dynamic nature in the demosponge Amphimedon queenslandica
4.1 Abstract
Stem cells are a hallmark of animal multicellularity, and are essential for developing and maintaining specialized cells and body plans. They are of particular interest in recent years because of their potential application in regenerative medicine. One of the many animal groups capable of extensive regeneration is the sponges. This capability appears to be largely via mesenchymal stem cells called archeocytes, which freely migrate throughout the sponge body. Choanocytes, the feeding cells of the sponge, have also been suggested as potential stem cells, although there is limited documentation on their capacity as stem cells. In this study, I document the involvement of choanocytes in the stem cell system of the juvenile demosponge
Amphimedon queenslandica using the cell-tracker CM-DiI and the cell proliferation marker
EdU. I demonstrate that choanocytes regularly dedifferentiate into archeocytes, a process observed in as little as 4 hours after labeling choanocytes with CM-DiI. These archeocytes are pluripotent, capable of differentiating into a range of cell types, including pinacocytes, sclerocytes, spongocytes as well as new choanocytes and choanocyte chambers. Using EdU, I show choanocytes in some chambers can be proliferative, although the level of cell division within chambers is highly variable, suggesting that different chambers are in different physiological states. This observation is substantiated by the comparison of CEL-Seq data from single choanocyte chambers, which indicates that chamber transcriptomes are either enriched in metabolic or proliferative gene sets. Although the dedifferentiation of choanocytes into archeocytes does not appear to be contingent upon cell proliferation, these findings suggest that choanocytes are a crucial part of the stem cell system, assisting in both the growth of the sponge as well as increasing and maintaining the archeocyte stem cell population.
4.2 Introduction
4.2.1 Stem cells and regeneration in metazoans
Development and maintenance in metazoans largely involves the use of stem cells (Fuchs and Segre, 2000; Sanchez Alvarado, 2000; Weissman, 2000; Gurley and Sanchez
Alvarado, 2008), with increasing interest in recent years, particularly after the discovery of induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006).
In animals, most cells in the adult body are stably or terminally differentiated, in that the cell cannot readily dedifferentiate back into an earlier cell state (Jopling et al., 2011). There are however, populations of stem cells with varying levels of stemness, including totipotent cells capable of differentiating into all cell types and giving rise to a whole organism (e.g. zygotes and blastomeres of early embryos) and pluripotent cells that are also capable of differentiating into a range of cell types, but do not have the organizing capacity to form an individual (de Vries et al., 2008; Mitalipov and Wolf, 2009). Additionally, there are usually populations of progenitor cells near or within specific organs or tissues; and these cells are generally multipotent or unipotent, and are capable of replenishing a restricted number of specific cell types (Anderson et al., 2001; Morrison, 2001; Mitalipov and Wolf, 2009). While most animals are not capable of extensive regeneration (i.e. whole limbs and body parts), there are a number of animals, and specific tissues, that are known for their remarkable regenerative capacity of whole body or specific tissues, - e.g. urodele amphibians (Reyer, 1954; Brockes and Kumar, 2005), frog tadpole tails (Beck et al., 2003), zebrafish fin and heart (Poss et al., 2003; Poss, 2007), mollusks (Lange, 1920; Needham, 1952), planarians (Reddien and Sanchez Alvarado, 2004), freshwater anemones (Holstein et al., 1991; Galliot, 2012) and sponges (Ayling, 1983). Within these examples of regeneration in animals, there is great variation in the extent of regenerative capacity, as well as in the mechanisms underlying this phenomenon (Gurley and Sanchez Alvarado, 2008; Tanaka and Reddien, 2011). Regeneration in many cases involves proliferation to increase and replenish the stem cell population, which will in turn differentiate and replace that which was lost (Gurley and Sanchez Alvarado, 2008; Li and Clevers, 2010; Buczacki et al., 2013). However, this is not always the case, with some regenerative mechanisms involving mainly transdifferentiation of nearby cells with little to no proliferation (Holstein et al., 1991; Bosch, 2007). As regeneration has been suggested to be an ancestral feature of all animals (Sanchez Alvarado, 2000), studying the development and regenerative mechanisms of a wide range of metazoan phyla is a stimulating avenue of research. Such studies can provide further understanding of the evolution of regeneration in metazoans, and have the potential to reveal ancestral mechanisms and pathways that could be applied in regenerative medicine.
4.2.2 Regeneration in sponges
One group of animals well known for their regenerative capacity is sponges, and observations on their regenerative characteristics date back to the early 1900s (Wilson, 1907a, b). In his classic experiment, Wilson cut a sponge tissue into small pieces and sieved these through a piece of cloth to dissociate the tissue into individual cells. When this cell suspension was left for some time, he found that these individual cells could reaggregate to form a functional sponge (Wilson, 1907b). This classic study has been revisited many times (e.g. Wilson and Penney, 1930; Humphreys, 1963; Custodio et al., 1998; Maldonado and Uriz, 1999; Perovic et al., 2003), with some recent multi-taxon comparative studies (Eerkes- Medrano et al., 2015; Lavrov and Kosevich, 2016) demonstrating that this ability to recreate a new individual from cell suspensions is not a universal trait among sponges.
In perhaps the most comprehensive study to date, Eerkes-Medrano et al. (2015) sampled seven sponge species including six demosponge species and one calcareous sponge and found that only two of these species (demosponges Spongilla lacustris and Haliclona cf.
permollis) were capable of regenerating a functional sponge from dissociated cells. Although
the mechanism underlying the difference between these species' regenerative capacities is yet to be discovered, it was suggested that environmental factors and the growth form of each species play decisive roles (Eerkes-Medrano et al., 2015). As such, it was proposed that those species that have branching growth forms, and live in areas with high water flow, are more susceptible to breakage of large portions of their body, and therefore have adapted a high regenerative capacity to enable settlement and growth of a new individual from the torn off piece (Wulff, 2006a; 2010). In contrast, species that are encrusting or cryptic and live in areas with relatively low water flow, are much more susceptible to predation and wounds rather than large portions of their body being misplaced and relocated. Therefore they have presumably faced selective pressure to develop high efficiency in wound healing rather than whole body regeneration from a small piece of tissue (Wulff, 2013). In a more recent study, the reaggregative capacities of three demosponge species (Halichondria panicea, Haliclona
aquaeductus and Halisarca dujardinii) were investigated, with the results again demonstrating
that not all sponge species are capable of forming functional sponges after (Leys and Degnan, 2002) (only H. dujardinii in this case) (Lavrov and Kosevich, 2016). This study also noted that life cycle, seasonality and even small changes in experimental setup could change the outcome