an evolutionarily conserved model system for regeneration? Dev Dyn, 226(2):257-67 (2003). 10.1002/dvdy.10227

[2] David, C. N. Interstitial stem cells in Hydra: multipotency and decision-making. Int. J. Dev. Biol., 56:489-497 (2012). https://doi.org/10.1387/ijdb.113476cd

[3] Holstein, T. W. The Hydra stem cell system – Revisited. Cells & Development, 174:203846 (2023). https://doi.org/10.1016/j.cdev.2023.203846

[4] Gahan, J. M., Bradshaw, B., Flici, H., Frank, U. The interstitial stem cells in Hydractinia and their role in regeneration. Current Opinion in Genetics & Development, 40:65-73 (2016). https://doi.org/10.1016/j.gde.2016.06.006

[5] Juliano, C. E., Swartz, S. Z., Wessel, G. M. A conserved germline multipotency program. Development., 137(24):4113-26 (2010). 10.1242/dev.047969

[6] Rojas-Ríos, P., Simonelig, M. piRNAs and PIWI proteins: regulators of gene expression in development and stem cells. Development, 145(17):dev161786 (2018). https://doi.org/10.1242/dev.161786

[7] Wang, Z., Lin, H. Nanos Maintains Germline Stem Cell Self-Renewal by Preventing Differentiation. Science, 303:2016-2019 (2004). 10.1126/science.1093983

Tags: cnidarian, i-cell, multipotent, nematostella, pluripotent, stem cell

Posted on: 30 January 2024

doi: https://doi.org/10.1242/prelights.36389

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Author's response

Ulrich Technau shared

(1) Do you have plans to assess other markers associated with i-cells in Hydra and/or Hydractinia or with pluripotency more generally?

Yes, we are currently exploring also other candidate gene markers that are markers in i-cells. Of note, the group of Patrick Steinmetz investigated the Vasa2 population in transgenic lines and while this population is only partially overlapping with the nanos2 cells, it also gave rise to somatic cells (neurons) and gametes.

(2) Would a rescue experiment be feasible in Nematostella as another means of assessing the functional nature of the nanos2-positive cell population?

We are in the process of FACSorting the nanos2 cells with the goal to sequence deeper into that population, but also to use them in cell transplantation experiments in non-transgenic cells. A rescue of the nanos2 mutant would be possible in principle, but quite tedious to do, as it would require a knockin of the wildtype into the mutant gene locus.

(3) While further study will be needed to completely tease out the characteristics of the nanos2-positive population, which hydrozoan i-cell population do you think it will bear more resemblance to?

If any, it seems to be more similar to the Hydra stem cells, as we see no evidence yet for a role in epithelial cell differentiation. However, we assume that like in Hydra and planarians, the stem cell population is probably more heterogeneous that may appear at first glance, with slow cycling and fast cycling cells and yet unknown relationships. Much more remains to be learned.

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