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Werner E. G. Müller, The Origin of Metazoan Complexity: Porifera as Integrated Animals, Integrative and Comparative Biology, Volume 43, Issue 1, February 2003, Pages 3–10, https://doi.org/10.1093/icb/43.1.3
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Abstract
Sponges [Porifera] are the phylogenetically oldest metazoan phylum still extant today; they share the closest relationship with the hypothetical common metazoan ancestor, the Urmetazoa. During the past 8 years cDNAs coding for proteins involved in cell-cell- and cell-tissue interaction have been cloned from sponges, primarily from Suberites domuncula and Geodia cydonium and their functions have been studied in vivo as well as in vitro. Also, characteristic elements of the extracellular matrix have been identified and cloned. Those data confirmed that all metazoan phyla originate from one ancestor, the Urmetazoa. The existence of cell adhesion molecules allowed the emergence of a colonial organism. However, for the next higher stage in evolution, individuation, two further innovations had to be formed: the immune- and the apoptotic system. Major defense pathways/molecules to prevent adverse effects against microbes/parasites have been identified in sponges. Furthermore, key molecules of the apoptotic pathway(s), e.g., the pro-apoptotic molecule comprising two death domains, the executing enzyme caspases, as well as the anti-apoptotic/cell survival proteins belonging to the Bcl-2 family have been identified and cloned from sponges. Based on these results—primarily obtained through a molecular biological approach—it is concluded that cell-cell- and cell-matrix adhesion systems were required for the transition to a colonial stage of organization, while the development of an immune system as well as of apoptotic processes were prerequisites for reaching the integrated stage. As the latter stage already exists in sponges, it is therefore likely that the hypothetical ancestor, the Urmetazoa, was also an “integrated colony.”
INTRODUCTION
The origin of Metazoa is to some extent still enigmatic despite the progress that has been achieved in the last years by molecular studies. The evolution of the Metazoa from unicellular/colonial organisms occurred some 1,300–600 Myr ago in the pre-Ediacaran period (Conway Morris, 1998). Morphological contributions to understanding of the transitional stages to the Metazoa suggest a colonial origin of Metazoa (see Dewel, 2000). This view implies that, based on the Beklemishev's cycles of duplication and individuation (Beklemishev, 1969), after duplication of an individual and the formation of a colony this entity has to undergo individuation again. It has been pointed out that two such cycles were necessary in early evolution for the emergence of Metazoa: first the transition to multicellular organisms, with the sponge grade of organization, and second the change to the modularized ancestor of the Bilateria (Dewel, 2000).
Recent phylogenies based on rDNA have suggested, that the Metazoa are polyphyletic. The proposal suggested that the Porifera/Cnidaria evolved separately from the Triploblasts; both having originated independently through aggregation of protists belonging to two different lineages (Christen et al., 1991). This view is based on the early idea that sponges (Porifera) are grouped with the Protozoa (Spencer, 1864, p. 302). Later, ontogenetic evidence provided the basis for sponges to be considered as metazoans (Haeckel, 1896, p. 18). However, until recently it was generally accepted that the choanoflagellates were the sister group to Metazoa (see Nielsen, 2001). Even though sponge choanocytes are similar to choanoflagellates and are composed of a single flagellum, surrounded by a microvillar collar, the long-standing view of a homology of these types of flagella/cilia (Kent, 1881; Lackey, 1959) could not be substantiated (Karprov and Efremova, 1994). Now, based on the study with protein molecules, Fungi are regarded as the nearest-neighbor kingdom of the Metazoa (Schütze et al., 1999), a conclusion which is supported by others (Baldauf et al., 2000).
During the past 10 years unequivocal support for the monophyly of metazoans has been presented (Müller, 1995) from molecular studies, from analysis of specific cell adhesion molecules and their receptors as well as of the extracellular matrix in sponges, and especially by combination of these data with morphological studies (see Dewel, 2000). The assumption that all metazoan phyla are of monophyletic origin has been widely accepted (see Borchiellini et al., 2001). For the molecular biological analyses the two demosponges Suberites domuncula and Geodia cydonium have been used. The facts compiled also imply that the ancestor of all metazoans was a sponge-like organism, which I termed Urmetazoa (Müller, 2001). This new step in understanding of the basal animal phylogeny is the platform for answering the next pressing question of the origin of individuation in Metazoa, again regarding Porifera as living fossils, descendants of a colonial ancestor. The step of individuation taken by sponges was also a prerequisite for the further progress in evolution of the Porifera to the level of Cnidaria (Dewel, 2000; Fig. 1).
In the following review I present a summary of the recent achievements in the understanding of principles of individuation in sponges, based on protein sequences functioning in the immune response and apoptosis, two processes which play central roles in the maintenance of development and homeostasis of metazoans.
METAZOAN ORIGIN: MONOPHYLY
The adhesion molecules in sponges provide solid grounds for the view that all metazoan animals originated from one ancestor, the Urmetazoa (reviewed in Müller, 2001 and 2003). These molecules were found to represent major metazoan autapomorphies (see Müller, 1995 and 1997).
Adhesion molecules
Already since their first use by Wilson (1907) sponges have been a traditional model for studies of cell-cell and cell-matrix adhesion (reviewed in Burger and Jumblatt, 1977; Müller, 1982). Primarily the two marine demosponges, Microciona prolifera and G. cydonium, have been the most thoroughly studied species. In 1973 two groups succeeded in isolating and purifying both from M. prolifera (Henkart et al., 1973) and G. cydonium (Müller and Zahn, 1973) the first extracellular particle, termed an aggregation factor (AF), which promotes the species-specific aggregation of sponge cells.
The G. cydonium AF is a complex particle, composed of several polypeptides. Three proteins associated with the AF from G. cydonium were identified in detail; a galectin, a 36 kDa putative AF as well as an 86 kDa AF-associated polypeptide. In addition, the proteoglycan-like core structure of the AF has been characterized from M. prolifera. The G. cydoniumgalectin was cloned; sequence analysis revealed that those aa residues which are involved in mammals in binding of galectins to galactose are conserved in the sponge sequence (Pfeifer et al., 1993). This observation was taken as evidence in support of the monophyly of Metazoa (Müller, 1995). The galectin links the AF-complex to the membrane-associated aggregation receptor (AR) (Wagner-Hülsmann et al., 1996). The 36 kDa putative AF was recently cloned (Schütze et al., 2001a). Its deduced aa sequence displays in the N-terminal portion high similarity to amphiphysin/BIN1 sequences found in Protostomia and Deuterostomia. In addition, an 86 kDa AF-associated polypeptide has been identified, whose predicted protein comprises nine short consensus repeats (SCR) (Müller, 2003). The core structure of the AF had been identified in the AF-complex from M. prolifera as a polymorphic proteoglycan-like molecule (Fernandez-Busquets et al., 1996).
Using the G. cydonium model sponge the putative aggregation receptor (AR) was cloned (Blumbach et al., 1998). It comprises fourteen scavenger receptor cysteine-rich (SRCR) domains, six SCR repeats, a C-terminal transmembrane domain and a cytoplasmic tail. Competition experiments using recombinant AR or antibodies raised against this receptor, suggested that the adhesion molecule present in the enriched AF binds to the AR. In addition, previous experiments also indicated that the strength of binding of the AF to the cell surface AR is augmented by galectin (Wagner-Hülsmann, 1996).
Extracellular matrix molecules
In sponges the space between the external pinacoderm and the internal choanoderm, the mesohyl, does not comprise a homogenous ground substance. It is composed, in addition to galectin, of the following main elements: collagen, fibronectin-like molecules, and a minor component, dermatopontin, was also recently identified, (Schütze et al., 2001b). These polypeptides form the extracellular matrix (ECM) which provides the platform for specific cell adhesion via the integrin receptor, as well as for signal transduction and cell growth. As an example, it has been summarized that in demosponges several cells are involved in spicule formation (Uriz et al., 2000). This process requires a series of complex pathways in which also the expression of silica-responsible genes is involved (Krasko et al., 2000 and 2002).
Collagen is an autapomorphic molecule that is present only in the Metazoa. In contrast to higher metazoan phyla, which contain approximately 20 different types of collagen, in sponges only two groups of collagen molecules have been identified, the fibrillar collagen and the type IV-related collagen (reviewed in Garrone, 1998). Exposito and Garrone (1990) were the first to sequence a collagen cDNA from a sponge. Recently, it was shown that cells of S. domuncula express a collagen gene in response to the growth factor myotrophin (Schröder et al., 2000). The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short with only 24 G-x-y collagen triplets (Schröder et al., 2000).
A further major component of the ECM is fibronectin. Evidence has been presented, suggesting that sponges also contain fibronectin. In 1981 Labat-Robert and colleagues described a protein in sponges, which cross-reacted with antibodies raised against vertebrate fibronectin (Labat-Robert et al., 1981). During our search for fibronectin we were able to demonstrate that in G. cydonium, there are protein(s) that cross-react immunologically with human anti-fibronectin antiserum (Müller, 1997). A subsequent screening for the respective cDNA revealed a protein which consists of three putative modules; a fibronectin module type-III, a SRCR unit and a SCR repeat; this sponge protein was called a “multiadhesive protein” (Pahler et al., 1998a).
One major class of receptors which interact with the ECM are the integrin receptors, membrane-anchored heterodimer receptors composed of α- and ß-subunits. After the identification of collagen in the ECM subsequent screening produced the integrin receptors in the marine sponges G. cydonium (Pancer et al., 1997) and S. domuncula (Wimmer et al., 1999).
One additional sponge membrane receptor that should also be mentioned here, is the receptor tyrosine kinase (RTK). RTKs are restricted to the Metazoa. The first RTK from lower metazoa was identified and cloned from G. cydonium (reviewed in Müller and Schäcke, 1996). As schematically outlined in Figure 2 (GC-RTK), the deduced polypeptide sequence comprises: (i) the extracellular part with a Pro/Ser/Thr-rich region, and two complete immunoglobulin-like (Ig-like) domains, (ii) the transmembrane domain, (iii) the juxtamembrane region and (iv) the catalytic tyrosine (TK)-domain. A ligand for this RTK, a mucus-like protein, was also identified (Schütze et al., 2001b).
Metazoan-fungal relationship
Using protein DNA sequences from sponges, especially those from adhesion molecules, the monophyletic origin of the Metazoa was established (Müller et al., 2001a). Using those molecules which function in signal transduction, growth control and defense, a common ancestry with Fungi was established (Schütze et al., 1999; Müller et al., 2001a); Figure 1. Already before such a relationship could be proposed (Borchiellini et al., 1998). The existence of cell-cell- and cell-matrix adhesion molecules was used as evidence of the evolution from the colonial stage to an integrated stage, the step towards individuation (Fig. 1).
METAZOAN INDIVIDUALITY: IMMUNE MOLECULES
Adhesion of cells is the basic property and prerequisite for a functional immune system. Therefore, it can be assumed that during evolution elements or molecules developed which became functional not only as adhesion molecules but also as elements of the immune response. One well known example is the immunoglobulin (Ig-) domains which are building blocks of polypeptides that participate in cell adhesion, muscle contraction, and immune defense (Nezlin, 1998). Later in evolution these domains served as immune molecules.
The early metazoans, the hypothetical Urmetazoa, lived in an aquatic environment and consequently were exposed to a massive load of both pro- and eukaryotic organisms trying to invade and destruct them. It is amazing that sponges have the capacity to process their own volume of water every 5 seconds in order to extract edible material (Vogel, 1977); this fact supports the notion that they are exposed to a huge number of bacteria and also viruses present in the seawater (see Gonzales and Moran, 1997). To cope with these threats sponges have developed an efficient chemical defense system (Proksch, 1994) as well as humoral and cellular defense mechanisms (Müller et al., 1999a). Studies on the immune system in sponges have been performed with the focus on the mechanisms by which (i) these animals react against microbes/parasites and (ii) respond to non-syngeneic tissue.
In recent studies it has been reported that there are pathways which control fusion and rejection during histo-(in)compatibility reaction in the Porifera (Müller et al., 1999a, 2001b). Although this had been expected from the precise historecognition reactions that were described on tissue level (see: Hildemann et al., 1979 and 1980), it was very surprising to discover that key molecules involved in allo/auto-immunity in sponges share high sequence and functional similarity with those molecules which had been found to control historecognition in deuterostomes. Among those are the molecules comprising polymorphic Ig-like domains (present in the sponge adhesion molecules [SAMs]), the allograft inflammatory factor (a sponge cytokine) as well as the (2–5)A system (control of infection) (see below), whose existence had not been reported in protostomes (Gamulin et al., 2000; Müller et al., 2001a). This fact, that sponges have molecules/pathways in common only with deuterostomes (i) strongly supports the monophyly of Metazoa, (ii) underscores that the degree of individuality of sponge species is high and (iii) suggests that sponges might/will become model organisms to understand the origin of vertebrate immunity and diseases connected with it.
METAZOAN INDIVIDUALITY: APOPTOTIC MOLECULE
Apoptosis in sponges
Until recently (Wiens et al., 2000a, b) it was proposed that the physiological cell death is restricted to multicellular organisms, which have separate germ and somatic cells (Vaux et al., 1994). Originally it was suggested to divide the process of physiological cell death into (a) “programmed cell death,” describing the developmentally regulated elimination of specific cells during embryogenesis (Lockshin and Williams, 1964), and (b) “apoptosis”, describing morphological changes of dying cells (Kerr et al., 1972). At present, these terms are used interchangeably; therefore, we use the term apoptosis. In the last three years it has become apparent that apoptosis is not restricted to metazoans that have separate cell lines, but came about during the transition from the common ancestor of all metazoan phyla to the phylogenetically oldest metazoan taxon, the Porifera (reviewed in Müller et al., 1998).
Two lines of evidence led us to assume that sponges are also provided with complex apoptotic pathways. In 1992 Pfeifer and others found that a factor could be identified in xenografts from G. cydonium that cross-reacted immunologically with an antibody raised against a mammalian tumor necrosis factor (TNF). The Mr was determined to be 30 kDa hinting at a relationship to the mammalian TNF. Furthermore, it was shown that sponge cells have a high level of telomerase activity, when they are present in the state of cell-cell contact [both in intact organism and in primmorphs] (Koziol et al., 1998). Consequently we postulated that, in order to maintain a defined “Bauplan,” sponge cells in tissue organization must undergo apoptosis (Wagner et al., 1998). Recently, we could identify in sponges homeobox genes, e.g., a LIM/homeobox encoding protein, which are involved in organogenesis in higher metazoan phyla (Wiens et al., submitted). However, in spite of intense efforts, the gene encoding the potential TNF as well as the receptor interacting in mammalian systems, the TNF-receptor, were only recently cloned. The first potential gene involved in apoptosis of sponge cells, the MA-3 gene from S. domuncula was identified (Wagner et al., 1998); the corresponding mouse MA-3 cDNA is assumed to encode an apoptotic molecule (Shibahara et al., 1995). Subsequently both pro- and anti-apoptotic proteins have been cloned from both S. domuncula and G. cydonium, and their functions have been analyzed to some extent.
Metazoan pro-apoptotic molecules
As the most promising segment to screen for a pro-apoptotic molecule, we selected the death domain part which is found in the mammalian apoptosis controlling proteins Fas, tumor necrosis factor-α or its receptor, and FADD (Cleveland and Ihle, 1995); it is absent in the nematode (Ruvkun and Hobert, 1998). This approach was successful; the molecule isolated from G. cydonium even comprises two death domains (Wiens et al., 2000a). Sequence comparisons revealed that the two domains found in the sponge molecule are to be grouped within the death domain family. It was claimed before that the death domain found in humans comprises relationship to ankyrin motifs (Boldin et al., 1995), an assumption which could be substantiated also experimentally (Müller et al., 2001a). Functional assays were performed with allografts from G. cydonium which revealed that in rejecting tissue a strong increase of the expression of the death domain-comprising gene (GCDD2) occurs (Wiens et al., 2001).
Caspases
In vertebrates, the death domain containing receptors/adapter molecules interact intracellularly with the caspase-8 proenzyme through the death-effector domain with a similar region in the caspase (Grütter, 2000). An adapter-mediated oligomerization causes an activation of the procaspase(s) which undergo cleavage and finally heterodimerization (Cory and Adams, 1998). Finally, upstream caspase(s) activate pro-caspase-3 which in turn is split into the large and small subunits that activate after heterodimerization a factor necessary for the DNase activity to degrade chromatin into the nucleosomal fragments the sign of apoptosis (Fig. 3B).
In Bilateria a series of caspases are involved in the tuned control of apoptosis, starting with caspase-8 in the cascade and ending with caspase-3. Interestingly enough, until now, only one gene has been identified in G. cydonium which encodes two transcript forms, both for caspase-8 and for -3 equivalents (Fig. 3A). Two deduced procaspases have been identified, which were termed CAS3l_GEOCY [long form] and CAS3s_GEOCY (short form) (Wiens, to be published). CAS3l_GEOCY can be considered as the procaspase-8 equivalent, due to the presence of the CARD domain (Hofmann et al., 1997). The—probably alternatively spliced product—procaspase-3 equivalent (CAS3s_GEOCY) lacks CARD, but like the CAS3l_GEOCY sequence, comprises the cleavage sites for the formation of the subunits as well as the two caspase family active sites (Fig. 3A). Functional studies indicate that the two forms of the sponge caspases act in G. cydonium in the apoptotic pathway.
Metazoan anti-apoptotic/cell survival proteins
In addition to the activation of the apoptotic process through TNF/TNF-receptor further pathways have been described in Bilateria, which include activation through growth factor deprivation, heat shock or bacterial infection (Nicholson and Thornberry, 1997), pathways which have also been described in sponge systems (Wagner et al., 1998). The signal transduction pathway initiated by those factors can be blocked by the function of molecules belonging to the Bcl-2 family (Nicholson and Thornberry, 1997).
In line with the biological evidence that in both S. domuncula and in G. cydonium apoptosis can be initiated by environmental stress factors, e.g., bacterial load (Wagner et al., 1998) or cadmium (Wagner et al., 1998) an intense screening for members of the Bcl-2 family was started. This effort resulted in the functional analysis of the anti-apoptotic/cell survival proteins from these two sponge species (Wiens et al., 2000a, b, 2001). The proof that the sponge gene product acts as a cell survival protein was performed by transfection studies using mammalian cells. It could be shown that mammalian cells transfected with the sponge Bcl-2 related gene confer resistance against heat shock and growth factor deprivation (Wiens et al., 2001).
Besides the molecules of the Bcl-2 family other polypeptides are also known to prevent apoptotic pathways, among them is 14–3–3 molecule which, under “cytoprotective” conditions, interacts with the pro-apoptotic Bad after this protein has undergone phosphorylation through Akt. If Bad is bound to 14-3-3 it has lost its binding capacity to the Bcl-2 related molecule Bcl-xL (Zha et al., 1996). Also this pathway, where 14-3-3 is involved in protection against man-made pollutants, e.g., PCB, has been demonstrated in G. cydonium (Wiens et al., 1998); Fig. 3B.
Taken together, the bulk of evidence shows that sponges have a complex apoptotic machinery, which allows the elimination of unwanted tissue (e.g., in allo-transplantation) and very likely also in the establishment of an organized body plan.
CONCLUSION: URMETAZOA AS COMPLEX AND INTEGRATED ANIMALS
The question from which organism the Urmetazoa evolved remains open. Frequently, the choanoflagellates have been considered as the sister group of the Metazoa. However, cytological data contradict this view (Karprov and Efremova, 1994), and molecular sequence data from proteins are not available from choanoflagellates. In view of existing data it appears more likely that the Urmetazoa share a common ancestry with the Fungi (Schütze et al., 1999).
An integrated organism, like the Porifera, requires as a prerequisite cell-cell adhesion systems which allow the transfer of signals between cell assemblies. The consequence of this level of integration are activation of intracellular signal transduction pathways that result in differential gene expression and cell specialization. One example has been described in primmorphs: only if cell-cell adhesion is allowed the cells undergo cell division and cell differentiation (Müller et al., 1999b). In addition, in the primmorph system, morphogen-like molecules, e.g., iron, initiate the complex synthesis of spicule formation (Krasko et al., 2002).
At the next level of integration, the cell-matrix adhesion system supports the integration of the functional units of sponges. The major extracellular molecules in sponges are collagen fibrils, which interact with integrin receptors on the cell surface, followed by G-protein and kinase-mediated signal transduction processes (Wimmer et al., 1999).
Cell-cell- and cell-matrix adhesion are the basis for the colonial stage of the metazoans and prerequisites for the establishment of integrated systems. These adhesion systems alone are not sufficient for individuation. The stage of individuation can only be reached after the acquisition of an immune system which is paralleled or complemented by a mechanism that eliminates unwanted, which accumulates during development in a multicellular organism; this process is termed apoptosis. Sponges have an amazingly complex immune system, which acts against invading microbes or parasites. Furthermore, the immune system is the basis for the individualization; mechanisms have been formed during evolution, which allow for discrimination between self/self and self/non-self.
Apoptosis is the mechanism in Metazoa that guarantees homeostasis. The characteristic apoptotic molecules have been identified in sponges which, together with the functional studies performed, demonstrate that these earliest of metazoans are provided with key regulatory elements for controlled development, tissue homeostasis and defense against pathogens.
Taken together, the phylogenetic oldest, extant metazoan phylum, the Porifera, are provided with complex immune and apoptotic systems that allow the formation of an integrated system (Fig. 1). Considering the fact that the different sponge species are not “amorphous, asymmetrical creatures” as suggested (Pechenik, 2000), but comprise a defined phenotype, a sponge might be defined as “integrated colony” or an individual, composed of functional units, allowing the formation of a defined body plan.
From the Symposium New Perspectives on the Origin of Metazoan Complexity presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3–6 January 2002, at Anaheim, California.
E-mail: WMUELLER@mail.UNI-Mainz.DE
This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany [Center of Excellence “BIOTECmarin”] and the International Human Frontier Science Program (RG-333/96-M).
References