Nikolai P Melnikov

Fyodor V Bolshakov

Veronika S Frolova

Kseniia Skorentseva

Alexander V Ereskovsky
email: alexander.ereskovsky@imbe.fr

Alina A Saidova

Andrey I Lavrov

Ksenia V Skorentseva

Tissue homeostasis in sponges: quantitative analysis of cell proliferation and apoptosis

Keywords: cell turnover, cell proliferation, apoptosis, Porifera, Calcarea, Demospongiae

Introduction

Morphogenetic processes underlying the development, growth and regeneration of multicellular animals have long been issues of scientific interest. These phenomena seem to be closely related to a process of physiological regeneration, also known as cell turnover [START_REF] Galliot | Cell plasticity in homeostasis and regeneration[END_REF][START_REF] Tanaka | The Cellular Basis for Animal Regeneration[END_REF] . The cell turnover consists of three key elements: 1) the elimination of old, non-functional cells by programmed cell death; 2) the proliferation of somatic stem cells; 3) the differentiation of newly generated cells along with their integration with the preexisting tissue 3 . Such tissue renewal through the physiological regeneration is an essential process for multicellular animals, as it allows reparation of minor defects and damages of tissues occurring during their normal functioning [START_REF] Galliot | Cell plasticity in homeostasis and regeneration[END_REF][START_REF] Sharpless | Telomeres, stem cells, senescence, and cancer[END_REF] . Abnormalities of tissue homeostasis are associated with numerous malfunctions, e.g., oncological and degenerative disorders [START_REF] Sharpless | Telomeres, stem cells, senescence, and cancer[END_REF][START_REF] Pardal | Applying the principles of stem-cell biology to cancer[END_REF] .

Sponges (phylum Porifera) is an early-branching group of metazoans comprising aquatic, mostly filter-feeding sedentary organisms [START_REF] Wörheide | Deep Phylogeny and Evolution of Sponges (Phylum Porifera)[END_REF] . These animals differ substantially from other metazoans in their body organization: sponges lack tissues and organ systems characteristic of other animals [START_REF] Leys | The Physiology and Molecular Biology of Sponge Tissues[END_REF][START_REF] Ereskovsky | The Comparative Embryology of Sponges[END_REF] .

The central anatomical structure in a sponge body is the aquiferous system, a complex network of canals and chambers through which sponges pump surrounding water for food and oxygen uptake 9 .

The rest of the sponge body is composed of the mesohyl, a tissue consisting of an extensive extracellular matrix and numerous wandering cells of different types and functions 9 . Tissues of sponges seem to be structurally and functionally less specialized than tissues of Eumetazoa. The main characteristic of sponge tissues is high plasticity: in particular, sponge tissues are multifunctional, and their cells show broad capabilities of transdifferentiation into other cell types [START_REF] Gaino | Organizational plasticity as a successful conservative tactics in sponges[END_REF][START_REF] Korotkova | Regeneration in animals[END_REF] . The tissue plasticity underlays many aspects of sponge biology, participating in constant reorganization of the aquiferous system 10 , sexual and asexual reproduction 8 , regeneration 12-17 and even movement of sponges [START_REF] Bond | Continuous cell movements rearrange anatomical structures in intact sponges[END_REF][START_REF] Bond | Locomotion of sponges and its physical mechanism[END_REF][START_REF] Lavrov | Stolonial movement: A new type of whole-organism behavior in Porifera[END_REF] .

At the same time, sponges lack a single category of somatic stem cells. Choanocytes (flagellated cells of the aquiferous system) and some amoeboid cells of mesohyl (e.g., archaeocytes of Demospongiae) are both considered to be the stem cells of sponges [START_REF] Ereskovsky | The Comparative Embryology of Sponges[END_REF][START_REF] Funayama | The cellular and molecular bases of the sponge stem cell systems underlying reproduction, homeostasis and regeneration[END_REF][START_REF] Ereskovsky | Stem cells cell in sponges (Porifera): an update[END_REF] . As has been shown for a number of species, choanocytes and archaeocytes proliferate in intact tissues, (trans)differentiate into other cell types, take part in regeneration and gametogenesis [START_REF] Ereskovsky | The Comparative Embryology of Sponges[END_REF][START_REF] Borisenko | Transdifferentiation is a driving force of regeneration in Halisarca dujardini (Demospongiae, Porifera)[END_REF][START_REF] Funayama | The cellular and molecular bases of the sponge stem cell systems underlying reproduction, homeostasis and regeneration[END_REF][START_REF] Funayama | The stem cell system in demosponges: Suggested involvement of two types of cells: Archeocytes (active stem cells) and choanocytes (food-entrapping flagellated cells)[END_REF][START_REF] Sebé-Pedrós | Early metazoan cell type diversity and the evolution of multicellular gene regulation[END_REF][START_REF] Sogabe | Pluripotency and the origin of animal multicellularity[END_REF] . They also express some stem cell-specific genes, such as the members of the germline multipotency program [START_REF] Funayama | The stem cell system in demosponges: Suggested involvement of two types of cells: Archeocytes (active stem cells) and choanocytes (food-entrapping flagellated cells)[END_REF][START_REF] Fierro-Constaín | The conservation of the germline multipotency program, from sponges to vertebrates: A stepping stone to understanding the somatic and germline origins[END_REF][START_REF] Alié | The ancestral gene repertoire of animal stem cells[END_REF][START_REF] Okamoto | The active stem cell specific expression of sponge Musashi homolog EflMsiA suggests its involvement in maintaining the stem cell state[END_REF] . Yet, choanocytes are usually interpreted as a minor component of sponge stem cell systemi.e., the stock of archaeocyte progeny that is able to transform back to archaeocytes 9,23 .

For the proper cell turnover, cell proliferation should be balanced by programmed cell death.

The most common path of programmed cell death in intact tissues of animals is apoptosis 3,29-32 .

However, immunohistochemical studies reveal few apoptotic cells in intact sponge tissues. Most of them are amoeboid cells located in the mesohyl; caspase-positive choanocytes are extremely rare [START_REF] Degoeij | Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding[END_REF] .

Meanwhile, some sponges are able to discard certain cell types (e.g., choanocytes, archaeocytes, lophocytes, spherulous and granular cells) into the environment through the aquiferous system [START_REF] Degoeij | Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding[END_REF][START_REF] Alexander | Cell turnover and detritus production in marine sponges from tropical and temperate benthic ecosystems[END_REF][START_REF] Maldonado | Sponge waste that fuels marine oligotrophic food webs: a re-assessment of its origin and nature[END_REF] .

Expelled cells show various signs of degradation, but the nature of this process remains obscure [START_REF] Degoeij | Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding[END_REF] .

Considering these peculiarities of the tissue organization, sponges can be promising objects for studies regarding the origin and evolution of physiological regeneration mechanisms in metazoans.

The aim of this study is the comparative and quantitative analysis of the proliferation and apoptosis in intact tissues of two sponge species, Halisarca dujardinii Johnston, 1842 (Demospongiae) and Leucosolenia variabilis Haeckel, 1870 (Calcarea). These species belong to different Porifera classes and represent fundamentally different types of the aquiferous system organization [START_REF] Lavrov | Sewing up the wounds : The epithelial morphogenesis as a central mechanism of calcaronean sponge regeneration[END_REF][START_REF] Ereskovsky | Molecular and morphological description of a new species of Halisarca (Demospongiae: Halisarcida) from Mediterranean Sea and a redescription of the type species Halisarca dujardini[END_REF] . We have combined confocal laser scanning microscopy and flow cytometry to identify proliferating and apoptotic cells, as well as evaluate their number and distribution in the sponge body. Altogether, these data are able to complement the concept of tissue dynamics in these basal metazoans.

Results

Cell proliferation in intact sponge tissues: quantitative analysis and spatial distribution

Halisarca dujardinii (class Demospongiae) is a thick globular sponge with 1-2 oscular tubes and a leuconoid aquiferous system (Figure 1A,B). Its body is subdivided into two regions: the ectosome and endosome (Figure 1C). The ectosome represents a superficial region of the body devoid of choanocyte chambers. The external parts of T-shaped exopinacocytes are underlined with a thick layer of diffuse collagen which contains rare spherulous cells (Figure 1C). A dense layer of amoeboid cells (e.g., spherulous and granular cells) and cell bodies of the exopinacocytes are located beneath the diffuse collagen layer (Figure 1C). The internal region of a sponge, the endosome, contains large, tubular choanocyte chambers surrounded by the mesohyl (Figure 1C,D). The mesohyl contains numerous cells (e.g., archaeocytes, lophocytes, spherulous, vacuolar and granular cells) and occupies a significant volume of the endosome (Figure 1C). The structure of the aquiferous system is similar in regions of endosome adjacent to the oscular tube and at some distance (approximately half the body radius) from it (Figure 2A,B). The oscular tube is a principally different region of a sponge, representing a thin-walled outgrowth composed mainly of exo-and endopinacoderm (Figure 2C). A thin layer of mesohyl lies between exo-and endopinacoderm; choanocyte chambers are absent.

To evaluate the number and distribution of proliferating cells in intact tissues of Halisarca dujardinii, we have utilized two markers: 5-ethynyl-2'-deoxyuridine (EdU) and Ser10-phosphorylated 1). All EdU-positive cells belong to the mesohyl/pinacoderm of the oscular tube, as this region is devoid of choanocyte chambers (Figure 3C). The fraction of EdU-positive cells in the oscular tube is significantly lower than the total fraction of EdU-positive cells in the regions of endosome (p=0.0157 and 0.005 compared to the endosome distant from the oscular tube and adjacent to it, respectively) (Figure 3A). However, the fraction of EdU-positive cells in the oscular tube shows no difference with the fraction of EdU-positive mesohyl cells in the regions of endosome (p-values>0.9999 in both cases) (Figure 3C).

Leucosolenia variabilis (class Calcarea

) is an asconoid sponge; its body is a plexus of branching and anastomosing thin-walled hollow tubes (Figure 4A). This plexus -the cormus -gives rise to the oscular tubes and blind outgrowths, the diverticula. The body wall is composed of the 2). In the distal parts of the diverticula, the proportion of pH3-positive cells remains the same (0.50±0.20%), while the proportion of EdU-positive cells decreases (6.53±2.08%) (Figure 6A, B; Table 2). The decrease is significant compared to the distal and middle parts of oscular tubes (p-values=0.0068 and 0.0022, respectively); in other pairwise comparisons differences are insignificant, but p-values are slightly higher than 0.05.

The oscular rim has the lowest fraction of proliferating cells compared to other body parts: it contains only 0.40±0.96% of EdU-positive and 0.17±0.20% pH3-positive cells (Figure 6A, B; Table 2). In case of EdU, the decrease in the number of labeled cells is significant compared to all body regions, except the distal part of the diverticula; the difference in the number of pH3-positive cells in significant, excluding the distal part of the diverticula and cormus tube.

Most EdU-and pH3-positive cells of L. variabilis are choanocytes. However, few EdU-positive and pH3-positive cells belong to the mesohyl/pinacoderm layer (Figures 5A-G; 6C, D). The proportion of the choanocytes in the total number of EdU-positive cells remains almost the same (95% on average) in different parts of the oscular tube, cormus and diverticula (Figure 6C; Table 2). In the case of pH3-positive cells, choanocytes usually represent approximately 75% of all late G2/M cells (Figure 6D; Table 2). In the distal parts of the diverticula, their proportion decreases to 40.64±22.29% (Figure 6D; Table 2). This decrease is significant in comparison with the cormus, middle and proximal parts of the oscular tube (p=0.0023, 0.0003 and 0.0002, respectively).

EdU tracking experiment in H. dujardinii

Since labeled nucleotides are transferred to daughter cells after mitosis, EdU allows us to track proliferating cells or their progeny. We treated individuals of H. dujardinii with 200 µM EdU for 12 hours and transported them to the running seawater aquarium. After the transportation, EdU incorporation is ceased, and the distribution of EdU-positive cells in intact tissues of H. dujardinii gradually changes: the contribution of the choanocytes to the total amount of EdU-positive cells decreases, while contribution of the mesohyl cells increases (Figure 7А,В). At the beginning of the experiment, mesohyl cells account for only 7.92±6.11% of labeled cells; 7 days after, their fraction significantly increases, reaching 55.12±23.15% (p=0.0026) (Figure 7С).

Cell cycle distribution in H. dujardinii and L. variabilis intact tissues

Cell cycle distribution was assessed by the quantitative DNA staining of single-cell suspensions obtained via mechanical dissociation in calcium and magnesium-free seawater (CMFSW-E). The major part of H. dujardinii cells (38.94±5.90%) form a diploid G0/G1 population peak (Figure 8А; Table 3). The tetraploid cells in the G2/M phase form a less distinguishable peak characterized by a 2-fold higher fluorescence intensity than G1/G0 cells (Figure 8A). The G2/M population contains 9.28±1.78% of all cells (Table 3). Cells in the S phase are located between the G0/G1 and G2/M populations and account for 20.18±6.14% of the total population (Table 3). The SubG1 population is characterized by lower DNA content than G1/G0 cells (Figure 8A). Scatter plots indicate that the SubG1 population consists of small and highly granulated objects -possibly, dying cells and debris (Figure 8C).

CMFSW-E suspensions of L. variabilis do not provide reliable data: they contain too many SubG1 and too few G2/M cells to obtain an accurate cell cycle distribution histogram and were excluded from the further analysis. Instead, suspensions made of pre-fixed L. variabilis tissues were used. This method provides improved resolution: the suspensions contain fewer SubG1 cells and show well-distinguishable G0/G1 and G2/M peaks. The general appearance of the histogram is similar to H. dujardinii (Figure 8В). The proportion of the G0/G1 cells is 53.88±2.59%, S cells -12.58±0.76% and G2/M cells -4.63±1.79% (Table 3).

Investigation of apoptotic activity

To study the apoptosis, individuals of H. dujardinii and L. variabilis were vitally stained with fluorescent substrate for effector caspases -CellEventfor two hours. Subsequent CLSM studies reveal an insignificant number of apoptotic cells in intact tissues of these sponges (Figure 9A,D).

Most of these cells exhibit weak CellEvent fluorescence. Nonetheless, they are easily resolved from autofluorescent cells by nuclear localization of the signal characteristic of CellEvent (Figure 9B-F). In L. variabilis, the proportion of CellEvent-positive cells does not exceed 1% in any studied body region (Table 4). There are choanocytes as well as mesohyl cells among these apoptotic cells. The endosome of H. dujardinii demonstrates even less apoptotic activity: for 6-9 thousand normal cells, there are usually 1-2 CellEvent-positive ones (Table 4).

We also studied suspensions obtained via mechanical dissociation in CMFSW-E. 5). TMRE+ CE+ population lies in the area of higher CellEvent fluorescence, Q2. This population reaches 7.51±0.87% in H. dujardinii and 8.58±0.77% in L. variabilis (Figure 11C, D; Table 5). TMRE-CE+ cells reside in Q3. They do not exceed 1% of the general population and do not form a distinguishable cluster (Figure 11C, D; Table 5). All CellEventpositive cells (Q2 and Q3) seem to be small and highly granulated objects, as indicated by light scatter parameters (Figure 11B). TMRE-CE-population is located in Q4 and accounts for 1.67±0.35% in H. dujardinii and 13.70±3.25% in L. variabilis (Figure 11C, D; Table 5). Although these cells are also smaller than TMRE+ CE-cells, they tend to separate from TMRE+ CE+ cells on scatter plots (Figure 11B).

Discussion

In multicellular animals, the long-term maintenance of tissue homeostasis is achieved by cell turnover 1,4

. Cell turnover is composed of three complementary processes: cell proliferation, cell differentiation and programmed cell death . Sponges are basal metazoans well known for their unique tissue dynamics. In this study, we mainly focus on cell proliferation and apoptosis in intact tissues of sponges, providing promising data on the cell turnover in sponges.

Cell proliferation in intact tissues of H. dujardinii and L. variabilis

Halisarca dujardinii is a leuconoid demosponge that demonstrates a significant number of cycling cells under the steady-state condition. The presence of EdU-and pH3-positive cells in intact tissues is consistent with flow cytometry data, as the distribution of H. dujardinii cells by the DNA content reveals the classic cell cycle distribution curve 37 . However, the estimates of proliferating cell fractions are slightly different. The proportion of EdU-and pH3-positive cells is about 8% of the total cell number, while S plus G2/M cells together reach 28% -possibly because our CLSM staining is not able to detect G2-phase cells.

Different regions of the endosome display active proliferation and do not differ significantly in the proportions of EdU-and pH3-positive cells; the proliferation activity seems to be evenly distributed throughout the sponge's endosome. The oscular tube possesses a different pattern of proliferation and contains few labeled cells. We attribute such difference to the absence of choanocyte chambers and choanocytes in the oscular tube. In other demosponges, choanocytes have already been shown

to incorporate labeled nucleotides [START_REF] Borisenko | Transdifferentiation is a driving force of regeneration in Halisarca dujardini (Demospongiae, Porifera)[END_REF][START_REF] Degoeij | Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding[END_REF][START_REF] Alexander | Cell turnover and detritus production in marine sponges from tropical and temperate benthic ecosystems[END_REF][START_REF] Kahn | The role of cell replacement in benthic-pelagic coupling by suspension feeders[END_REF] ; however, they might have retained the BrdU signal as the progeny of other proliferating cells. Here we demonstrate that choanocytes are also labeled with anti-pH3 antibodies. Thus, choanocytes do not only synthesize DNA but also divide, being the main proliferating fraction of cells in H. dujardinii.

Leucosolenia variabilis is a calcareous asconoid sponge. Despite that, the pattern of proliferation is the same as in H. dujardinii. A significant number of EdU-and pH3-positive cells and cell cycle distribution curve prove the fact of constant proliferation. As in other calcareous sponges, the major part of DNA synthesizing cells are choanocytes [START_REF] Lavrov | Sewing up the wounds : The epithelial morphogenesis as a central mechanism of calcaronean sponge regeneration[END_REF][START_REF] Kahn | The role of cell replacement in benthic-pelagic coupling by suspension feeders[END_REF] . Most pH3-labeled cells are also choanocytes, although the proportion of choanocytes among all pH3-labelled cells is lower than in H.

dujardinii. Body regions containing choanocytes display a high number of cycling cells and do not differ in the rate of proliferation. The only exception is the distal parts of the diverticula. Fewer EdUpositive cells are found there; interestingly, the proportion of pH3-positive cells remains almost the same. This body region also differs in the proportion of choanocytes among the pH3-positive cells;

nearly half of the G2/M cells belong to the mesohyl/pinacoderm layer. It could be evidence of potential growth since new sclerocytes, pinacocytes and porocytes are obviously needed to lengthen the diverticula. In the oscular rim, which is devoid of choanocytes, the intensity of cell proliferation is very low. There are, however, few labeled cells, indicating either slow cell turnover or growth.

This study provides the first spatial analysis of cell proliferation in different body regions of sponges representing distinct phylogenetic lines and distinct types of aquiferous system. In general, proliferation activity appears to be evenly distributed throughout sponge tissues (excluding regions that do not contain choanocytes). The proportion of S-phase cells in H. dujardinii is lower than in other sponges -in particular, tropical Demosponges, in which this parameter reaches 20-25% on average 34,38 . Considering calcareous sponges, the proportion of S-phase cells in L. variabilis is slightly higher than in Sycon coactum

38

. It should also be noted that during regeneration studies, the proportion of EdU-labeled cells in L. variabilis has been rather overestimated compared to current data, possibly because of the counting technique 12 . Similar to the previous studies, we detect that DNA synthesis occurs primarily in choanocytes; additionally, we show that the distribution of mitotic cells in adult sponges have similar pattern. Overall, the proportion of proliferating cells in H. dujardinii and L. variabilis resembles those in animals characterized by fast cell turnover -platyhelminthes, cnidarians, bivalvians 39-42 .

The contribution of choanocytes to homeostasis

The physiological role of proliferation in intact tissues is either growth or renewal In fact, the boundary position of choanocytes might be advantageous since cell division requires significant amount of resources. As the primary food-entrapping sponge cells, choanocytes are provided with sufficient energy for constant proliferation and respond appropriately to environmental changes. Moreover, since choanoderm is obviously enriched in cell-cell interaction and at least partially compartmentalized by extracellular matrix 18 , it could maintain the proliferative potential of choanocytes through some signaling system resembling the stem cell niche. In some animals (e.g., Hydra and planarians), the concept of niche may be interpreted in a rather broad sense, regarding a whole organ/tissue as stem cell niche 69,70 . Choanoderm might be just the example of such a "tissue niche".

Since the flagellum is resorbed during the cell division, the filtration process becomes impaired; to resolve the conflict between proliferation and food uptake, choanocytes should a) maintain the proliferating fraction at a certain level or b) minimize the period of the absence of the flagellum. The short duration of the M phase is possibly the reason why the proportion of pH3-positive cells does not exceed 1% in any examined area of H. dujardinii and L. variabilis. Indeed, although the length of cell cycle phases varies significantly among different animals, the M phase is always the shortest (e.g., 3-5% of the total duration of the cell cycle in mammals)

51

.

It has been shown earlier that choanocytes are the main proliferative fraction of cells in intact tissues of demosponges and calcareous sponges: in most species, the proportion of EdU(BrdU)positive cells of mesohyl is significantly lower than the proportion of labeled choanocytes 33,34,38 . Still, it is usually stated that the proliferation of choanocytes is restricted to the self-renewal of the choanoderm, and the mesohyl contains an autonomous population of cycling cells (e.g., archaeocytes of Demospongiae) The second option considers an extensive shedding of choanocytes described in the previous works; in these studies, choanocytes were found to be massively discarded into the environment.

Nonetheless, there is little physiological sense in getting rid of newly produced (i.e., EdU-positive) cells. One should expect that old, non-proliferative choanocytes are predominantly shed. Finally, extensive cell shedding is characteristic of sponges having high microbial abundance in their tissues 34

; in turn, H. dujardinii should be categorized as the low microbial abundance sponge 36 .

In line with similar experiments on planarians, the label flux in H. dujardinii can also occur via cell migration

52

. Since choanocytes are the main DNA synthesizing population of cells, the increase in the proportion of EdU-positive mesohyl cells could be a consequence of the migration of choanocytes (or their progeny) into the mesohyl. Of course, the population of archaeocytes might be self-renewing to some extent, but the label flux can be an indirect evidence that choanocytes contribute to the pool of mesohyl cells. Even if archaeocytes take part in the renewal of the choanoderm, the general tissue dynamics leans towards the opposite direction.

The same process may occur in intact tissues of L. variabilis. We have not performed pulsechase experiments of this species; however, the spatial heterogeneity of the proliferation might serve as potential evidence of cell migration. For instance, it is doubtful that DNA synthesis occurs in the oscular rim. In this body region, the decrease in the proportion of EdU-positive cells outstrips the decrease of pH3-positive cells. The observed variation in the ratio of EdU/pH3-positive cells could be explained by the alterations in the cell cycle lengthe.g., the presence of fastly cycling cells.

However, it is unlikely since we would have seen much more EdU-positive cells that retain the label after the S phase. We suppose that pH3-positive cells in the oscular rim actually might have migrated from the more proximal parts of the oscular tube possessing well-developed choanoderm. In the distal part of the diverticula, there are fewer pH3-positive choanocytes than in other parts of the body; however, the proportion of EdU-positive choanocytes and mesohyl cells remains the same. This can be a result of cell migration: choanocytes occasionally leave the choanoderm and contribute to the growth of the diverticula, giving rise to mesohyl/pinacoderm cells.

To sum up, choanocytes are apparently the major part of cycling cells not only in calcareous sponge L. variabilis but also in demosponge H. dujardinii. It seems that the proliferation of choanocytes is not solely limited to the renewal of the choanoderm. It has been previously shown that choanocytes contribute to gametogenesis 8,53-55

, regeneration 12,14,16 , and express certain stem cell markers (at least in demosponges and homoscleromphs) 26,28,56,57 . Moreover, it seems that choanocytes as the main proliferating fraction of cells may also participate in cell turnover through cell migration 57

. Altogether, these data suggest that the role of choanocytes in the stem cell system of sponges can be underestimated. They may be the major source of new cells (including cells of mesohyl) in both calcareous sponges and demosponges during regular cell turnover.

Apoptotic activity in intact tissues of sponges

Under the steady-state condition, cell proliferation is counterbalanced by the process of cell elimination. Apoptosis is considered to be the most common way of cell death in animals . Given the fact that choanocytes are highly proliferative, we would expect a significant amount of dying cells.

CLSM studies using CellEvent staining indicate that apoptotic cells are almost absent in intact tissues of H. dujardinii and L. variabilis: less than 1% of cells contain active effector caspases. We consider this result to be the most relevant -in particular, it fits well with other known studies 16,33,34 .

Surprisingly, there are significantly more CellEvent-positive cells in the CMFSW-E suspensions analyzed with the flow cytometer. These cells are small, highly granulated and contain active effector caspases; thus, they demonstrate classic features of apoptosis [START_REF] Vorobjev | Temporal Heterogeneity Metrics in Apoptosis Induced by Anticancer Drugs[END_REF][START_REF] Barteneva | Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting[END_REF] . Suspensions of H. dujardinii contain few apoptotic cells. The case of L. variabilis, in which CellEvent-positive cells comprise up to 60% of the general population, is more intriguing.

SubG1 cells detected by means of DNA content are also to be mentioned here. These small, highly granulated cells characterized by fragmented DNA should be considered apoptotic. Since cell debris and bacteria might also be detected as a SubG1 population, it does not represent an accurate estimation of the number of apoptotic cells. However, SubG1 cells may serve for indirect rough evaluation of apoptotic activity. The presence of SubG1 cells seems to be an inevitable consequence of sample preparation 65 . Yet, the proportion of SubG1 cells differ drastically between species:

CMFSW-E dissociation does not significantly affect H. dujardinii, but leads to a massive transition of Apparently, H. dujardinii and L. variabilis cells maintain functional mitochondria during apoptosis.

Thus, there is a possibility that the apoptosis in sponge cells is MOMP-independent. Therefore, in contrast to mammalian cells, the TMRE-CE-population may contain not early apoptotic cells but represents a population of damaged or dead cells and cell debris. Rare TMRE-CE+ cells might be interpreted as non-viable, late apoptotic cells with non-functional mitochondria.

Conclusions

Here we provide quantitative and spatial analysis of cell proliferation and apoptosis in two The number of apoptotic cells in intact tissues of both species is insignificant; however, there is a possibility that a short duration of apoptosis might compensate for the low proportion of apoptotic cells. Regarding that apoptotic response is developed rather quickly under certain conditions (e.g., dissociation of L. variabilis) it could still be involved in the maintenance of the tissue homeostasis. For instance, choanocytes might undergo some kind of anoikis, the detachment-induced form of apoptosis [START_REF] Günther | Apoptosis, necrosis and necroptosis: Cell death regulation in the intestinal epithelium[END_REF][START_REF] Bullen | Characterization of epithelial cell shedding from human small intestine[END_REF] . In tissues exposed to the environment, cell extrusion may serve as an alternative to the engulfment of apoptotic bodies. It is commonly thought that cell extrusion promotes anoikis via loss of survival signaling through cell-cell and cell-matrix interactions 75,76 , although the activation of caspases may occur prior to cell shedding

74

. There is a possibility that choanocytes actually undergo apoptosis, but the activation of caspases takes part mostly after the detachment.

Obtained data will serve as a basis for further detailed researches of tissue homeostasis in H. dujardinii and L. variabilis. While we have an information about spatial distribution and intensity of cell proliferation and apoptosis for a number of sponge species, many other processes and mechanisms contributing to the tissues homeostasis in sponge remains uncharacterized, e.g., the behavior and fate of proliferating cell progeny, temporal characteristics of the proliferation, apoptosis and differentiation, and, finally, the signaling systems maintaining proliferative potential of cells. Altogether, these data would help us to establish a comprehensive model of the tissue dynamics in these basal metazoans.

Experimental Procedures

Sampling and laboratory maintenance

Histological studies of intact tissues

For histological studies, sponge tissues were fixed for 2 hr at 4°C with 2.5% glutaraldehyde (Electron Microscopy Science, 16020) and post-fixed for 1 hr at room temperature (RT) with 1% OsO 4 (Electron Microscopy Science, 19100). Fixation and post-fixation were done in modified 0.1M Na-Cacodylate buffer (0.1M Na-Cacodylate, 85.55 mM NaCl, 5 mM CaCl 2 , 5 mM MgCl 2 ; рН 7.0-7.5).

After the post-fixation, specimens were dehydrated and embedded in Epon/Araldite epoxy embedding media (Electron Microscopy Science, 13940) according to the standard protocol described earlier (Lavrov et al.,

Semi-thin sections (1 µm) were cut using LKB V and Leica UC6 ultramicrotomes. The sections were stained with 1% toluidine blue -0.2% methylene blue mixture for 1-1.5 min at 60°C and studied with Carl Zeiss Axioplan 2 microscope (Carl Zeiss) equipped with digital camera AxioCam HRm (Carl Zeiss) and software AxioVision 3.1 (Carl Zeiss).

Study of cell proliferation with CLSM

We used 5-ethynyl-2'-deoxyuridine (EdU) and Ser10-phosphorylated histone 3 (рН3) monoclonal antibodies as the markers of cell proliferation. EdU is a nucleoside analog of thymidine, which incorporates into nuclei of proliferating cells during DNA synthesis marking cells in the Sphase 77

. Antibody against phosphorylated histone 3 labels cells in the late G2-phase and M-phase 78 .

To mark choanocytes' flagella, we used antibodies to acetylated-α-tubulin.

The EdU (Lumiprobe, 10540) stock solution was prepared in dimethyl sulfoxide (DMSO, MP Biomedicals 196055). Optimal EdU concentration and incubation time were found out during the preliminary studies. H. dujardinii individuals were incubated in 5 ml of FSW with 200 μM EdU for 12 hours, L. variabilis -in 5 ml of FSW with 20 μM EdU for 6 hours. Sponges kept in 5 ml FSW with 100 or 10 μM DMSO served as negative controls. We examined 8 individuals of H. dujardinii and 6 individuals of L. variabilis.

After the EdU incubation, all individuals were fixed with 4% paraformaldehyde in phosphatebuffered saline (4% PFA PBS, Carl Roth 0335.2) for 12 hours at +4°С. Fixed specimens were rinsed with PBS several times. Then they were incubated in 1-3% bovine serum albumin in PBS (BSA, MP Biomedicals 0216006980) and permeabilized by 0.5% Triton X-100 (Sigma-Aldrich T8787) in PBS.

Click-reaction (visualization of EdU) was performed for 1 hour at RT in a solution of 4 mM CuSO 4 , 10 μM Sulfo-Cyanine3 Azide (Lumiprobe A1330), 20 mg/ml sodium L-ascorbate (Sigma-Aldrich 11140) in PBS. Click-reaction and subsequent manipulations were carried out in the dark. Specimens were blocked with a blocking solution of 1% BSA, 0.1% gelatin from cold-water fish skin (Sigma-Aldrich G7041), 0.5% Triton X-100, 0.05% Tween-20 (Sigma-Aldrich P1379) in PBS and then incubated in the primary antibodies (Rabbit Anti-phospho-histone H3, Sigma-Aldrich H0412 and Mouse Antiacetylated-α-tubulin, Sigma-Aldrich T6793, both diluted 1:1000 in blocking solution) overnight at +4°С.

After that, specimens were rinsed twice with the blocking solution and treated with the secondary antibodies (Donkey anti-Mouse IgG Alexa Fluor 488, ThermoFisher Scientific A21202 and Donkey anti-Rabbit IgG Alexa Fluor 647, ThermoFisher Scientific A-31573 in blocking solution, both final concentration 1 μg/ml) overnight at +4°С. Cell nuclei were stained with 4′, Cell proliferation was studied in several body regions of sponges. Three regions were studied in H. dujardinii: 1) the endosome adjacent to an oscular tube (n=8), 2) the endosome at some distance (approximately half the body radius) from an oscular tube (n=8), 3) the oscular tube (n=4).

Seven regions were studied in L. variabilis: 1) the oscular rim (n=10), 2) the distal part of the oscular tube (n=10), 3) the middle part of the oscular tube (n=10), 4) the proximal part of the oscular tube (n=10), 5) the distal part of the diverticulum (n=14), 6) the proximal part of the diverticulum (n=14), 7)

the cormus tube (n=11). Studied fields of view within each region were chosen randomly. For subsequent analysis, a Z-stack was obtained from each field of view.

Z-stacks were obtained via CLSM Nikon A1 (Nikon, Shinagawa, Japan) using lasers of wavelength 405 nm (DAPI), 488 nm (Mouse Anti-acetylated-α-tubulin ABI + DAM IgG Alexa Fluor 488 ABII), 561 nm (EdU + Sulfo-Cyanine3 Azide) and 648 nm (Rabbit Anti-phospho-histone H3 ABI + DAR IgG Alexa Fluor 647). All Z-stacks were obtained with a 1-μm Z-step, 48 μm (H.

EdU tracking experiment in H. dujardinii

Intact H. dujardinii individuals were incubated in 5 ml of FSW with 200 μМ EdU for 12 hours and then transferred into a running seawater aquarium. Sponges incubated in 5 ml of FSW with 100 µl of DMSO were used as negative controls. Sponges were fixed with 4% PFA PBS at 1, 5 and 7 days after transferring into the aquarium (3, 3, and 5 individuals, respectively). Individuals fixed immediately after the EdU incubation served as the initial time point -0 days. All specimens were treated for

To obtain cell cycle distribution, we used the internal FlowJo CellCycle plugin. The Watson model approximates DNA content curve as a sum of two Gaussian distributions represented by G0/G1 and G2/M cells, considering cells between them as cells in the S phase. In most cases, standard parameters were used; when the G2/M peak was hard to distinguish, we constrained its position depending on the relative positions of the G0/G1 and G2/M peaks. No CV constraint was performed.

Study of apoptotic activity in intact tissues

Tetramethylrhodamine ethyl ester (TMRE, ThermoFisher Scientific, T669) and CellEvent

Statistical analysis and plotting

Statistical analysis and plotting were performed in GraphPad PRISM v8.0.

histone 3 (

 3 рН3) monoclonal antibodies. After 12 hours of treatment with 200 µM EdU, intact tissues of H. dujardinii contain numerous EdU-positive nuclei belonging to a population of cycling cells (Figure 2A, B). Fewer cells have nuclei labeled with pH3-antibodies, representing a fraction of cycling cells in the late G2 phase or M phase. Very few pH3-positive cells also contain EdU in the nuclei. The endosome adjacent to the oscular tube and the endosome distant from the oscular tube display similar fraction of proliferating cells: they contain 8.44±3.08% and 8.10±1.64% of EdU-positive cells, respectively. Each region also has 0.11±0.07% of pH3-positive cells (Figure 3A, B;

  exopinacoderm and choanoderm layers separated by thin mesohyl containing few cells, mostly amoebocytes and sclerocytes (Figure 4C-G). The cell bodies with nuclei of T-shaped exopinacocytes and porocytes are located near mesohyl cells (Figure 4E). Still, choanocytes are easily distinguished from other cells as they form a continuous layer of tightly packed pyriform nuclei associated with flagella (Figure 4E-G). Such structure of the body wall is characteristic of most regions of Leucosolenia body: diverticula, cormus tubes, and the most part of oscular tubes. Only the oscular rim, the most distal part of the oscular tube, differs in the body wall structure: in this region, the choanocytes are substituted by the flat endopinacocytes (Figure 4C). Intact tissues of L. variabilis display a significant number of cycling cells in S and G2/M cells after 6 hours of treatment with 20 µM EdU (Figure 5A-G). Few pH3-positive cells display EdU signal in the nuclei (Figure 5H). Regions containing choanocytes exhibit high proliferation rate. Most of these regions (cormus tubes, proximal parts of diverticula, oscular tubes) show similar fractions of proliferating cells (p-values>0.9999 in each pairwise comparison), each containing about 10% of EdUpositive and 0.6% of рН3-positive cells (Figures 5B-G; 6A, B; Table

  They were stained with CellEvent, fixed, and analyzed via flow cytometer. Cells of H. dujardinii form a single peak with low/negative CellEvent fluorescence (Figure 10А, B). The fraction of apoptotic cells is low: CellEvent-positive cells account only for 1±1.15% of the total population. In contrast, cells of L. variabilis are clearly divided into two subpopulations by the level of CellEvent fluorescence (Figure 10C, D). CellEvent-positive cells with elevated autofluorescence (as evidenced by increased shortwave fluorescence in the PacificBlue channel) comprise 60.8±13.46% of the total cell population (Figure 10C, D). We consider this result as a sampling artifact (see the "Discussion" section) Suspensions were also studied in vivo; during this experiment, non-fixed suspensions were stained for 30 min with CellEvent and TMRE (TMRE indicates the presence of the mitochondrial outer membrane potential -i.e., the viability of the mitochondria). These suspensions were studied both by CLSM and flow cytometry. Four cell types can be distinguished via CLSM (Figure 11A): 1) TMRE+ CE-cells possessing functional mitochondria (as indicated by TMRE fluorescence) and no active effector caspases (as clear nuclear CellEvent signal is absent); 2) TMRE-CE-cells characterized by the absence of viable mitochondria or active effector caspases (i.e., no CellEvent or TMRE fluorescence observed); 3) TMRE+ CE+ cells possessing both functional mitochondria and active effector caspases (bright TMRE and CellEvent signals are present); 4) TMRE-CE+ cells distinguished by high activity of effector caspases and the loss of the mitochondrial outer membrane potential; these cells display bright nuclear CellEvent signal but no TMRE fluorescence. The same types of cells have been observed via flow cytometer (Figure 11B-D). Both species demonstrate similar cell distribution. The major part of cells belongs to the TMRE+ CE-population, which resides in quadrant Q1. It accounts for 89.67±1.06% cells in H. dujardinii and 70.55±1.48% cells in L. variabilis (Figure 11C, D; Table

3

 3

  dynamics of sponge tissues, we should figure out the fate of proliferating cells. In intact sponges, the proliferation takes place in the choanoderm. Since the choanoderm is a) metabolically active and b) directly exposed to the environment, we should expect high rate of choanocyte turnover Despite constantly facing a potentially hostile environment (e.g., toxic and mutagenic factors dissolved in seawater), choanocytes themselves undergo constant proliferation and represent the main source of new cells in the choanoderm. It should be noted here that the proliferation of morphologically differentiated cells is fairly common among metazoans, including vertebrates 3,46-49 . Particularly interesting is the case of Cnidaria, in which ecto-and endodermal epithelial cells do not only act as barrier and contractile cells but also retain multipotency and the ability to proliferate 50 .

8 .homoscleromorph sponges 8 .

 88 According to Funayama, choanocytes and mobile mesohyl cells both represent distinct types of stem cells 21 ; however, choanocytes display broad stem cell activity only in calcareous 12 and In demosponges and hexactinnelids, archaeocytes are thought to be active stem cells; choanocytes are the minor component of the stem cell system, transforming into archaeocytes only under specific circumstances 23 .Indeed, we have observed EdU-and pH3-positive cells outside the choanoderm in intact tissues of H. dujardinii and L. variabilis. However, the fraction of labeled mesohyl cells is too low and variable to assume that the mesohyl contains a large population of cycling cells. Instead, we suppose that at least part of labeled mesohyl cells originates from the choanoderm. Since we have used prolonged incubation in EdU, not all observed EdU-positive cells are actually in the S phase. For instance, few pH3-positive cells also contain the EdU signal, indicating that these cells have finished the S phase and passed through the G2 phase by the moment of fixation. Since DNA synthesis occurs predominantly in the choanoderm, it is tempting to assume that these pH3-positive cells have migrated to the mesohyl from the choanoderm. In such case, some of the EdU-only-positive cells in the mesohyl could be choanoderm-derived cells that have not yet reached the M phase.EdU tracking experiment in H. dujardinii provides us with more clues. After the removal of the EdU from the medium, the proportion of labeled mesohyl cells continuously increases. There are several ways to interpret this result. It could well be that the proliferation of mesohyl cells drive the increase in the proportion of labeled mesohyl cells. Comparing to choanocytes, cell cycle of archaeocytes might be way shorter. If so, EdU-labeled progeny of archaeocytes would easily outnumber slowly proliferating choanocytes by the final time point. Apparently, this is not the case with H. dujardinii: assuming that the S and M phases of archaeocytes are very short, we would have seen much more EdU-positive cells in mesohyl immediately after the treatment with EdU (i.e., at the initial time point). Thus, there is no evidence that archaeocytes proliferate faster than choanocytes.On the other hand, the proportion of labeled mesohyl cells can increase only due to the loss of EdU-positive choanocytes -e.g., label dilution or cell shedding. The first option assumes that labeled nucleotides are equally distributed between daughter cells; after the division, the intensity of the signal decreases by two. Continuous proliferation may cause the intensity of the fluorescence to drop beyond the registration threshold, making it hard to detect some EdU-positive cells. Since choanocytes are actively cycling, they are more affected by the label dilution than mesohyl cells. On the other hand, the division of an EdU-positive cell should also result in appearance of two EdU-positive daughter cells. This process could at least partially compensate for the decrease in the number of EdU-positive cells through label dilution.

L 66 . 67 . 63 . 68 . 69 . 70 . 72 .

 66676368697072 . variabilis cells to the SubG1 population. Obviously, CMFSW-E suspensions of L. variabilis indicate massive cell death caused by sample preparation; the apoptotic response develops quicklywithin one or two hours. More importantly, this is not the case of suspensions made of pre-fixed tissues. It seems dissociation itself results in the activation of the apoptotic machinery in cells of L. variabilis. Thus, the observed number of CellEvent-positive cells and SubG1-cells in the suspensions of L.variabilis cannot be considered an adequate estimation of the apoptotic activity.The observed interspecific variability of the apoptotic response might be associated with the phylogenetic position and type of body organization. While the major part of H. dujardinii (Demospongiae) body is occupied by mesenchymal tissue, L. variabilis (Calcarea) is mainly composed of epithelial-like layers of choanoderm and pinacoderm. Overall, it seems that calcareous sponges represent more epithelial-like organization relying on tighter cell-to-cell interactions. For example, the regeneration occurs via blastema formation and mesenchymal morphogeneses in demosponges, while in calcareous sponges, it depends on the morphallactic reorganization of adjacent intact tissues through epithelial morphogeneses11,12,The disruption of the natural system of cell interactions during the CMFSW-E dissociation could lead to the massive induction of apoptotic activity in calcareous sponges.Certainly, some degree of apoptosis upregulation should be expected in non-fixed suspensions. Indeed, apoptotic cells account for 7-8% of the general population in both species during in vivo studies; however, despite the apoptosis upregulation, in vivo experiment sheds some light on the mechanisms of apoptosis. In mammalian cells, double staining with TMRE and CellEvent usually reveals the presence of three populations: 1) intact TMRE+ CE-cells characterized by intact mitochondria; 2) early apoptotic TMRE-CE-cells, in which mitochondrial transmembrane potential has vanished; 3) late apoptotic TMRE-CE+ cells, which contain non-functioning mitochondria and active effector caspases This situation is typical for mammalian cells since the permeabilization of the inner mitochondrial membrane is shown to be tightly associated with the mitochondrial outer membrane permeabilization (MOMP) In turn, MOMP is the critical event during the apoptotic response, resulting in the release of some pro-apoptotic proteins from the mitochondrial intermembrane space into the cytoplasm. The most crucial pro-apoptotic protein is cytochrome c, which is able to bind to cytoplasmic protein APAF1, forming the apoptosome and beginning to recruit effector caspases32,In flatworms (Dugesia dorotocephala and Schistosoma mansonii) and echinoderms (Strongylocentrotus purpuratus and Dendraster excentricus), the activation of effector caspases is also cytochrome-c dependent Thus, active effector caspases in their cells should be detected only after the inner membrane permeabilization and MOMP. Some invertebrates, however, do not fit into this scenario In common model objects, Caenorhabditis elegans and Drosophila melanogaster, the activation of effector caspases is not associated with the MOMP. In such case, the promotion of apoptotic cell death does not depend on the presence of cytochrome c in the cytoplasm; even if the MOMP is present, it is more a consequence than a reason for apoptotic response 71,Our data indicate that the apoptosis in sponges might resemble the scenario of Caenorhabditis elegans and Drosophila melanogaster: a significant amount of TMRE+ CE+ cells has been observed in H. dujardinii and L. variabilis suspensions. TMRE+ CE+ cells contain active effector caspases but possess well-functioning mitochondria. Unstained negative control suspensions indicate that the high level of TMRE fluorescence in these presumably early apoptotic cells is specific.

  sponges belonging to different classes of Porifera. Intact tissues of Halisarca dujardinii (Demospongiae) and Leucosolenia variabilis (Calcarea) are highly proliferative, indicating either high rate of growth or cell turnover. In both species, the most proliferating fraction of cells are choanocytes, and the proliferation occurs evenly throughout the areas of the body containing these cells. The proliferation of choanocytes maintains the tissue homeostasis in the choanoderm and, possibly, in the mesohyl/pinacoderm. Further investigations with in vivo cell tracking and in situ hybridization are needed to prove if the choanocytes or their progeny actively differentiate into other cell types.

  Halisarca dujardinii (Demospongiae, Chondrillida) and Leucosolenia variabilis (Calcarea, Leucosolenida) were collected in the Kandalaksha Bay of the White Sea near the Pertsov White Sea Biological Station of Lomonosov Moscow State University (66°34′ N, 33°08′ E) during August 2019-2020. Sponges were collected in the upper subtidal zone with the substrate (brown algae Fucus and Ascophyllum) and cleared of epifauna. Specimens were kept in laboratory aquariums with running natural seawater of ambient temperature. Each experiment was carried out on individuals of the same size collected the same day. Sponges were kept in open plastic Petri dishes filled with 5 ml of 0.22 μm-filtered seawater (FSW) during the experiments. Petri dishes were kept at 8-12°C on a shaker for better stirring and aeration of FSW.

  dujardinii) and 68 (L. variabilis) μm thick on average.During subsequent Z-stack analysis, we estimated the total fractions of EdU-and pH3positive nuclei (as a proportion of EdU-/pH3-positive nuclei to all nuclei stained with DAPI), the fractions of EdU-and pH3-positive choanocytes and mesohyl cells (as a proportion of EdU-/pH3positive choanocytes/mesohyl cells to all nuclei stained with DAPI) and the fraction of choanocytes among all EdU/pH3-positive cells (as a proportion of EdU-/pH3-positive choanocytes to all EdU-/pH3positive cells). Z-stack analysis was performed with Bitplane Imaris v7.2.1 software. Each stack of H. dujardinii and L. variabilis contained 6000-7000 and 2000-4000 cells, respectively. Choanocytes were identified by the presence of flagella and the characteristic shape of choanoderm/choanocyte chambers. Mesohyl cells were separated from choanocytes via the «Surface» tool. Cells of interest (e.g., EdU-or pH3-positive cells) were counted by the «Spots» tool; we combined automatic identification of nuclei via Quality/Intensity Median thresholds with manual correction of spots. Such counting procedure implies 3D visualization of a whole Z-stack and allows us to precisely distinguish between labeled cells of choanoderm and mesohyl.

Caspase- 3 / 7 63 .

 3763 Green ReadyProbes Reagent (ThermoFisher Scientific, R37111) (CellEvent) were used as markers of apoptotic activity for both CLSM and flow cytometry studies. The absence of TMRE fluorescence serves as a sign of the mitochondrial inner membrane permeabilization, which is characteristic of the early stages of apoptosis. CellEvent is an indicator of activated effector caspases present in cells during the middle and late stages of apoptosis In each experiment, we used unstained specimens as negative controls.CellEvent staining of intact tissues for CLSM. Specimens (3 sponges per species) were incubated in 5 ml of FSW with CellEvent (dilution 1:100) for 2 hours at +10°С in the dark. After the incubation, they were fixed with 4% PFA PBS. Then specimens were fixed with 4% PFA PBS, washed with PBS and blocking solution for several hours, and stained with DAPI as described above (see "Study of cell proliferation with CLSM" section). Specimens of L. variabilis were stained with antibodies to acetylated-α-tubulin to distinguish choanocytes. In case of H. dujardinii, no antibody staining was performed, and choanocyte chambers were identified by their characteristic shape visualized by DAPI staining. During this experiment, we used lasers of 405 nm (DAPI), 488 nm (CellEvent) and 648 nm (Mouse Anti-acetylated-α-tubulin ABI + DAM IgG Alexa Fluor 647 ABII) wavelengths. Details of data acquisition and nuclei counting are provided in the "Study of cell proliferation with CLSM" section. Any nuclei displaying bright CellEvent staining was considered an apoptotic; during the analysis, we estimated the total fraction of CellEvent-positive cells (as a proportion of CellEvent-positive nuclei to all nuclei labeled with DAPI) and the proportion of of cell cycle distribution in intact tissues" section); the final quadrant gating to distinguish between different cell populations was performed according to unstained control. Any event exceeding the autofluorescence of unstained control was considered a TMRE/CellEvent-positive cell.

Figure 1 .

 1 Figure 1. Anatomy and histology of Halisarca dujardinii. Ageneral appearance; Bschematic drawing of the aquiferous system representing regions of interest; Csection through the endosome and ectosome, light microscopy; Darea of endosome distant from the oscular tube, CLSM (maximum intensity projection of several focal planes); blue color -DAPI, cell nuclei; greenacetylated α-tubulin, flagella. Black dotted line marks the border between the ecto-and endosome. White dotted lines mark the borders of several choanocyte chambers. ararchaeocyte, ccchoanocyte chamber, cfcollagen fiber, enendosome, ecectosome, flflagella, grgranular cell, mesnucleus of mesohyl cell, sphspherulous cell. Scale bars: A -1 mm, C, D -50 µm.

Figure 2 .

 2 Figure 2. Cell proliferation in different body regions of Halisarca dujardinii, CLSM. A -endosome distant from the oscular tube, B -endosome adjacent to the oscular tube, Cthe oscular tube. All images are maximum intensity projections of several focal planes. Insets schematically show position of the studied regions in the sponge body. Blue color -DAPI, cell nuclei; red -EdU, nuclei of DNA synthesizing cells; yellow -pH3, chromatin of late G2/M cells; green -acetylated α-tubulin, flagella. Dotted lines mark the borders of choanocyte chambers. White arrowheads mark cells in the M-phase of the cell cycle; red arrowheads mark EdU-positive cells outside the choanocyte chambers. ссchoanocyte chambers (outlined by the dotted lines). Scale bars: A, B, C -50 μm.

Figure 3 .

 3 Figure 3. Quantitative analysis of cell proliferation in intact tissues of Halisarca dujardinii. A, Bfractions of EdU-(A) and рН3-positive (B) cells in different body regions. Data are shown with median values (thick horizontal lines), interquartile ranges (boxes), total ranges (whiskers), and outliers (dots). Asterisks mark significantly different regions of the body. C, Dfractions (mean values) of choanocytes/mesohyl cells among the total number of EdU-(C) and рН3-positive (D) cells.

Figure 4 .

 4 Figure 4. Anatomy and histology of Leucosolenia variabilis. Аgeneral appearance; Bschematic drawing of the aquiferous system representing regions of interest; Clongitudinal section through the distal part of the oscular tube (including the oscular rim), light microscopy; D, Ecross sections through the middle part of the oscular tube, light microscopy; Fthe proximal part of the divericulum, CLSM (maximum intensity projection of several focal planes); Gorthogonal XZ projection of the same Z-stack, CLSM (maximum intensity projection of several focal planes); blue color -DAPI, cell nuclei; green -acetylated α-tubulin, flagella. Osc. rimthe oscular rim; Osc. dist -distal part of the

Figure 5 .

 5 Figure 5. Cell proliferation in different body regions of Leucosolenia variabilis, CLSM. Аoscular rim; Bthe distal part of an oscular tube; C -the middle part of an oscular tube; D -the proximal part of an oscular tube; E -the proximal part of a diverticulum; F -the distal part of a diverticulum; G -a cormus tube; Emetaphase plate in the choanoderm of the middle part of the oscular tube. Images with the "xz" index represent XZ orthogonal projections of the respective Z-stack. All images are maximum intensity projections of several focal planes. Insets schematically show position of the studied regions in the sponge body. Blue color -DAPI, cell nuclei; red -EdU, nuclei of DNA synthesizing cells; yellow -pH3, chromatin of late G2/M cells; green -acetylated α-tubulin, flagella. Arrowheads mark cells in the M-phase of the cell cycle. Scale bars: A, B, C, D, E, F, G -50 μm.

Figure 6 .

 6 Figure 6. Quantitative analysis of cell proliferation in different body regions of Leucosolenia variabilis. A, B -fractions of EdU-(A) and рН3-positive (B) cells in different body regions. Data are shown with median values (thick horizontal lines), interquartile ranges (boxes), total ranges (whiskers), and outliers (dots). C, Dfractions (mean values) of choanocytes/mesohyl cells among the total number of EdU-(C) and рН3-positive (D) cells, respectively. Asterisks mark significantly different regions of the body. Osc. rim -oscular rim; Osc. dist -distal part of an oscular tube; Osc. mid. -middle part of an oscular tube; Osc. prox. -proximal part of an oscular tube; Div. dist. -distal part of a diverticulum; Div. prox. -proximal part of a diverticulum; Cormus -cormus tube.

Figure 7 .

 7 Figure 7. EdU tracking experiment in Halisarca dujardinii, CLSM. A -0 days after the incubation in EdU (maximum intensity projection of several focal planes); B -7 days after the incubation in EdU (maximum intensity projection of several focal planes); blue color -DAPI, cell nuclei, red -EdU, nuclei of DNA synthesizing cells. C -fractions of labeled choanocytes and mesohyl cells among all EdU-positive cells (mean values). Dotted lines mark the borders of choanocyte chambers. Arrowheads in B mark EdU-positive nuclei in mesohyl. Scale bars: A, B -50 μm.

Figure 8 .

 8 Figure 8. Single-parameter histograms of PI fluorescence (DNA content) in Halisarca dujardinii (A) and Leucosolenia variabilis (B) сell suspensions. C, Dan example of light-scatter primary gating to distinguish single cells in a suspension of L. variabilis. G0/G1 (red) are diploid cells, G2/M (blue) are tetraploid cells, S (green) are DNA-synthesizing cells. SubG1 cells are cells with fragmented DNA.

Figure 9 .

 9 Figure 9. Apoptosis in intact tissues of Halisarca dujardinii and Leucosolenia variabilis, CLSM. Athe endosome of H. dujardinii; B, Cenlarged single-channel images of the zone in the white rectangle in A; Dthe proximal part of the oscular tube of L. variabilis; E, F, Genlarged single-channel images of the zone in the white rectangle in D. Several focal planes are combined into one picture. Blue color -DAPI, cell nuclei; magenta -CellEvent, nuclei of apoptotic cells; green -acetylated α-tubulin, flagella. Magenta arrowheads mark CellEvent-positive cells. Scale bars: A, D -50 μm; B, C, E, F, G -25 μm.

Figure 10 .

 10 Figure 10. Apoptosis in cell suspensions of Halisarca dujardinii (A, B) and Leucosolenia variabilis (C, D), flow cytometry. А, C -Pacific Blue-A (autofluorescence) vs FITC-A (CE fluorescence) dot plots; B, Dsingle-parameter histograms of CellEvent fluorescence. CE(+) population gate is set according to the negative control.

Figure 11 .

 11 Figure 11. In vivo TMRE and CellEvent staining of Halisarca dujardinii and Leucosolenia variabilis suspensions. А -CLSM of L. variabilis suspension, scale bar 10 μm; blue color -Hoechst 33342, cell nuclei; orange -TMRE, functional mitochondria; magenta -CellEvent, nuclei of apoptotic cells; Bscatter plot of FSC-A (cell size) vs SSC-A (cell granularity), an example of primary gating; C, D -TMRE vs CellEvent dot plot of L. variabilis (C) and H. dujardinii (D) suspensions. The borders of quadrants (Q1-Q4) are set by the negative controls.

  Each fraction is given as mean value and SD. Six individuals have been examined; n is the number of analyzed stacks.

Table

  ).

	The oscular tube differs significantly from the regions of endosome. There are only
	0.61±0.64% of EdU-positive cells in the oscular tube, and pH3-positive cells are absent (Figures 2C;
	3A, B; Table

  1. Mean value and standard deviation were calculated for every parameter (the proportions of EdU-positive cells, pH3positive cells, etc.). To compare these parameters between different body parts or time points, we used Kruskal-Walles test followed by Dunn test since it is a distribution-free test appropriate for multiple comparisons between small data subsets. The significance level was 0.05. All p-values represented in the text are Dunn test p-values. Tables with exact p-values for all tests are provided in the Mendeley Data root directory (see "Availability of data and material" section).apoptosis evaluation in cell suspensions, and performed the statistical analysis. AL, NM, AE and AS prepared the manuscript with contributions from all authors. All authors reviewed and approved the final manuscript.

Table 2 .

 2 Fractions of proliferating cells in different body regions of Leucosolenia variabilis, CLSM.

		Oscular rim		10	0.40±0.96%				0.17±0.20%
	Distal part	of the	oscular tube	10	10.58±1.66%	10.19±1.57%	0.39±0.38%	96.36±2.96%	0.74±0.17%	0.52±0.16%	0.22±0.09	70.25±10.94%
	Middle part	of the	oscular tube	10	11.01±1.76%	10.62±1.55%	0.40±0.73%	96.68±5.11%	0.69±0.18%	0.54±0.16%	0.15±0.10%	78.70±11.22%
	Proximal	part of the	oscular tube	10	9.76±1.91%	9.43±1.95%	0.33±0.25%	96.48±2.91%	0.71±0.36%	0.56±0.30%	0.15±0.07%	79.09±7.06%
	Distal part	of the	diverticulum	14	6.53±2.08%	6.07±2.16%	0.46±0.55%	92.08±9.89%	0.50±0.20%	0.21±0.15%	0.29±0.15%	40.64±22.29%
	Proximal	part of the	diverticulum	14	9.84±2.60%	9.14±2.33%	0.71±0.68%	93.00±4.73%	0.64±0.24%	0.41±0.17%	0.22±0.13%	65.30±15.43%
	Cormus	tube	11	9.44±1.58%	9.00±1.59%	0.44±0.22%	95.20±2.61%	0.56±0.18%	0.43±0.16%	0.13±0.07%	78.31±12.93%

Table 3 .

 3 Fractions of cells in different phases of the cell cycle, flow cytometry.

	Species	N	SubG1	G0/G1	S	G2/M
	Halisarca	5	23.41±12.22% 38.94±5.9%	20.18±6.14%	9.28±1.78%
	dujardinii
	Leucosolenia	5	26.42±3.43%	53.88±2.59%	12.58±0.76%	4.63±1.79%
	variabilis
	Each proportion is given as mean value and SD.

Table 4 .

 4 Fractions of apoptotic cells in the intact tissues of H. dujardinii and L. variabilis, CLSM.

				CE-positive cells
	Species	Body region	n
				(% of all cells)
	H. dujardinii	Distant from the	7	0.03±0.05%
		oscular tube
	L. variabilis	Oscular tube	9	0.22±0.19%
		Cormus tube	6	0.44±0.66%
		Diverticula	3	0.21±0.16%

Each proportion is given as mean value and SD. Three individuals per species have been examined; n is the number of analyzed stacks.

Table 5 .

 5 Fractions of cell populations revealed during the in vivo apoptosis investigation, flow cytometry.

	Species	N	TMRE-CE-	TMRE+ CE-	TMRE+ CE+	TMRE-CE+
	Halisarca	3	1.67±0.35%	89.67±1.06%	7.51±0.87%	0
	dujardinii
	Leucosolenia	2	13.7±3.25%	70.55±1.48%	8.58±0.77%	0.06±0.01%
	variabilis

. Similarly to feeding structures of other suspension feeders, constant replacement of old, damaged cells is crucial for the long-term maintenance of the choanoderm[START_REF] Strahl | Cell turnover in tissues of the long-lived ocean quahog Arctica islandica and the short-lived scallop Aequipecten opercularis[END_REF][START_REF] Shunatova | Proliferating activity in a bryozoan lophophore[END_REF][START_REF] Rinkevich | Whole-body protochordate regeneration from totipotent blood cells[END_REF][START_REF] Martínez-Expósito | Proliferation kinetics of mussel (Mytilus galloprovincialis) gill cells[END_REF] .

Acknowledgments

The authors acknowledge the support of Lomonosov Moscow State University Program of Development (FACSAria SORP flow cytometer/sorter, Nikon A1 CLSM) and Center of microscopy WSBS MSU. Authors sincerely thank Darya Potashnikova, Lomonosov Moscow State University, for operating the flow cytometer, Daniyal Saidov, Lomonosov Moscow State University, for statistical analysis tips, Vitaly Kozin and Alexandra Shalaeva, Saint-Petersburg State University, for helping with Bitplane Imaris learning, Olga Vorobjeva, Lomonosov Moscow State University, for collection and transporting L. variabilis individuals for the in vivo apoptosis investigation, Igor Kosevich, Lomonosov Moscow State University, and Ilya Borisenko, Saint-Petersburg State University, for helpful tips and advice.

Funding information

Fund of President of the Russian Federation, Grant/Award Number: MK-1096.2021.1.4; Russian Foundation for Basic Research, Grant/Award Numbers: 19-04-00545 and 19-04-00563; Russian Science Foundation, Grant/Award Number: 17-14-01089

Availability of data and materials

The draft dataset supporting the conclusions of this article is available in the Mendeley Data repository, https://data.mendeley.com/datasets/hhmc8t6gv7/draft?a=ee1a45f9-2b2d-4b65-b863-ec4e84785263 (H.dujardinii; doi:10.17632/hhmc8t6gv7.1) and https://data.mendeley.com/datasets/vbk6h8vcyy/draft?a=97e2ee7f-68e0-455f-b36b-2ad25872ff9a (L. variabilis; doi:10.17632/vbk6h8vcyy.1).

CLSM studies as described above (see "Study of cell proliferation with CLSM" section), but no antibody staining was performed in this experiment. We studied areas of the endosome at some distance from an oscular tube. One randomly chosen field of view within the region was analyzed in each individual. For subsequent analysis, a Z-stack was obtained from each field of view. We estimated the fractions of choanocytes and mesohyl cells among all EdU-positive cells (as a proportion of EdU-positive choanocytes/mesohyl cells to all EdU-positive cells). As a result, the main parameter of the analysis was the mean proportion of choanocytes and mesohyl cells in the total number of EdU-positive cells. Details of data acquisition, nuclei counting, and further analysis have been described above (see "Study of cell proliferation with CLSM" section). In this experiment, choanocyte chambers were identified by their characteristic shape visualized by DAPI staining.

Evaluation of cell cycle distribution in intact tissues

We combined calcium and magnesium-free seawater (20 mM KCl, 300 mM NaCl, 10 mM Tris-HCl and 15 mM EDTA in distilled water) (CMFSW-E) and mechanical tissue dissociation to obtain single-cell suspensions. Sponges were cleaned from epifauna, soaked in CMFSW-E for 30 min and squeezed into CMFSW-E through a sterile gauze with a mesh size of 30 μm. After 30 min, suspensions were centrifuged and resuspended in a new portion of CMFSW-E. After another 30 min, suspensions were fixed with 70% cold ethanol at -20°С. Such dissociation technique resulted in poor quality of the cell cycle curve in case of L. variabilis. An alternative fixation method was used: intact pieces of L. variabilis were first fixed in cold 70% ethanol. After the fixation, tissues were soaked in CMFSW-E and mechanically dissociated through gauze with a mesh size of 30 μm.

Cell suspensions from five H. dujardinii and five L. variabilis were studied for cell cycle distribution, using propidium-iodide (PI) staining since PI fluorescence is proportional to DNA content 37 . Cell suspensions were resuspended in PBS and stained with 1 μg/ml PI (P1304MP, ThermoFisher Scientific) for at least 12 hours. Unstained suspensions served as a negative control.

Suspensions were analyzed by a FACSAria SORP instrument (BD Biosciences, USA) using an excitation laser wavelength of 561 nm (PI excitation); 50 000 events were collected per sample. The data were processed via FlowJo V10. Primary gating was performed according to forward (FCS-A) versus side scatter (SSC-A) parameters; secondary gating on singlet cells -according to FSC-W versus FSC-H parameters.

choanocytes/mesohyl cells among all CellEvent-positive cells (as a proportion of CellEvent-positive choanocytes/mesohyl cells to all CellEvent-positive cells).

CellEvent staining of suspensions for flow cytometry. We took samples of 250 ml containing 1.5×10 6 cells from non-fixed CMFSW-E suspensions of H. dujardinii and L. variabilis (4 samples from 4 sponges per species). The suspensions were obtained as described in the "Evaluation of cell cycle distribution in intact tissues" section. The samples were incubated in CellEvent (dilution 1:100) at +10°С for 30 min in the dark. The samples were then centrifuged and fixed with 4% PFA PBS at +4°С and stored for 2 months. Before the flow cytometry analysis, cells were washed thrice with PBS.

Suspensions were analyzed by a FACSAria SORP instrument (BD Biosciences, USA) using an excitation laser wavelength of 488 nm (CellEvent excitation). Primary gating was described above (see the "Evaluation of cell cycle distribution in intact tissues" section); the final quadrant gating to distinguish CellEvent-positive cells was set according to unstained control. Any event exceeding the autofluorescence of the unstained control was considered a CellEvent-positive (apoptotic) cell.

Subsequent flow cytometry studies showed that L. variabilis suspensions contained an anomally high number of CellEvent-positive (apoptotic) cells. Thus, the data obtained from these samples were not used in the main analysis.

Vital staining of suspensions for CLSM and flow cytometry. For vital CLSM study, the suspensions were obtained as described in the "Evaluation of cell cycle distribution in intact tissues" section. The suspensions were stained with TMRE (final concentration -100 μM), CellEvent (dilution 1:100), and Hoechst 33342 (dilution 1:100) at +10°С for 30 min in the dark. During this experiment, we used lasers of wavelength 405 nm (DAPI), 488 nm (CellEvent), and 561 nm (TMRE). Data acquisition has been described above (see "Study of cell proliferation with CLSM" section). No quantitative analysis was performed; acquired images were used to illustrate flow cytometry data.

For vital flow cytometry study, three individuals of H. dujardinii (August 2020) and two individuals of L. variabilis (February 2020) were transported to Moscow in thermoses with natural seawater at +10°С. Sponges were dissociated in CMFSW-E for 30 min and stained with TMRE and CellEvent as described above. After the staining, the suspensions were analyzed by a FACSAria SORP instrument (BD Biosciences, USA) using excitation laser wavelength of 488 nm (CellEvent excitation) and 561 nm (TMRE excitation). Primary gating was described above (see the "Evaluation


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