ISSN 10623590, Biology Bulletin, 2016, Vol. 43, No. 3, pp. 216–225. © Pleiades Publishing, Inc., 2016. Original Russian Text © V.V. Kozin, R.P. Kostyuchenko, 2016, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2016, No. 3, pp. 265–275.
CONFERENCE MATERIALS
Evolutionary Conservation and Variability of the Mesoderm Development in Spiralia: A Peculiar Pattern of Nereid Polychaetes V. V. Kozin and R. P. Kostyuchenko St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg, 199034 Russia email:
[email protected] Received August 26, 2015
Abstract—An analysis of the comparativemorphological, moleculargenetic, and evolutionary aspects of the teloblastic mesoderm formation in the Spiralia representatives was performed. The conservative and the most expressive varying features of morphogenesis and genetic developmental programs of the mesodermal germ layer were considered. Using nereid polychaetes, we revealed peculiarities of their developmental pat terns, related to the role of inductive interactions and the dynamics of the molecular profile in the formation of mesodermal derivatives. DOI: 10.1134/S1062359016030079
MESODERM FORMATION IN ONTOGENESIS AND PHYLOGENESIS OF MULTICELLULAR ORGANISMS During embryonic development, germ layers are formed, each of which makes a predominant contri bution to the formation of distinct organs and tissues. Current data on the developmental genetics and genomics of multicellular animals with various levels of organization—Porifera and Placozoa (the exist ence of germ layers in which is rejected), diplo and triploblastic animals—attest to the surprising conclu sion that the body plan and ontogenesis evolved on the basis of one and the same set of families of transcrip tion factors (TFs) and types of cell signaling (Dondua and Kostyuchenko, 2013; Nakanishi et al., 2014). According to the principle of specificity, the germ layers in different animals have the same sets of deriv atives. The invariant relative position of the outer, inner, and middle layers after gastrulation corresponds with the principle of topography. Based on the theory of germ layers, cnidarians and ctenophores have two layers: the outer ectodermal and inner endodermal. The latter is sometimes called the “endomesoderm” (Martindale, 2005). This term corresponds to the con cept of the Russian “mesendoderm”, i.e., the com mon precursor of mesodermal and endodermal struc tures. The anlage of the parenchyma in Acoela can be also termed mesendoderm (IvanovaKazas, 1995). In other eumetazoans all three layers are distinguished, hence the name “Triploblastica.” However, both pro tostomes and deuterostomes almost always have a bipotential mesendodermal germ layer in their early development with conservative mechanisms of specifi cation (Rodaway and Patient, 2001). The most important evolutionary acquisition in early development of Triploblastica (Bilateria) was the
separation of the individual mesodermal layer. The molecular basis of this innovation is considered to be the segregation of myogenic factors from the common mesendodermal program and acquired independence and selfsufficiency followed by divergence of the cur rent genetic mesodermal regulatory network (Martin dale, 2005). Being evolutionarily younger, the meso dermal layer has the highest variability in the mecha nisms of its formation in different phylogenetic lineages. According to a popular opinion, the appear ance of the mesoderm in the embryogenesis of Bilateria largely provided for the explosive increase of body plans number in the Early Cambrian (Martindale et al., 2004; Burton, 2008). However, fundamental issues such as the cellular and molecular mechanisms of the meso derm formation in embryogenesis and postlarval development (during metamorphosis and regenera tion) of many animals have not yet been solved. Thus, a comprehensive comparative study of mesoderm development becomes even more important, as it should bring us closer to understanding the ways of evolution and emergence of modern biodiversity. The morphological separation of the mesoderm during gastrulation is, as a rule, carried out by one of two alternative modes: teloblastic or enterocoelic. The former is implemented in annelids and crustaceans, in which the mesodermal blastomeres are determined quite early in the development. The proliferation of these mesodermal teloblasts gives rise to mesenchymal clusters, which form a cavity inside by schizocoely. The cells lining the cavity constitute the epithelium of the coelomic sac. In the course of enterocoely, which is found in echinoderms and hemichordates, the arch enteron forms local protrusions, so the forming pri mary coeloms detach and enter inside the blastocoel. The primary coelomic pouches are then divided into a
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number of coelomic sacs, which is characteristic for this species. We concentrated here on the teloblastic mesoderm formation. It is usually considered that this morphogenesis is quite uniform in a large number of species of different animal phyla, but its mechanisms are not clearly understood. MESODERM CELL LINEAGES IN SPIRALIA In an embryological sense, the group Spiralia includes annelids, mollusks, nemerteans, and flat worms. They are all mainly characterized by a stereo typical pattern of cleavage with early segregation of germ layers and cell lineages. This feature is mani fested in the teloblastic mode of forming various rudi ments, including the mesoderm. Traditionally, two mesodermal components are distinguished in the embryo of spiralians: the ecto and endomesoderm. It is assumed that the ectomesoderm or the larval mesenchyme, which ingress during gastrulation inside the embryo as single cells, derives ontogenetically from the ectoderm, while the endomesoderm origi nates from the endoderm (Hyman, 1951). The embry onic source of the ectomesoderm in different spiralian species (table) is the cells of the second and/or third quartet of micromeres (Anderson, 1973; Ivanova Kazas, 1977). For a long time, it was assumed that the ectomesoderm differentiates into the larval muscles, which are not retained in adulthood, whereas the endomesoderm is the main source of definitive meso dermal structures. However, in light of new data this assumption was reconsidered. The only ectomesodermal source in turbellarian Hoploplana inquilina—the 2b cell—as well as the 4d blastomere, gives rise to the muscles of the Müller’s larva, which are retained after metamorphosis (Boyer et al., 1996). In the mollusk Patella vulgata, the descendants of micromeres of the third quartet and, probably, 2b differentiate into the ectomesoderm and ectoderm of the larval hyposphere (Dictus and Damen, 1997). In the polychaete Platynereis dumer ilii, the ectomesoderm is comprised of micromeres of the third quartet, the derivatives of which are the mus cular envelope of the pharynx (the descendants of 3c and 3d) and head muscles (the descendants of 3a and 3b) (Ackermann et al., 2005). In leeches, the ectome soderm (3a–3d cells and, possibly, 1a–1d) forms the muscles of the proboscis, connective tissue elements and a network of fibers in the body wall with unidenti fied function (Huang et al., 2002). Spiralia is characterized by an extremely high con servatism of the endomesoderm cell lineage. With few exceptions, this component develops from the blas tomere 4d, called the second somatoblast (table). In most spiralians, 4d is a mesendoblast; i.e., it contains the potential for development in the meso and endo dermal directions. For some spiralians, the origin of primordial germ cells (PGCs) from the 4d was also proved (Rebscher et al., 2007; Gline et al., 2011; Lyons BIOLOGY BULLETIN
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et al., 2012). The temporal order of separation of these cell fates varies greatly in different taxons. In polyclad turbellarians, two bilaterally symmetric mesoblasts (4d212 and 4d222) are formed only after the third divi sion of 4d, the remaining descendants of which (4d1, 4d211, and 4d221) form the intestinal epithelium (IvanovaKazas, 1975). In mollusks and annelids, 4d almost always divides bilaterally into two Mcells (ML and MR, which can also be designated 4d1 and 4d2) (Fig. 1b). A detailed study of the 4d cell lineage in the mollusks Crepidula showed an alternating separation of endodermal (1m, 3m, 4m, 5m) and mesodermal (2m, 6m, 7m) descendants from the multipotent telo blasts ML and MR (Lyons et al., 2012). During these asymmetrical divisions, the remaining large Mcells produce small daughter cells in different directions: the descendants of the first and third generation (1m and 3m) are separated toward the vegetal pole, while the other traced cells (2m, 4m, 5m, and 6m) are pro duced toward the animal pole. The mesendodermal bands of the gastropod Ilyanassa obsoleta embryo are organized in a similar way (with the central position of the teloblast) (Rabinowitz et al., 2008). The character of divisions of Mcells in the poly chaete P. dumerilii is much more reminiscent of that in mollusks than in oligochaetes and leeches. According to Fischer and Arendt (2013), the mesoteloblastlike Mcells produce seven smaller offspring cells in a fan like manner. Then Mcells divide equally and become indistinguishable. Notably, there is no sequential dis tribution of the Mcells descendants along the ante rior–posterior axis of the mesodermal band at the tro chophore stage. On the contrary, the earliest mesote loblast daughter cells (the socalled secondary mesoblast) retain their posterior position at the basis of the mesodermal bands, giving rise to PGCs. The Mcells of polychaetes are traditionally referred to as mesoblasts (Wilson, 1892), as data on the involvement of 4d in the construction of the gut are contradictory. Note, however, that after the injection of the TRITC dextran dye into the 4d blastomere in P. dumerilii, in addition to the marked mesodermal derivatives of the nectochaete body, a dense ball of tissue of unknown nature is observed at the posterior end of the develop ing midgut (Ackermann et al., 2005). Taking into con sideration the report on the participation of the sec ondary mesoblast in the formation of the endodermal epithelium in Nereis (Wilson, 1898), we can assume that, in polychaetes, 4d is a mesendoblast as well. Perhaps the only clearly proven example of meso dermal bands development in polychaetes not from the 4d blastomere is Capitella teleta (Meyer et al., 2010). In this case, the 3c and 3d cells are used instead of 4d. Nevertheless, 4d is involved in the formation of some muscles in the body and PGCs. Thus, a transi tion of the segmental mesoderm origin from endome soderm to ectomesoderm is observed in Capitella. Capitella is closely related phylogenetically to the Cli
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Fig. 1. Participation of MAP kinase and transcription factors Twist and Mox in mesoderm development in the nereid polychaete Alitta virens. (a) The stage of eight blastomeres, view from the dorsoanimal side; (b) an embryo at the stage of transition from the spiral to the bilateral cleavage (shortly before the start of gastrulation), the cells of the blastopore edge are outlined, view from the dorsal side; (c) MAP kinase cascade; (d) disruption of morphogenesis of mesodermal bands affected by an inhibitor of MAPK signaling U0126, applied at the early development (from the stage of eight blastomeres till the stage of early gastrula); (e, f) normal pattern of expression of the gene Avitwist; (g, h) normal pattern of expression of the gene Avimox; (d, e, g) the larva at the stage of middle trochophore, view from the ventral side; (f, h) the larva at the stage of early metatrochophore, view from the ventral side. (bl) Blastopore, (M) mesoteloblast, (mb) mesodermal band (endomesoderm), (pt) prototroch, (Raf, Mek, Erk) kinases of MAPK cascade in an inactive form, (RafP, MekP, ErkP) phosphorylated (active) form of kinases, (st) stomodeum (anlage of the foregut), (1a, 1b, 1c, 1d, 1A, 1C, 1D, 2d112) designations of blastomeres, (1) distribution of the active form of MAP kinase Erk1/2, (2) localization of mRNA of Avitwist, (3) mRNA localization of Avimox.
tellata branch (i.e., clitellate annelids: oligochaetes and leeches) and shows a rapid divergence with them from the rest of the annelids. The fast evolution explains the secondary modifications of development of annelids from the classes Clitellata and Echiura, as well as representatives of the family Capitellidae. The early development of the clitellate annelids is spiral, but much altered in many ways: the precision of the spiral cleavage is quickly lost, and the embryo becomes bilaterally symmetrical early on. Further more, the development of Clitellata is direct and has no larval stages or metamorphosis (IvanovaKazas, 1977). It was generally assumed that in clitellates 4d is not directly involved in endoderm development. It is known that a few of the latest descendants of Mcells, like the cells themselves at the late segmentation stages, merge with the socalled yolk syncytium (the material of the fused macromeres), whose subdivision into distinct cells (cellularization) gives rise to the intestinal lining (Liu et al., 1998). More recently, it has been shown that the earliest progeny of Mcells (not involved in the segmental mesoderm development em1 and em2 cells) in the leech Helobdella provide material for the intestinal epithelium, inter alia (Gline
et al., 2011). Based on these data Gline et al. (2011) suggested that the mesendodermal nature of the 4d blastomere is an ancestral (evolutionarily primary) feature of all Spiralia. As in polychaete, the segmental mesoderm of cli tellates is derived from the 4d cell. The result of the symmetrical division of 4d is the formation of two mesodermal teloblasts, whose further proliferation produces the bilaterally symmetrical mesodermal bands (Anderson, 1973; IvanovaKazas, 1977). In oli gochaetes and leeches, the highly asymmetrically dividing mesoteloblasts give rise to a serial number of small primary blast cells. The birth of all primary blast cells occurs at the same pole of the mesoteloblast, so that its early descendants take a position at the anterior end of the germ band, and the later ones–at the poste rior end. The teloblasts themselves (both mesodermal and ectodermal) retain the terminal position, which is specific only of clitellates and outside Spiralia only of higher crustaceans (Malacostraca). It should be noted that a comparison of the teloblastic growth in these two remotely related groups of protostomes leaves no doubts as to the independence of the evolutionary acquisition of this feature (Scholtz, 2002). BIOLOGY BULLETIN
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3D, micromeres; specification of mesendod23
3D; specification of mesendod19
3D; specification of mesendod14,15,*
Endomes, Blastopore edge11 ectod, gut, GZ10
Endomes, Endomes, Micromeres, macromeres; ectomes, ectod, morphogenesis ectod, endod, GZ5,* of endomes* PGCs, GZ4
nanos
For several species of annelids and mollusks, blastomeres with ectomesodermal and endomesodermal fate are listed. The tissue specificity of expression domains of certain regulatory genes, which manifest mesodermal expression (columns 4–9), is described. The localization of the active form of MAP kinase and its functions during early development (column 10) is given. (GZ) Growth zone; (mesendod) mesendoderm, (PGCs) primary germ cells, (ectod) ectoderm, (ectomes) ectomesoderm, (endod) endoderm, (endomes) endomesoderm, (–) no data. * Our data; 1 Ackermann et al., 2005; 2 Steinmetz, 2006; 3 Gillis et al., 2007; 4 Rebscher et al., 2007; 5 de Rosa et al., 2005; 6 Meyer et al., 2010; 7 Dill et al., 2007; 8 Boyle and Seaver, 2008; 9 Dill and Seaver, 2008; 10 Seaver et al., 2012; 11 Amiel et al., 2013; 12 Dictus and Damen, 1997; 13 Nederbragt et al., 2002; 14 Lartillot et al., 2002; 15 Kozin et al., 2013; 16 van den Biggelaar, 1993; 17 Hinman and Degnan, 2002; 18 Kranz et al., 2010; 19 Koop et al., 2007; 20 Render, 1997; 21 Swartz et al., 2008; 22 Rabinowitz et al., 2008; 23 Lambert and Nagy, 2001.
Ilyanassa obsoleta (Caenogas tropoda)
Haliotis asinina (Vetigastropoda)
Patella vulgata, Testudinalia testudinalis (Patellogastropoda)
Capitella teleta (Sedentaria, Capitellidae)
Alitta virens, Platynereis dumerilii 3a, 3b, 3c, 4d1,* (Errantia, Nereididae) 3d1,*
Organism
Cell lineages and molecular determinants of the mesoderm in Spiralia
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Each primary blast cell in clitellates undergoes a series of stereotypical (i.e., oriented in a similar man ner) divisions, thereby forming a separate mesodermal compartment (Shimizu and Nakamoto, 2001). In leeches, a clone of one primary blast cell is the meso dermal material of a distinct body segment (if its bor ders are defined by the dissepiments). Thus, the metamerization of the mesoderm in Clitellata occurs at the earliest stages of its development. According to some researchers (Shimizu and Nakamoto, 2001), at the moment of separation from the mesoteloblast, the primary blast cells have polarity and segmental iden tity, which indicates very autonomous development of the clitellate mesodermal anlage. An analysis of published data suggests that the cel lular composition and details of morphogenesis of the mesodermal germ layer in Spiralia show high variabil ity. The general rule for Spiralia empryos is the forma tion of specific blastomeres with a purely meso or mesendodermal fate. Via varying gastrulation mor phogeneses, whose type is greatly determined by the amount of yolk in the egg, the mesodermal progenitors are internalized into the embryo. Apparently, the peculiarity of the teloblasts division pattern is defined by the mechanisms of further segregation of different cell lineages (endodermal, mesodermal, and PGCs), as well as the characteristics of the life cycle and the body plan of the organism emerging from the egg: the larva or juvenile animal. SOMATOBLAST 4d AND ORGANIZER IN SPIRALIA In Spiralia, the lineage of blastomeres leading to the 4d somatoblast and 4d itself are intimately con nected with the embryonic organizer. The existence of the organizer in mollusks has been proven in experi ments on removing the cells of quadrant D. In these experiments on I. obsoleta, besides the impaired devel opment of derivatives of the removed blastomere, Clement (1962) also observed the absence of organs originating from other cell lineages, such as the eyes and statocyst. Removing the blastomere 3D, the num ber of anomalies depended on the lifetime of the intact 3D cell: if it was removed immediately after its birth, most organs of the larva did not develop, but if the operation was performed later, only mesendodermal derivatives were lacking, whereas the bilateral body sym metry was retained. It led to conclusion that 3D induces certain cell fates in the neighboring micromeres. In addi tion, in the absence of the organizer cell (early 3D), radi alized (i.e., without bilateral symmetry) larvae are formed. The existence of mutual inducing signals between the future organizer blastomere and the animal micromeres was also shown for the mollusks with the homoquadrant spiral cleavage (van den Biggelaar and Guerrier, 1979; Henry et al., 2006). It is generally accepted that in the mollusks Ilya nassa, Patella, and Lymnaea the organizer blastomere
is 3D, but some capacity is retained in its daughter 4d cell (Henry, 2014). The organizer in the gastropod Crepidula fornicata is different since its role is played exclusively by the second somatoblast 4d (Henry et al., 2006). In the oligochaete Tubifex tubifex, the organizer activity, apparently, is produced by the combined action of two cells: the first somatoblast 2d and the sec ond somatoblast 4d (Nakamoto et al., 2011). In another annelid, polychaete C. teleta, the ability to induce cell fates and to establish a secondary (dorsoven tral) axis of the body belongs to the blastomere 2d, but disappears after its division (before the formation of the third quartet of micromeres) (Amiel et al., 2013). Until recently, the specific molecular components of embryonic induction in Spiralia remained a mys tery. The first established mechanism of intercellular interaction in mollusk embryos was signaling via MAP kinase (Lambert and Nagy, 2001). MAP kinase signal ing cascade (Fig. 1c) consists of three conserved pro teins with the kinase domain (Raf (MAPKKK), Mek (MAPKK), and Erk (MAPK)), which successively phosphorylate each other, and a number of regulatory factors. The activated MAP kinase Erk (extracellular signalrelated kinase), in turn, phosphorylates the tar get proteins, which belong to the systems of the cell cytoskeleton, metabolism, proliferation, and differen tiation. These effectors are localized in the cytoplasm, mitochondria, endoplasmic reticulum, and especially the nucleus (Yang et al., 2013). The MAPK cascade is differentially activated in the embryos of gastropods Ilyanassa, Patella, Tectura, Testudinalia, Haliotis, Lymnaea, and chiton Chaetopleura exactly at the stage of interaction between the 3D cell and the animal micromeres (Lambert and Nagy, 2001, 2003; Koop et al., 2007; Kozin et al., 2013). For the first time the phos phorylated form of MAPK in mollusks was found in the 3D macromere (table), but in the case of Ilya nassa, the signal soon appeared in the animal blas tomeres as well, indicating the distribution of the inductive influence emanating from the 3D organizer. This agrees with the fact that the elimination of the 3D macromere disrupts the normal activation of MAPK in the micromeres neighboring 3D (Lambert and Nagy, 2001). In experiments with the pharmacological inhibi tion of MAP kinase signaling, at the cleavage stage, the development of a number of structures is deficient in these mollusks, up to full recurrence of the phenotype of larvae with the removed organizer. Thus, MAPK is required for the acquisition of identity by the Dquad rant and for ensuring its activity as an organizer (Henry, 2014). It is noteworthy that in the gastropod Crepidula, the earliest and very weak MAPK activation was shown in the micromeres of the first quartet, before the formation of 3D. The results on Crepidula and another gastropod with homoquadrant spiral cleavage Testudinalia testudinalis demonstrated that the severest developmental disorders occur from the suppression of MAPK phosphorylation at the stages BIOLOGY BULLETIN
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before the formation of the 3D macromere (Kozin et al., 2013). The possibility of early interactions between macromeres and micromeres that determine the Dquadrant has been actively discussed recently (Henry, 2014). It also becomes apparent that the MAPK cascade is not the only pathway of intercellular communication involved in determining cell fates and body axes during early development in spiralians. It is important to note that, in the polychaete Hydroides, the activation of MAPK was found not in the 3D macromere, but in the 4d somatoblast (Lam bert and Nagy, 2003). Across the abovementioned spiralians, the worm C. teleta is noteworthy, whose phosphorylated form of MAP kinase appears around the blastopore relatively late, in the period of gastrula tion (Amiel et al., 2013). In the embryos of the nereid polychaete Alitta virens, we found another, not previously described pat tern of activation of MAPK (Figs. 1a, 1b). For the first time (at the stage of eight cells), the specific antibodies revealed phosphorylated Erk1/2 in the nuclei of the dorsal blastomeres: micromeres 1c, 1d, and mac romere 1D. Further MAPK activation is observed in the majority of embryonic cells, including the lineages of the first and second somatoblasts and macromere 3D. In the experiment with total suppression of this signaling by the inhibitor U0126, the mesodermal material is formed retaining the expression of the spe cific markers twist and mox (Fig. 1d). However, if the inhibition started before the formation of the second somatoblast 4d, the morphogenesis of the endomeso derm was significantly disturbed: shorter mesodermal bands or dense cellular conglomerates at the vegetal pole were developed in trochophores. Thus, we con clude that, during cleavage, the MAP kinase signaling in A. virens is responsible for realization of the normal morphogenesis of the endomesoderm. At the same time MAPK does not determine endomesoderm spec ification and patterning; i.e., it does not primarily gov ern the formation of the mesodermal germ layer and its molecular profiling. DIFFERENTIATION INTO THE MUSCLETYPE CELLS: THE ORIGIN OF THE MESODERM AND MOLECULAR MECHANISMS OF MYOGENESIS The embryological, morphological, and molecular data suggest that the most ancient mesodermal deriv atives were muscle cells (Rieger and Ladurner, 2003; SchmidtRhaesa, 2007). The primary function of myocytes may have been control over the directed locomotion and maintenance of body flexibility. Pro totypes of such a primitive organism with ciliary loco motion are basal Bilateria, such as Acoelomorpha. The myocytes in Nemertodermatida and Acoela are derived from the intestinal cell lines of mesendoder mal origin (Rieger and Ladurner, 2003). BIOLOGY BULLETIN
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Free muscle cells, as considered by many modern researchers, originated from the myoepithelium, which is found in cnidarians, protostomes, and deu terostomes (Seipel and Schmid, 2005; Burton, 2008). In polyps and jellyfish, the myoepithelial cells are present in the epidermis and gastrodermis; in bilateri ans, they compose the coelomic lining in whole or in part. Based on the results of microanatomical compar ative studies, a gradual evolutionary transformation of the homogeneous myoepithelium into a stratified one has been repeatedly assumed, in some cells of which the contractile apparatus was shifted basally and/or taken out of the layer. Later, these cells were com pletely separated from the epithelium, giving rise to individual muscle fibers adjacent to the coelom wall. Such morphological transformation sequences were revealed, for example, in echinoderms and annelids. Since the histological organization and the develop mental stage of formation of the coelomic epithelium vary greatly in annelids, we must admit the possibility of an independent and repeated origin of the separated peritoneal layer and the muscles even within this type of animals (Rieger and Purschke, 2005). The earliest origin of muscletype cells is con firmed by molecular genetic data as well. It was found that the conservative set of structural genes, on the basis of which the evolution of the muscle cells began, appeared before the divergence of fungi, choanoflagel lates, and multicellular organisms being retained in the genomes of Opisthokonta (Steinmetz et al., 2012). The analysis of the development of lower Metazoa (Technau and Scholz, 2003; Martindale et al., 2004; Burton, 2008) have allowed researchers to come to a number of conclusions that we share: “mesodermal” genes appeared earlier than the mesoderm; in bilateri ans, they participated in the development of the endo derm; the mesodermal layer appeared in evolution due to the segregation of groups of cells at the blastopore area (gastrulation center); the primary mesodermal differentiation was the muscle elements; free myocytes and striated muscles emerged as adaptations during the evolution several times, and, in some cases, it took place without the preceding formation of the meso dermal germ layer. The transcriptional regulation of myogenesis in Bilateria—the formation of the skeletal muscles of vertebrates and the muscle wall of the body in inverte brates—has become one of the key models of genetic regulatory networks from the time of the discovery of MRF proteins (myogenic regulatory factors). This genetic developmental program is well developed for higher vertebrates, but in other animals, in particular in Spiralia, it is almost unexplored. The MRF molecules are the TFs of the bHLH (basic helix–loop–helix) superfamily. They have a remarkable ability to transform nonmesodermal deriva tives such as melanoblasts, hepatoblasts, or neuroblasts, into muscle cells. This property, together with their exceptional importance for normal muscle development,
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makes MRF true master genes of myogenesis (Tapscott, 2005). In vertebrates, there are four paralogous MRF: MyoD, Myf5, and Mrf4 are involved in myoblast specifi cation, while MyoG (Myogenin) ensures the differentia tion of myoblasts (Buckingham and Rigby, 2014). On the contrary, in Drosophila the master regulator of myogenesis is considered to be another TF of the bHLH superfamily—Twist—as it is required and suf ficient to launch the program of muscle development (Baylies and Bate, 1996). Unlike the MRF of verte brates, Twist in insects performs a much broader role in mesoderm development. In Drosophila, Twist is responsible for the specification and patterning of the mesoderm, its gastrulation morphogenesis, differenti ation, and specification of different muscle groups, including the precursors of the embryonic and defini tive muscles of the body wall (Leptin, 1991; Ciglar and Furlong, 2009). The molecular mechanisms of such a diverse specificity are determined by the possibility of direct interaction of the Twist protein with the enhancers (which have different sensitivities to its concentration) of hundreds of regulatory genes, including the tinman. The homeodomain TF Tinman (Nkx2.5 homolog) is consid ered the second most important for myogenesis in Droso phila and is responsible for the formation of the heart and dorsal musculature (Ciglar and Furlong, 2009). The only homolog of MRF genes in Drosophila nautilus is similar in the primary sequence (i.e., orthologous) to the gene myoD. The dynamics of its expression and role in normal development are very similar to the MRFs in vertebrates (Wei et al., 2007). Nautilus also has genuine activity of a master regulator of myogenesis as it is able to induce the cell culture to myogenic differentiation and its ectopic expression throughout the embryo mesoderm is sufficient for transforming cardioblasts into the body wall muscles (Ciglar and Furlong, 2009). Although the regulation of transcription of nautilus by Twist has not been studied specifically, the fact that it is launched much later than the beginning of expression of twist and tinman sug gests that nautilus is subordinate to these TFs in the network. The identity of myoblasts of Drosophila is deter mined by the socalled iTFs (muscle identity tran scription factors): Eve, S59, Kr, Msh, Col, Ap, Runt, Lb, Lms, Nau, Poxm, Vg, Sd, DPtxl, and Tey (de Joussineau et al., 2012), whereas TFs from completely different families, i.e., Pitx, Lbx, Msx, Sim, Mox, Tbx, Lhx, etc., are responsible for the patterning (molecular profiling) of the myogenic material in var ious parts of the embryo in vertebrates (Buckingham and Rigby, 2014). The final stage of myogenesis comprises the termi nal differentiation, in which myocytes merge into myotubes and a specific contractile apparatus and neuromuscular junctions appear. In the vast majority of animals studied, the main driver of this process is the MADSboxcontaining TF Mef2 (Ciglar and Fur long, 2009). The enhancers of mef2 are the direct tar
get of the MRFs (primarily MyoD) in vertebrates and Twist in Drosophila, which provides a fairly early start of mef2 expression. Mef2 itself supports the transcrip tion of these myogenic specifiers through positive feedback loop, which further amplifies the signal and stabilizes the chosen direction of differentiation (Pot thoff and Olson, 2007). Furthermore, Mef2 activates in myocytes multiple structural genes, such as actins, myosins, tropomyosins, and others that are specific for muscles. Mef2 is incapable of ectopical launching myogenesis, highlighting the requirement of coopera tion with other regulators, in particular, homologs of MRFs, CF2, and Twist. The latter TF can physically interact with Mef2 and they both have adjacent cis regulatory sites in the enhancers of subordinate genes (Sandmann et al., 2007). Summarizing the analysis of the myogenic program of development, it can be argued that the set of its components (classes of regulatory and structural genes) is mainly constant even in remotely related bilaterians, but the network topology and the specific functions of the homologous factors have evolved independently in different clades. The mesodermal bHLHfactors (MRF paralogs altogether and Twist) occupy a similar position in the myogenesis program in vertebrates and ecdysozoans, launching a large number of gene batteries with a variety of functions: from proliferation to metabolism and biogenesis of the contractile apparatus. Regulatory axis myoD–mef2 and twist–mef2 together with the inferior modules also demonstrates certain conservation when comparing the myogenic programs in mice and Drosophila. EXPRESSION OF MESODERMAL MARKERS IN SPIRALIA In contrast to the welldesigned model of tran scriptional regulation of myogenesis in mice and Drosophila, the full set of mesodermspecific genes and the function of at least one of them remain a mys tery for representatives of Spiralia. Until recently, this was due to lack of suitable methods and convenient model objects. The role of the latter is now claimed by several species of planarians, gastropods, leeches, and nereid polychaetes. Researchers succeeded in reveal ing the expression of homologs of some important reg ulatory genes in the mesoderm derivatives of these ani mals. Conventionally, these genes can be divided into four groups: factors of the myogenic program (twist, myoD, mef2, mox); blastoporal/mesendodermal genes (foxA, goosec oid, brachyury, gata); markers of germline cells and multipotent/stem cells (GMP genes (germline multipotency program)— vasa, piwi, nanos); segmentation genes (delta, notch, hairy, eve, runt). BIOLOGY BULLETIN
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Each of these groups of genes requires special anal ysis. The basic information about the localization of expression of the majority of these genes is summa rized in the table. We will review only the first group of factors as the most specific and, apparently, related to the most ancient part of the developmental program of mesoderm derivatives. In the gastropod P. vulgata, the mRNA signal of twist acquires reliable cellular localization at the early trochophore stage (Nederbragt et al., 2002). In accor dance with the fate map, the authors associate the expression of twist with some of the descendants of ectomesodermal micromeres 2b, 3a, and 3b. Based on this, we can conclude that the twist homolog is not involved in the processes of the mesoderm specification, but this gene participates in the differentiation of some mesodermal derivatives, presumably in myogenesis. Research on the leech Helobdella robusta showed the presence of maternal mRNA and protein product of the twist gene in the oocyte (Farooq et al., 2012). In the course of development, these products are local ized in the teloplasm and are mostly inherited by the D blastomere lineage, but further, no tissuespecific distribution of them in mesodermal blastomeres is observed. In the genome of the polychaete Capitella, two homologs of the twist gene were revealed, but none of them is expressed before the completion of gastrula tion (Dill et al., 2007). In trochophore larvae, the expression is present in the mesodermal bands, head mesoderm (ectomesoderm), and some ectodermal derivatives. The results are interpreted by Dill et al. (2007) as evidence that the twist homologs in Capitella cannot participate in mesoderm specification, but play a role in its further patterning and differentiation. In the nereid P. dumerilii, the homologs of twist and myoD have solely mesodermal specificity, while mef2, in addition to myogenic cells, is expressed in the head ectoderm (Steinmetz, 2006). The only representative of Spiralia in which the expression of homeodomain TF Mox has been studied is the gastropod Haliotis. The spatial distribution of mox mRNA in the trochophore of Haliotis is limited to the internal mesodermal cells, organized in the form of two bilaterally symmetrical bands (Hinman and Degnan, 2002). Unfortunately, owing to the poor knowledge of the embryology of Haliotis, the authors failed to link the expression pattern to a specific cell lineage (ecto or endomesoderm). According to our data, obtained for A. virens and P. dumerilii, the homologs of twist and mox are tissue specific solely for mesodermal cells, and expressed in both ecto and endomesoderm (Figs. 1e–1h). During gastrulation mRNA of twist was detected in the cells descendants of 4d and 3a–3d, arranged around the blastopore. In the larval development of the nereids, the expression of twist and mox in the endomesoderm assumes a metameric character. In trochophores, the BIOLOGY BULLETIN
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expression domains of twist and mox overlap greatly, whereas in metatrochophore larvae twist transcripts are localized in the developing parapodial muscles (laterally), while mox transcripts are localized in the ventrally arranged anlagen of oblique muscles and/or coelomic sacks. A similar refinement of the expression domains, evidently reflecting the progressive determi nation, is observed during caudal regeneration. Thus, the molecular profile of the mesodermal derivatives, which differentiate in the muscle direc tion, is largely similar in the representatives of all three branches of bilateral animals (Spiralia, Ecdysozoa, and Deuterostomia). However, to understand the degree of conservation of the entire myogenic pro gram, the role of each factor and its position in the hierarchy has yet to be determined using functional methods. ACKNOWLEDGMENTS This study was supported by St. Petersburg State University (grant 1.38.209.2014) using the equipment of the RRC MCT SPbSU. REFERENCES Ackermann, C., Dorresteijn, A., and Fischer, A., Clonal domains in postlarval platynereis dumerilii (annelida: poly chaeta), J. Morphol, 2005, vol. 266, no. 3, pp. 258–280. Amiel, A.R., Henry, J.Q., and Seaver, E.C., An organizing activity is required for head patterning and cell fate specifi cation in the polychaete annelid Capitella teleta: new insights into cell–cell signaling in Lophotrochozoa, Dev. Biol., 2013, vol. 379, no. 1, pp. 107–122. Anderson, D.T., Embryology and Phylogeny in Annelids and Arthropods, Oxford: Pergamon Press, 1973. Baylies, M.K. and Bate, M., Twist: a myogenic switch in Drosophila, Science, 1996, vol. 272, no. 5267, pp. 1481– 1484. van den Biggelaar, J.A.M. and Guerrier, P., Dorsoventral polarity and mesentoblast determination as concomitant results of cellular interactions in the mollusk patella vulgata, Dev. Biol., 1979, vol. 68, no. 2, pp. 462–471. van den Biggelaar, J.A.M., Cleavage pattern in embryos of Haliotis tuberculata (Archaeogastropoda) and gastropod phylogeny, J. Morphol., 1993, vol. 216, no. 2, pp. 121–139. Boyer, B.C., Henry, J.Q., and Martindale, M.Q., Dual ori gins of mesoderm in a basal spiralian: cell lineage analyses in the polyclad turbellarian Hoploplana inquilina, Dev. Biol., 1996, vol. 179, no. 2, pp. 329–338. Boyle, M.J. and Seaver, E.C., Developmental expression of foxA and gata genes during gut formation in the polychaete annelid, Capitella sp. I, Evol. Dev., 2008, vol. 10, no. 1, pp. 89–105. Buckingham, M. and Rigby, P.W.J., Gene regulatory net works and transcriptional mechanisms that control myo genesis, Dev. Cell, 2014, vol. 28, pp. 225–238. Burton, P.M., Insights from diploblasts; the evolution of mesoderm and muscle, J. Exp. Zool., 2008, vol. 310B, no. 1, pp. 5–14.
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Translated by N. Smolina
