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01 August 2005

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Charles N. David, Nikola Schmidt, Marsha Schade, Barbara Pauly, Olga Alexandrova, Angelika Böttger, Hydra and the Evolution of Apoptosis, Integrative and Comparative Biology, Volume 45, Issue 4, August 2005, Pages 631–638, https://doi.org/10.1093/icb/45.4.631
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Abstract

Programmed cell death occurs in most, if not all life forms. It is used to sculpt tissue during embryogenesis, to remove damaged cells, to protect against pathogen infection and to regulate cell numbers and tissue homeostasis. In animals cell death often occurs by a morphologically and biochemically conserved process called apoptosis. A novel group of cysteine proteases, referred to as caspases, constitute the central component of this process. Caspases are activated following the induction of apoptosis and cleave a variety of cellular substrates, thus giving rise to the characteristic morphological events of apoptosis. Apoptosis is rapid and cell corpses are removed by phagocytosis. Recent work has shown that apoptosis also occurs in Cnidaria and Porifera, thus extending the origin of this evolutionary innovation down to the first metazoan animal phyla. Here, we review several examples of the role of apoptosis in cnidarians and then summarize new results on the subcellular localization of caspases and the control of apoptosis in Hydra. We show by immuncytochemistry that caspases in Hydra are localized in mitochondria. Following induction of apoptosis caspases are released from mitochondria as proenzymes and then activated by proteolytic cleavage in the cytoplasm. We also present evidence that apoptosis in Hydra is dramatically stimulated by inhibitors of PI3-kinase. Since PI3-kinase is a central component of growth factor signaling cascades in higher metazoans, this result suggests that control of apoptosis by growth factors is also evolutionarily conserved. We speculate on the role of growth factors in the evolution of apoptosis.

INTRODUCTION

Programmed cell death by apoptosis is a prominent feature of all multicellular animals. During embryogenesis it removes specific cells unneeded for development. In adult organisms, it is involved in cell number regulation, protection from pathogens and the removal of damaged cells (see Jacobson et al. [1997] for review). The morphological features of apoptosis are highly conserved. Cells undergoing apoptosis round up and lose contact with neighboring cells. The nucleus becomes pycnotic and the cytoplasm appears to condense. Apoptotic cells are then phagocytized by neighboring cells. The process is rapid, often being completed in less than one hour. During apoptosis the cell membrane remains intact and, as a result, intracellular contents are not released into tissue.

The highly conserved morphological features of apoptosis suggest that it is under genetic control and this has been elegantly demonstrated by the genetic analysis of apoptosis in the nematode C. elegans. By isolating mutants defective in apoptosis during embryogenesis Ellis and Horvitz (1986) defined a core pathway of genes regulating apoptosis. Ced-3 and ced-4 are essential for apoptosis; loss of function mutations in these genes completely blocks apoptosis in C. elegans. A third gene ced-9 is epistatic to ced-3 and ced-4. Mutants in ced-9 exhibit massive apoptosis indicating that the wild type function of ced-9 is to inhibit ced-3 and ced-4 activity. Molecular cloning of these genes revealed that ced-3 encodes a novel cysteine protease whose activation in cells causes apoptosis (Yuan et al., 1993). Ced-4 encodes a molecular chaperone required for activation of ced-3 (Yuan and Horvitz, 1992) while ced-9 encodes a homolog of bcl-2, an oncogene regulating cell death in mammalian tissue (Hengartner and Horvitz, 1994). Following the molecular identification of these three genes in C. elegans, it has been shown that homologs of these genes orchestrate apoptosis in Drosophila and mammals (Meier et al., 2000).

Results to be discussed below have now extended the occurrence of apoptosis and homologs of caspases and bcl-2 proteins to the most basal metazoan phyla: Porifera and Cnidaria. By contrast apoptosis has not been observed in protozoans or in plants nor have essential genetic components of apoptosis been identified in these organisms (see Böttger and David [2003] and Golstein et al. [2003] for reviews). This suggests that apoptosis is a unique feature of multicellular animals and raises the question of what led to the evolution of this process. Once evolved, apoptosis has been remarkably well conserved throughout metazoan phyla both in terms of morphological features and in terms of the genes controlling the process.

In this brief summary we will concentrate on results from Hydra where most of the work on apoptosis in basal metazoans has been done. Both Hydra vulgaris and Hydra magnipapillata have been used in these investigations and yield similar results. We begin with a description of the process of apoptosis in Hydra and discuss three examples illustrating the biological roles of apoptosis in Hydra and Hydractinia. We then proceed to a closer examination of the biochemical mechanism of apoptosis in Hydra. We also include results on the isolation of apoptosis genes from sponges. Finally, we demonstrate that inhibition of PI3-kinase induces apoptosis in Hydra and speculate on the role of growth factor signaling in the evolution of apoptosis.

BASIC FEATURES OF APOPTOSIS IN HYDRA

Apoptosis in Hydra exhibits basic features characteristic of apoptosis in higher metazoans (Cikala et al., 1999). Figure 1A and B show an interstitial stem cell undergoing apoptosis. The apoptotic cell is rounded up and condensed due to loss of cytoskeletal integrity and the nuclear DNA (stained with DAPI) is pycnotic. Nuclear DNA is degraded to nucleosome size fragments and hence such nuclei stain strongly with the TUNEL reagent (see below; Gavrieli et al., 1992). Finally apoptotic cells are phagocytized by neighboring epithelial cells, both epidermal and gastrodermal (Fig. 1C and D). The process of apoptosis is rapid and, as a consequence, few apoptotic cells are observed in Hydra tissue.

BIOLOGICAL FUNCTION OF APOPTOSIS IN HYDRA AND HYDRACTINIA

Apoptosis regulates growth in Hydra

Hydra polyps grow continuously due to proliferation of epithelial and interstitial stem cells throughout the body column. However, polyps do not increase in size since cells are continuously transferred to asexual buds, which form on the lower body column, and lost at the tentacle tips and in the basal disk. Budding is dependent on feeding: well-fed polyps produce roughly one bud per day; starved polyps cease to form buds after 1–2 days. This striking dependence of budding on feeding is not due to a change in cell proliferation, as initially anticipated, but rather to apoptosis (Bosch and David, 1984). Figure 2B shows that rapid cell proliferation detected as an increase in the 3H-thymidine labeling index occurs in both well-fed and starved animals. The increase in cell numbers, however, is dramatically different: cell numbers increase exponentially in fed animals but do not change in starved animals (Fig. 2A). This difference is due to an increased rate of apoptosis in starving polyps. Bosch and David (1984) observed a 7-fold increase in epithelial cells containing phagocytized apoptotic bodies in starving polyps compared to well-fed polyps.

Apoptosis during oogenesis and spermatogenesis in Hydra

Oogenesis in female hydra occurs by fusion of large numbers of germ line cells to form an oocyte (Zihler, 1972; Honegger et al., 1989; Miller et al., 2000). Germline cells proliferate in the ectoderm of female polyps giving rise to a mass of roughly 5,000 cells localized in a so-called egg patch. A single germline cell develops into an oocyte precursor while the remaining germline cells differentiate to form nurse cells. The nurse cells grow in size and their cytoplasm fills with mitochondria, membranous vesicles and various storage granules (Fig. 3, large nurse cell). Subsequently, these large nurse cells fuse with the developing oocyte and transfer cytoplasm to it. As a result of this transfer nurse cells decrease in size giving rise to a new population of small nurse cells (Fig. 3). These small nurse cells initiate apoptosis: TOPRO staining reveals clumps of chromatin in the nucleus and TUNEL staining indicates that the DNA is fragmented. Following the onset of apoptosis, the small nurse cells are phagocytized by the oocyte. These phagocytised apoptotic bodies (Fig 3, “Schrumpfzelle”) fill the cytoplasm of the mature oocyte. Apoptosis is, however, arrested at this point and the apoptotic bodies remain in the cytoplasm for months (Technau et al., 2003). During cleavage the apoptotic bodies are partitioned to blastomeres. After hatching of the young polyp, the apoptotic bodies are digested and presumably provide nourishment to the young polyp.

There are striking parallels between the process of oogenesis in Hydra (Honegger et al., 1989; Miller et al., 2000) and oogenesis in Drosophila and Caenorhabditis (Gumienny et al., 1999; Foley and Cooley, 1998). Also in these cases nurse cells contribute cytoplasm directly to the developing oocyte. Since nuclei of both nurse cells and the oocyte itself contribute to the formation of cytoplasm for the oocyte, the process of ooogenesis is greatly accelerated. The residual cell bodies of nurse cells, following cytoplasmic transfer, undergo apoptosis. In the case of Hydra these apoptotic cells are phagocytized by the oocyte; in Drosophila and Caenorhabditis they are phagocytised by follicle cells surrounding the developing oocyte or by epithelial cells forming the gonad.

Apoptosis also occurs during spermatogenesis in Hydra (Kuznetsov et al., 2001). The function of apoptosis in this process is not understood, although it could be the result of recombination defects during meiosis.

Apoptosis during metamorphosis of Hydractinia larva

The marine hydroid Hydractinia echinata forms colonies of polyps growing up from a dense mat of interconnected stolons. The colonies develop from a single primary polyp which initiates formation of the stolon network. As the stolon network grows additional polyps form as lateral branches. Polyps are specialized for feeding or gametogenesis. The gametes are released into sea water and the fertilized egg develops to form a planula larva. These larvae are motile and, in the natural environment, seek out suitable sites and undergo metamorphosis to form primary polyps.

Seipp et al. (2001) have recently shown that metamorphosis in Hydractinia is accompanied by massive apoptosis of larval cells. Figure 4 shows a planula larva and a metamorphosing larva stained for apoptotic cells with the TUNEL reagent. The planula larva has very few apoptotic cells. Shortly after the onset of metamorphosis apoptosis increases dramatically and the larva undergoes extensive morphological change to form a primary polyp. Estimates suggest that up to 50% of larval cells undergo apoptosis during the course of metamorphosis. Thus, as in other organisms, the morphogenetic changes accompanying metamorphosis in Hydractinia are mediated by apoptosis.

CASPASE GENES AND CASPASE ACTIVITY DURING APOPTOSIS IN HYDRA

The striking similarity between apoptosis in Hydra (Fig. 1) and apoptosis in higher metazoan phyla suggested that the biochemical mechanism is conserved. Using PCR with primers to conserved sequence motifs Cikala et al. (1999) isolated two caspase cDNAs from Hydra. Figure 5 compares the sequence organization and conserved motifs between the hydra caspases, CED-3 from C. elegans, and human caspase 3. The sequences of the large and small subunits show 30– 40% overall identity to the nematode and mammalian sequences and exhibit conserved sequences surrounding the active site cysteine and the substrate binding domain. Caspase cleavage sites are present between the large and small subunits and between the prodomain and the large subunit. In contrast to the strong conservation in the large and small subunits, the size and sequence of the prodomains show little similarity among the three species. Partial sequences for three additional caspases have now been found and are listed in the recently available Hydra expressed sequence tag (EST) collections (www.hydrabase.org), suggesting that the complexity of the caspase gene family in Hydra is similar to that in higher metazoans. Furthermore, Wiens et al. (2003) have identified caspase sequences in the marine sponge Geodia cydonium indicating that caspases are also present in the Phylum Porifera. In contrast, caspase homologs have not been identified in single celled eukaryotes or in plants.

Induction of apoptosis by treatment of Hydra tissue with colchicine leads to a strong increase in caspase enzyme activity (Cikala et al., 1999). This caspase activity cleaves caspase 3 substrates but not caspase 1 substrates, in agreement with caspase activities identified during apoptosis in higher metazoans. Biochemical purification of the caspase activity from apoptotic Hydra revealed that the active enzyme is caspase 3B. Caspase 3A, in contrast, had a different substrate specificity and was not activated in apoptotic hydra. Its biological function is presently unknown.

LOCALISATION OF HYDRA CASPASES IN MITOCHONDRIA AND REGULATION OF CASPASE ACTIVITY DURING APOPTOSIS

To investigate the regulation of caspase activity in hydra, we made antibodies to the bacterially expressed recombinant hydra caspases and used them to localize the caspases in hydra cells by immunocytochemistry. Figure 6 shows confocal images of fixed polyps stained with antibodies to caspase 3A and caspase 3B. Both hydra caspases localize to numerous small particles in the cytoplasm of all cells. The size and distribution of the stained particles suggested that they were mitochondria. To confirm this we co-immunostained animals with an antibody to the mitochondrial ATP/ADP carrier (AAC). Figure 6 shows that this mitochondria specific antibody stained the same cytoplasmic particles as the antibody against caspase 3A. Additional coimmunstaining experiments with caspase 3B and mitochondria antibodies confirmed that the caspase 3B positive cytoplasmic particles are also mitochondria.

The localization of caspases to mitochrondria in Hydra suggests the need to release the enzymes to the cytoplasm during apoptosis in order to cleave target proteins. The results in Figure 7A show that caspase 3B is released into the cytoplasm as a 37 kD proenzyme following induction of apoptosis. The 37 kD procaspase is subsequently processed to yield an active caspase, which could be identified with an active site label (Figure 7B). This caspase activity is also inhibited by the caspase 3 specific inhibitor DEVD-fmk and thus has the same substrate specificity as the caspase isolated from apoptotic Hydra (see above).

BCL-2 PROTEINS IN HYDRA

The release of inactive caspase proenzymes from mitochondria during apoptosis suggests that this process is central to the control of apoptosis in Hydra. In higher metazoans mitochondria play a central role in the initiation of apoptosis by controlling the release of cytochrome c and additional factors required for the activation of caspase proenzymes (Li et al., 1997; Zou et al., 1997; Zou et al., 1999). This release is controlled by members of the bcl-2 family of proteins (see Adams and Cory, 1998 for review). Bcl-2 itself is antiapoptotic while Bax is proapoptotic. Searching the newly available Hydra EST collections (see above) has revealed partial sequences of bcl-2 family members. We have now isolated and sequenced full length clones corresponding to these partial sequences. The results confirm that both an antiapoptotic bcl-2 homolog and a proapoptotic Bax homolog are present in Hydra. A bcl-2 homolog has also been identified in sponges (Wiens et al., 2000). In analogy to their role in higher metazoans, these proteins could control the release of caspase proenzymes from mitochondria in Hydra and in sponges. However, this still has to be demonstrated.

GROWTH FACTORS AND THE CONTROL OF APOPTOSIS

Our results have demonstrated the occurrence of apoptosis in Hydra and the presence of caspase and bcl-2 homologs. They also suggest that mitochondria play a central role in the control of apoptosis in Hydra. Together with the recent identification of caspase and bcl-2 homologs in the sponge Geodia cydonium (Wiens et al., 2000; Wiens et al., 2003) this brings the occurrence of caspase-mediated apoptosis down to the earliest metazoan phyla. A similar process does not appear to be present in single- celled eukaryotes nor in plants, although programmed cell death has been well documented in these kingdoms. Thus the specific process of apoptosis first appeared with the evolution of metazoan animals and has been well conserved in all animal phyla.

What led to this evolutionary innovation and how was it controlled? As with most evolutionary questions, it is not possible to give a definitive answer. It is, however, interesting to speculate about the selective forces which might have supported the origin of apoptosis in multicellular animals. Multicellular animals contain a variety of different cell types. These cell types do not compete with each other but rather cooperate to form a multicellular tissue. Populations of single celled organisms, by comparison, are characterized by competition between cells or cell clones. This suggests that the transition from single- celled to multicellular animals required the control of competition between individual cell populations making up an animal. An attractive mechanism to achieve such control is to make the different cell types in a multicellular tissue mutually dependent on each other. Results from higher metazoans indicate that this can be achieved with the help of growth factors, such as epithelial growth factor (EGF), nerve growth factor (NGF) or insulin. These factors are, in fact, better termed survival factors since one of their prime functions is to prevent apoptosis (Raff, 1992).

Figure 8 shows a schematic model demonstrating how two growth factors can stabilize a simple multicellular tissue consisting of two cell types. Cell type A produces a growth factor required by cell type B. Similarly, cell type B produces a growth factor required by cell type A. This mechanism stabilizes the relative proportions of each cell type in the AB tissue. Any attempt by one cell type to overgrow the other and hence destroy the multicellular tissue will be counteracted since each cell type is dependent for survival on the presence of the other.

In higher metazoans, such growth factor signaling is mediated by receptor tyrosine kinases (RTKs). Although RTKs have been been identified both in Hydra (Steele et al., 1996; Bridge et al., 2000; Miller and Steele, 2000; Reidling et al., 2000) and in sponges (Muller et al., 1999) by PCR cloning and more recently in EST collections, it is not known whether they play a role in growth factor signaling. Potential ligands have also not yet been identified.

To investigate whether RTKs might play a role in survival signaling and apoptosis in Hydra, we tested the effect of inhibitors of growth factor signaling on Hydra. In higher metazoans, the PI3-kinase is activated by RTKs and leads to formation of PI(3,4,5)P3 in the plasma membrane and subsequently to activation of the protein kinase PKB/Akt. Activated PKB/Akt phosphorylates the bcl-2 family member BAD, inactivating it and thus inhibiting apoptosis (Zha et al., 1996; Datta et al., 1997). In the absence of growth factors or following inhibition of this signaling pathway by treatment with wortmannin, apoptosis is induced. Figure 9 shows that brief treatment of Hydra with wortmannin induces massive apoptosis. This suggests that an RTK signaling pathway may indeed regulate apoptosis in Hydra. Identifying the ligands and receptors which constitute this pathway in Hydra is one of the current challenges.

SUMMARY

The results presented here indicate that the basic biochemical features of apoptosis are present in the cnidarian Hydra and in sponges. Caspases have been identified with conserved sequence motifs and conserved substrate specificity. Mitochondria, at least in Hydra, play a central role in the regulation of apoptosis, since caspases are sequestered in mitochondria in an inactive form and released into the cytoplasm following the initiation of apoptosis. Bcl-2 proteins, which regulate the release of proapoptotic proteins from mitochondria during apoptosis in higher metazoans, have also been identified in Hydra and in sponges, although their involvement in apoptosis has not yet been directly demonstrated. In contrast to the striking conservation of caspases and bcl-2 proteins in all animal phyla from Porifera and Cnidaria to mammals, these features of apoptosis are not present in yeast or plants. Since Hydra and sponges represent the earliest multicellular animals, this suggests a possible correlation between the origin of multicellularity and the origin of apoptosis.

In reflecting on possible scenarios which might have led to this close association of apoptosis with metazoan evolution, we are impressed by the need to reduce cell-cell competition in multicellular tissues. Growth factors, as shown schematically in the model in Figure 8, provide a mechanism to stabilize the proportions of different cell types in multicellular tissue. The role of growth factors in cell survival has been well documented in higher metazoans. A similar function in lower metazoans has not yet been demonstrated, although homologs of mammalian growth factor receptors have been identified in Hydra and sponges and preliminary observations presented here demonstrate that inhibition of growth factor signaling in Hydra induces apoptosis. Thus, an evolutionary scenario in which apoptosis evolved as a result of the need to control cell-cell competition in multicellular tissue appears plausible.

Finally, not only are the morphological and biochemical events of apoptosis well conserved from Hydra to mammals, but also the functional contexts in which apoptosis occurs. Hydra and Hydractinia use apoptosis for cell number regulation during growth, in oogenesis, and in morphogenesis (metamorphosis), in exactly the same way as in higher metazoans. Thus, both the mechanism of apoptosis and many of the biological functions of apoptosis arose at the beginning of metazoan evolution.

Fig. 1. Apoptotic cells in Hydra. (A, B) Two normal interstitial stem cells and one apoptotic stem cell (lower left). (C, D) Ectodermal epithelial cell with three phagocytized apoptotic cells. Cells were prepared from normal tissue by maceration in acetic acid and fixed with formaldehyde (David, 1973). A, C: phase contrast; B, D: DAPI stained. Scale bar: 10 μm

Fig. 2. Growth rate and cell proliferation in fed and starved Hydra. Animals were adapted to feeding and starvation regimes 2 days before the start of the experiment. (This gives rise to the different sizes and labeling indices at t0.) Animals were continuously labeled for three days with 3H-thymidine. At the times indicated animals were macerated, cell numbers were counted and the labeling index was determined by autoradiography of cell spreads (Data from Figure 1 in Bosch and David, 1984)

Fig. 3. Nurse cell differentiation and apoptosis during oogenesis in hydra. Egg patches were macerated (David, 1973) at various times during development of the oocyte. Preparations were stained with the DNA specific fluorochrome TOPRO (Molecular Probes) to visualize nuclear morphology and with TUNEL to identify DNA fragmentation (Gavrieli et al., 1992). Large nurse cell: before cytoplasmic transfer to the oocyte. Small nurse cell: after cytoplasmic transfer to the oocyte. “Schrumpfzelle:” phagocytized small nurse cell in an apoptotic vacuole, released by gentle squashing of the developing oocyte. Scale bar 5 μm

Fig. 4. Apoptosis during metamorphosis of Hydractinia larva to primary polyp. The schematic drawing illustrates the process of metamorphosis, which takes about 24 hours to complete. The micrographs show a TUNEL stained larva before metamorphosis and a metamorphosing larva at 3 hr. The bright spots are apoptotic cells stained with TUNEL. A planula larva contains approximately 104 cells. Scale bar: 20 μm. (Data from Seipp et al., 2001)

Fig. 5. Caspase genes in Hydra. Schematic diagram of Hydra caspase 3A and 3B compared to CED-3 from C. elegans and human caspase 3. The prodomain, large and small subunits are indicated. The highly conserved sequences LSHG and QACRG form the active site; GSWFI represents a conserved sequence in the substrate binding site. Thin lines represent known or potential cleavage sites between the subunits. (Data from Cikala et al., 1999)

Fig. 6. Hydra caspases are localized in mitochondria. The images represent confocal sections of fixed Hydra stained with specific antibodies to the two hydra caspases 3A and 3B (green) and an antibody to the ATP/ADP carrier (AAC) (red) in the inner membrane of mitochondria. Nuclei are stained with TOPRO (false color, blue). Scale bar: 10 μm

Fig. 7. Hydra caspase 3B is released from mitochondria and activated in the cytoplasm during apoptosis. Western blots of the cytoplasmic fraction from control animals (−) and from animals treated with colchicine (4 mg/ml) for 8 hours to induce apoptosis (+). (A) Caspase 3B antibody recognizes a 37 kD band in the cytoplasm of apoptotic hydra. The band corresponds to the full length unprocessed procaspase 3B. (B) Active site labeling with FITC-VAD-fmk. The reagent binds covalently to the large subunit of active caspases and can be identified in Western blots with antibodies to FITC. The strongly labeled band at 25 kD represents the large subunit of the activated caspase 3B

Fig. 8. Mechanism to stabilize the cell composition of a multicellular tissue containing two cell types A and B using survival factors. See text for details

Fig. 9. Induction of apoptosis in Hydra by treatment with the PI3-kinase inhibitor wortmannin. Animals were treated for 4.5 hours with the indicated concentrations of wortmannin, macerated and stained with DAPI and TUNEL to score apoptotic cells. The number of apoptotic cells per epithelial cell is given in percent

1

From the Symposium on Model systems for the Basal Metazoans: Cnidarians, Ctenophores and Placozoans presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.

Research from the authors' laboratory was supported by the German Science Foundation. Dr. Steffanie Seipp provided the photographs in Figure 4.

Note Added In Proof

The results on apoptosis during oogenesis in Hydra (Fig. 3) have recently been published in Alexandrova et al. Developmental Biology 281: 91–101 (2005).

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