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Background: Cell lineage studies in amphipods have revealed an early restriction of blastomere fate. The mesendodermal cell lineage is specified with the third cleavage of the egg. We took advantage of this stereotyped mode of development by fluorescently labeling the mesodermal precursors in embryos of Orchestia cavimana and followed the morphogenesis of the mesodermal cell layer through embryonic development. Results: The mesoderm of the trunk segments is formed by a very regular and stereotypic cell division pattern of the mesoteloblasts and their segmental daughters. The head mesoderm in contrast is generated by cell movements and divisions out of a mesendodermal cell mass. Our reconstructions reveal the presence of three different domains within the trunk mesoderm of the later embryo. We distinguish a cell group median to the limbs, a major central population from which the limb mesoderm arises and a dorsolateral branch of mesodermal cells. Conclusions: Our detailed description of mesodermal development relates different precursor cell groups to distinct muscle groups of the embryo. A dorsoventral subdivision of mesoderm is prepatterned within the longitudinal mesodermal columns of the germ-band stage. This makes amphipods excellent crustacean models for studying mesodermal differentiation on a cellular and molecular level. Developmental Dynamics 241:697–717, 2012. © 2012 Wiley Periodicals, Inc.
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- EXPERIMENTAL PROCEDURES
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Recently, several studies have demonstrated the advantages of amphipods as model organisms for comparative developmental biology. The feasibility of gene expression studies (e.g., Browne et al., 2006; Price and Patel, 2008), transgenesis (Pavlopoulos and Averof, 2005) and the availability of cell lineaging techniques (Gerberding and Scholtz, 1999; Gerberding et al., 2002; Wolff and Scholtz, 2002, 2006, 2008; Ungerer and Scholtz, 2008), and the cell ablation technique (Price et al., 2010; Alwes et al., 2011) are a unique combination of factors that qualify amphipods as a crustacean model for the study of morphogenesis and differentiation processes. The origin of all germ layers has been traced back to identified blastomeres of the 8- and 16-cell stage in Orchestia cavimana (Scholtz and Wolff, 2002; Wolff and Scholtz, 2002) and in a corresponding way (only the nomenclature of the blastomeres differs) in the marine species Parhyale hawaiensis (Gerberding et al., 2002). These studies reveal that the early cleavage pattern is holoblastic with early cell fate restriction. Part of this developmental pattern is that all mesodermal tissues descend from three different blastomeres of the eight-cell stage. Another notable characteristic of later amphipod mesoderm development is the occurrence of eight mesoteloblasts, four on each body half. These are large posteriorly situated cells that give rise to segmental mesodermal founder cells by repeated asymmetric divisions. It is assumed that the presence of eight mesoteloblasts is characteristic for all malacostracan crustaceans (Dohle and Scholtz, 1997; Scholtz, 2000). Furthermore, a formation of homonomous segments from a posterior growth zone consisting of mesoteloblasts and ectoteloblasts has previously been discussed as a feature shared between annelids and arthropods, supporting a clade “Articulata.” Nowadays the common consensus based mainly on molecular data is that these two groups are not closely related but fall into the two main protostome lineages Lophotrochozoa and Ecdysozoa (Aguinaldo et al., 1997). Until today the occurrence of ectoteloblasts and mesoteloblasts has only been shown in subgroups of annelids and arthropods. These are clitellates and, as mentioned above, malacostracan crustaceans (with a secondary loss of ectoteloblasts in amphipods; Scholtz, 1990). Therefore, a common phylogenetic origin of the teloblastic developmental mode in the two taxa must be doubted (reviewed in Scholtz, 2002). Nevertheless a developmental mode comprising a posterior growth zone can be assumed to be basal in arthropods as it is found in crustaceans as well as in myriapods and chelicerates (Anderson, 1973).
In O. cavimana the A macromere of the eight cell stage gives rise to the naupliar mesoderm while the b and d micromeres (Fig. 1A) are the mesendodermal precursors that give rise to the somatic mesoderm of the morphological right (d) and left (b) body half and some endodermal tissues (Wolff and Scholtz, 2002). Thus not only the germ layers but also the head and the somatic mesoderm are separated at a very early stage of embryonic development. With the following synchronous division to the 16-cell stage an extra-embryonic cell lineage is separated from the somatic mesendodermal lineage. Only the anterior daughters of d and b (cells da and ba of the 16-cell stage, see Fig. 1A) will give rise to mesendoderm (Scholtz and Wolff, 2002; Wolff and Scholtz, 2002). During formation of the germinal disc the embryo gastrulates through the sinking of mesendodermal cells beneath the ectodermal cells on the surface (Scholtz and Wolff, 2002; Wolff and Scholtz, 2002). Afterward, when the germ bands arise out of the germinal disc by transversal row formation within the ectoderm, four mesoteloblasts will appear on each body half. They subsequently produce the segmental mesodermal founders (Scholtz, 1990, 2002; Gerberding et al., 2002; Scholtz and Wolff, 2002; Wolff and Scholtz, 2002).
Figure 1. A: First cleavages of Orchestia cavimana. The mesodermal precursors are specified with the third cleavage. The post-naupliar mesoderm arises from the cells d (red) and b (green) of the micromere quartet. The A macromere (yellow) gives rise to the naupliar segmental mesoderm. With the next cleavage to the 16-cell stage an extra-embryonic lineage (bp and dp) is separated and the somatic mesoderm is only produced by the anterior daughters ba (green) and da (red). Both the left and right daughters of A (Ar and Al, yellow) give rise to naupliar mesoderm. Modified from Wolff and Scholtz (2002). B: A first indication of germ disc formation is a sickle-shaped arrangement (dotted line) of specific cells. The descendants of micromere a are slightly subsided. B′: Schematic illustration of the clonal composition of the sickle-shaped arrangement with the same color coding as used in A. Arrows indicate the cell migration process during germ disc formation. C: Early germ disc with a still-open gastrulation centre (Gc). C′: Schematic illustration of the clonal composition of an early germ disc using the same color coding as in A. The internalized mesendodermal cells are overgrown by ectodermal cells coming from blastomeres Ba and Da (grayish translucent). Arrows indicate the migration process during germ disc formation. D: Advanced germ disc with a closed gastrulation centre. Dashed line indicates the germ disc area where the tissue is multi-layered. Arrow indicates the anteroposterior axis. D′: Schematic illustration of the clonal composition of the mesendodermal cells underneath the ectoderm (not drawn) with the same color coding as used in A. Arrows indicate the migration process of descendants of bae and dae.
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In amphipods and all other peracarid groups investigated until now the post-naupliar germ band, including the mesoteloblasts and their segmental offspring, is formed on the egg surface (see Dohle, 1970, 1972). It is assumed that the peracarid germ band has evolved from a tubular structure as it is found in other malacostracan crustaceans (Scholtz, 2000, 2002). The germ band is characterized by a grid-like arrangement of ectodermal cells that undergo a well-understood series of cleavages before segments are formed (Weygoldt, 1958; Scholtz, 1990). This arrangement gives us the possibility to study the propagation of the somatic mesodermal cells starting from a well-defined cellular context. Insights gained into the following spatial patterning of the mesoderm will offer the possibility to set it into context of what is known about ectodermal patterning, for example during limb formation in the Orchestia embryo (see Wolff and Scholtz, 2008) and how the pattern relates to later distribution of muscle precursors (as described for isopods by Kreissl et al., 2008) and the architecture of the differentiated musculature of the O. cavimana embryo itself.
Understanding the mesodermal patterning process in O. cavimana will allow comparisons on different phylogenetic levels. Within the cleavage pattern of the mesoteloblast daughter cells, which are the mesodermal founder cells of each segment, some deviations in the orientation and timing of the divisions between different peracarid groups have already become evident (Dohle, 1970, 1972). Comparing closely related species and identifying differences enables us to evaluate how much species-specific variation is possible within a division pattern and what the remaining conserved aspects are.
When considering the broader phylogenetic context among arthropods, we assume a segmental formation out of a posterior growth zone, as found in crustaceans and short germ insects (Fränsemeier, 1939; Ullmann, 1963; Sommer and Tautz, 1994) to be characteristic. In the insect model organism Drosophila melanogaster, a dorsoventral subdivision of the mesoderm during later development has been characterized (Baylies and Bate, 1996). But to understand the evolution of this patterning system within arthropods, studies on representatives of other arthropod lineages are essential. Our description of how the mesodermal units in O. cavimana give rise to different mesodermal organs along the dorsal-ventral axis will form the basis for understanding mesodermal subdivision in a crustacean representative.
To visualize the patterning process, we labeled the mesodermal precursors and raised the embryos up to the desired stages (as defined by Ungerer and Wolff, 2005; see Table 2). Embryos were fixed and analyzed up to just before muscle differentiation (stage 4). In addition, to obtain a picture of the differentiating musculature as the main mesodermal derivative we carried out a phalloidin staining of F-actin on late stage embryos.
Our morphological analysis complements first data on expression patterns of mesodermal genes as described in P. hawaiensis (Price and Patel, 2008, Vargas-Vila et al., 2010) and a combination of both will allow a more comprehensive comparative approach toward the understanding of mesodermal patterning in crustaceans and arthropods in general.