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Keywords:

  • cell lineage;
  • gastrulation;
  • germ layer differentiation;
  • embryogenesis;
  • mesoderm

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

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).

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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.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Gastrulation and Germ Disc Formation

During early development a characteristic sickle-shaped arrangement of distinct cells occurs (see Wolff and Scholtz, 2002). This first indication of embryonic differentiation starts with the cellular transformation of blastomeres ba and da at 16-cell stage.

Within the fifth cleavage cycle (from 16- to 32-cell stage) ba and da divide along the contact zone with the a-descendants into an inner cell bai/dai and outer cell dae/bae (Fig 1B,B′). During this division cycle they excorporate most of their yolk and the cells (bai/bae and dai/dae) become somewhat rectangular in shape. Due to the smaller amount of the purple yolk they are brighter than neighboring cells (at frame 250 of Supp. Movie S1, which is available online). This gives a distinct and clearly visible landmark consisting of bai/bae and dai/dae as a slightly convex-curved cell band posteriorly adjacent to the descendants of micromere a (at frame 250 of Supp. Movie S1). Supp. Movie S1 shows that the yolk excorporation can occur before the fifth division cycle at (blastomere ba, indicated by a green cube, at frame 60-230 of Supp. Movie S1) or after the cycle (blastomere da, indicated by a red cube, at frame 90-250). In contrast to the prominent daughters of ba and da, their sister cells bp and dp (see Fig. 1A) produce ordinary blastodermic cells which are slightly elongated (bp ′″ and dp ′″ in Fig. 2A,B).

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Figure 2. Early mesodermal row formation. A: In vivo labeling of micromeres b and d at eight-cell stage. Clones descending from the ba and da micromeres become overgrown by adjacent ectodermal cells (grayish lines), which start to form the germ disc. Descendants of bp and dp (white stars) remain as slightly elongated yolky cells anteriorly in front of the emerging germ disc. B: Light microscopical image of the same embryo as in A. The black lines indicate the cells which originate from micromeres ba and da (compare with A). Descendants of bp and dp (bp′″ and dp′″, encircled with white line) are bigger and contain much more yolk (purple color). C: From the most posterior situated bae- and dae- clones the mesoteloblasts (arrows) become specified. D: Two segmental daughters have been produced by the first divisions of the MT123, forming the most anterior row m(1). E: The most anterior row m(1) is produced by the MT progenitor cells and lies beneath the ectodermal unit E(2). Beneath E(1) only endodermal cells (en) are evident. F: During the proceeding teloblastic divisions MT2 is in general the first to enter mitosis. MT4 is the last to divide; divisions of this cell are delayed by up to one cell cycle. m(2) is filled up to a total number of four cells by the division (arrow) and posterior migration of one daughter cell of the median cell in m(1). The two lateral cells in m(2) both originate from MT4. G: The anterior rows start to drift apart from one another, in accordance with ongoing elongation of the overlying ectodermal germ band. A network of cytoplasmic bridges is retained between the mesodermal cells. At the shown stage cells of m(1) and m(2) are clustered in one group. The lateral cell of m(1) divides and gives rise to a lateral daughter (arrowhead). H: Separation of the anterior cell cluster originating from rows m(1) and m(2) (arrow). I: a–c show the generation of the mesoteloblasts as described by Dohle and Scholtz (1997) and Price and Patel (2008) and confirmed by our observations. d–f are based on E–H and additional microscopic observations. Segmental daughter cells are shown in light red and the mesoteloblasts in dark red. See text for further explanations. Anterior is up. A,C–H: Fluorescent pictures of DiI-labeled mesodermal cells in live embryos of early germ band stage. C and E Fluorescence images superimposed on light microscopy images. Sketches indicate labeled blastomeres. I: Schematic representation of early anterior mesodermal row formation. ml, midline; me, mesendoderm; en, endodermal cells; MT1-4, mesoteloblasts; m(1), mesodermal row 1 (posterior-following rows numbered accordingly); E(1), ectodermal genealogical unit 1 (posterior-following named accordingly); mc1-4, mesodermal column 1-4; produced by mesoteloblasts MT1-4.

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Subsequently, the descendants of macromeres Ba and Da which are adjacent to the A-quadrant also start to excorporate their yolk. They become smaller and extend the cell band of bai/bae and dai/dae to a sickle-shaped and strong convex-curved cell band (Fig. 1B,B′; at frame 280-380 of Supp. Movie S1). This sickle-shaped cell formation contains descendants of micromere a and macromere A inside its curvature. Both are easy to distinguish, a-descendants represent the smallest cells in this arrangement and are located directly posterior to the ba and da descendants (Fig. 1B). The A-descendants are, compared with a-descendants, larger cells with some purple yolk still visible at the cell surface, and in addition they are characterized by the smooth edge of their white periplasm compared with the more fringed periplasm of the surrounding cells (descendants of other macromeres) (e.g., yellow spheres at frame 300 of Supp. Movie S1).

Concurrently, more descendants from Ba and Da migrate toward the sickle formation until maximum extension has been reached. At the same time four a-descendants and 8 to 10 A-descendants are inside the sickle formation (Fig. 1B, frame 300 of Supp. Movie S1). Compared with neighboring cells, the a-descendants have a conspicuous depressed position (arrow in Fig. 1B). This ingression and change of relative positions within the forming germ disc represents the first indication for the gastrulation. This is followed by the essential step of gastrulation. Cells coming from macromeres B and D conduct an epibolic migration toward the virtual centre of the sickle shaped cell formation (Fig. 1C,C′,D,D′). Coming from a lateral position, descendants of Ba and Da (now ectoderm) overgrow the mesendodermal cell layer (clones of ba, da, and A) and the germ progenitors (a) (Fig. 1B,B′,C,C′). From the posterior some C-descendants (Cr and Cl) also have a minor posterior contribution to the formation of the germ disc.

The ectodermal cells continue their epibolic growth until the tissues coming from left (Da-descendants) and right (Ba-descendants) meet medially. At this time a slit-like gastrulation centre is visible (Fig. 1C). During the germ disc formation, the clones of ba and da divide and extend the cell band (Fig. 1D). Cells from the A-quadrant (A, a) are slightly delayed in division and undergo a maximum of one cell cycle and double their cell number (Fig. 1D).

More cells coming from Ba and Da, which are at the border of the early germ disc, excorporate their yolk and attach to the germ disc. The germ disc cells also divide so that its diameter is constantly growing. At this time, the open gastrulation centre gets closed by descendants of Ba and Da from posterior to anterior (Fig. 1D). Only a whitish area in the anterior part of the germ disc designates the double-layered construction of mesendoderm (lower level) and ectoderm (upper level) (black dashed line in Fig. 1D).

Descendants of ba and da migrate posterolaterally, passing the germ cells (a-clones) and lie at the same level as the A-descendants (Figs. 1D′, 2C). Some cells of bai and dai separate from the cell cluster and migrate slightly further than the remaining cells (Figs. 1D′, 2C). These cells are of particular interest because they are the precursors of the somatic mesoderm.

In summary, the gastrulation begins with an ingression of a-clones and is followed by epibolic migration of ectodermal clones of Ba and Da. The migration process also causes the condensation and formation of the germ disc. Finally, the mesendodermal cell layer (clones of ba, da, and A) and the germ cell progenitors (a) are internalized and covered by descendents of Ba (right) and Da (left). Descendants of C additionally contribute to the formation of the posterior part of the germ disc.

Mesodermal Patterning at Early Germ Band Stage: Post-naupliar Row Formation

The posterolaterally migrating cells (bae and dae) protrude further toward the posterior and finally give rise to two mesoteloblast mother cells on each side of the embryo (compare with Wolff and Scholtz, 2002). One gives rise to mesoteloblasts 1–3 (MT123) and the other is MT4 from the beginning onward (for nomenclature of the mesoteloblasts and their daughter cell rows see Fig. 2I). During our observations we found no indication of these two cells being sister cells, they most likely have a separate origin as already assumed by Dohle and Scholtz (1997). Somatic mesodermal patterning begins with the described specification of four mesoteloblasts on each body half of the embryo. Through iterated teloblastic divisions these cells will produce segmental rows, consisting of four cells each, which are the precursors of all segmental mesoderm (Scholtz and Wolff, 2002).

The first cleavage of MT123 leaves behind one big daughter cell that contributes to the most anterior mesodermal row M(1) (Fig. 2Ia). Then MT123 divides into MT3 and MT12 (Fig. 2Ib). One other segmental daughter cell, which will be assigned to the mesodermal row two, is produced by MT3 (Fig. 2D,E,Ic) and cell MT12 divides into MT1 and MT2. Simultaneously, MT4 starts its unidirectional divisions and leaves behind one segmental founder contributing to m(1). Hence m(1) initially consists of two cells (Fig. 2D,E,Ib-e). MT4 continues its teloblastic divisions producing a coherent longitudinal cell column. Notably there are two cells produced by MT4 that are assigned to mesodermal row two (m[2]). In addition to the one cell produced by MT3 (see above), m(2) initially comprises three cells (Fig. 2D,G,Ic). A little later this row is completed up to a number of four cells by a first division of the segmental daughter cell of MT123 in m(1) (cell m1 of row m(1), nomenclature of the segmental daughter cells after Price and Patel, 2008). This cell divides in the anterior–posterior direction (Fig. 2F,G,Ie). The posterior daughter cell m1p moves more posteriorly and attaches itself to row m(2) (Fig. 2Ie,f). At germ band stage the migration of this cell is the only example where an early mesodermal cell leaves the genealogical unit of its origin. The anterior daughter cell m1a remains in m(1), which at this point still consists of two cells. Hence row m(1) is the only row that is built up by only two founders, in contrast to four cells in every posteriorly following row. Four founders are also present in row m(2), although the origin of the cells is slightly different, as described above. Cells of the rows m(1) and m(2) form one cluster in the beginning (Fig. 2G,Ie). They are only separated from each other at a time in development when several (approx. 11) more mesodermal rows have been formed posteriorly (Figs. 2H, arrow, If).

The mesodermal row m(3) is the first that is generated by regular unidirectional divisions of the complete set of four mesoteloblasts. It underlies the ectodermal unit E(4) that corresponds to the second thoracomere (first walking leg). The same process is repeated to produce every posteriorly following row (see Wolff and Scholtz, 2002). This means that one longitudinal mesodermal column (mc 1-4, see Fig. 2I) is produced respectively by MT1, MT2, MT3, or MT4. When considering the divisions from the production of m(3) onward, MT4 is always delayed by one division with respect to the other mesoteloblasts. A second peculiarity of column mc4 (produced by MT4) is its initial position at some distance from the other three columns, which are arranged closely to each other (Fig. 2E–G). This phenomenon disappears during further development and mc4 also aligns more closely to the remaining columns (Fig. 2H). By the described process 16 mesodermal rows are formed. Each row, except for m(1), consists of eight mesoteloblasts (mesodermal founder cells; four on each body half) that will give rise to the segmental mesodermal organs during the ensuing development.

Mesodermal cells relatively close to the mesoteloblasts show an interesting feature. Cytoplasmic bridges make cell–cell connections to longitudinally adjacent cells (having their origin in the same mesoteloblast) and transversally adjacent cells (nonsister cells which form one transversal row/hemi-segmental unit). This means that every mesodermal cell within the left and the right body half is in contact with its direct neighboring mesodermal cell (see Fig. 2I). These plasma bridges are most likely needed for cell–cell communication (Figs. 2F–H, 4B, yellow arrows, 4C). Due to the process of germ band split and longitudinal extension of the embryo, the mesodermal rows drift apart from one another. However, there are still cytoplasmic bridges that connect the mesodermal cells (see Figs. 2G,H, 4B, yellow arrows, C). The connections between different rows dissolve on initiation of ectodermal segment differentiation. Between cells of one row they appear to be retained (Fig. 5, arrow), preceding the close contact of cells of one row that is observed later on.

In summary, the initial patterning of the most anterior mesodermal units m(1) (mesoderm of the second maxilla) and m(2) (mesoderm of the maxilliped) is peculiar as the complete set of mesoteloblasts is not differentiated at the time these rows are formed. The m(3) and posterior-following mesodermal units are formed very stereotypically by the four mesoteloblasts. A network of cytoplasmic bridges connects all early mesodermal cells with their lateral and longitudinal neighbors.

Clonal Split Between the Naupliar and Post-naupliar Mesoderm

The amphipod trunk segments are prepatterned ectodermally during germ band stage, first, by an alignment of cells forming the grid like pattern of the early germ band. This process proceeds from the anterior margin of the post-naupliar germ band toward the posterior. The primary rows undergo two mitotic waves with anterior–posterior orientated spindle axes. By this means the genealogical units of the germ band are produced, with each consisting of four cell rows (Scholtz, 1990). During the following segment formation the segmental furrows will be drawn in between the anterior and posterior daughters of ectodermal row b, whereas the anterior cells of the unit contribute to the posterior part of the segment and vice versa. This is true for the units E(2) and all posterior following units. The genealogical unit E(0) produces the mandibular segment in a direct manner (see Wolff and Scholtz, 2006). With the posterior following unit E(1) the shift from direct to parasegmental segment formation takes place. The segment of the first maxilla is produced by all four ectodermal cell rows of E(1) and the two anterior rows of E(2) (Dohle, 1976; Scholtz, 1990; Scholtz et al., 1994; Dohle et al., 2004; see Fig. 9A). The anterior-most mesodermal row m(1) is produced by division products of MT4 and the shared progenitor cell of MT1, 2, and 3 (MT123) beneath the ectodermal unit E(2). No clones of this cell lineage were found underneath E(1), which corresponds to the segment of the first maxilla (Figs. 2E, 3D). The cells of m(1) underlie the posterior portion of E(2) and are later integrated into the segment of the second maxilla. As DiI staining in later stages shows (see Fig. 3E,F), the mesoderm of the first maxilla does not stem from the mesoteloblasts but from the naupliar mesodermal lineage which goes back to the macromere A of the eight cell stage (Wolff and Scholtz, 2002).

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Figure 3. Development of the anterior (naupliar) mesoderm. A: Embryo at the stage of early germ band formation. First transverse ectodermal rows are evident (indicated by arrows). Underneath the ectoderm of the head region (borders indicated by white lines) a field of undifferentiated mesendodermal cells (nme) is present. Black arrows point to the beginning formation of transverse rows in the post-naupliar ectoderm. B: Slightly more advanced germ band stage than in A. The formerly homogenous sheet of naupliar mesendodermal cells becomes scattered into small groups. Some of the groups (arrowheads) populate the presumptive head area (medially to the yellow line). One single mesodermal cell situated in the centre of the head area (arrow). C: Labelled naupliar mesendodermal cells (nme) on the morphological left side, opposite labeled post-naupliar mesodermal cells (pm) on the right side at early germ band stage. Naupliar mesendodermal cells are found much more anteriorly than cells of the post-naupliar mesoderm, but areas where endodermal cells (en) descending from bp are found overlapping with the area of naupliar mesendodermal cells. D: Early stage 1 embryo. Mesodermal descendants of bp or dp are found underneath the second maxillary segment (mx2). No clones are found in the mandibular (md) and first maxillary segment (mx1). E: Stage 1 embryo, dorsal view with mesodermal cells lying on top (dorsal view). Naupliar mesendodermal cell groups protrude into the forming segment of the mandible (md) and first maxillary segment (mx1) (arrowheads). More mesendodermal cells are found laterally in the area of anlage of the midgut glands (mgg) and the anterior head segments (asterisks). A single cell is positioned posteromedially in the head region (arrow, compare with B). F: Late stage 1 embryo. This embryo represents an example in which large numbers of mesodermal cells are also found in the head area of the other body half (only the left precursor has been labeled). Mesodermal cells start to cluster within future head segments (white circles, a1, a2, md). Mesodermal cells in the mandibular and maxillary segments are not clearly subdivided and cross the midline. Laterally to the head segments the midgut gland (dashed line, mgg) of the left body half is mainly formed by descendants of the marked blastomere. Note that single clones are also found in the second maxillary segment (mx2). Anterior is up. Sketches indicate labeled blastomeres. A–C: Epifluorescence images of live embryos. A,B: Epifluorescence superimposed on light microscopy images. Approximate presumptive head area between yellow lines (A,B), start of post-naupliar germ band indicated by white lines. D–F: Confocal microscopy image stack reconstructions of dissected germ bands. nme, naupliar mesendoderm; ml, midline; pm, post-naupliar mesoderm; en, endoderm; MTs, mesoteloblasts; m(1), post-naupliar mesodermal row 1; md, mandibular segment; mx1, first maxillary segment; mx2, second maxillary segment; a1, first antennal segment; a2, second antennal segment.

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The mesoderm itself shows no parasegmental pattern. Each row produced by the mesoteloblasts gives rise to the mesodermal portion of one later segment. After the two mitotic waves within the ectodermal units each mesodermal row is normally found beneath row c of one unit. The mesodermal row is later incorporated in the segment to which the posterior ectodermal rows (c and d) will give rise to (Fig. 4B,C).

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Figure 4. Cleavage pattern of the mesodermal cells at germ band stage (#1). A: Overview of cleavage pattern of the mesodermal cells within a germ band. A developmental progression from posterior to anterior is evident. Founders in mesodermal segments m(1) and m(2) have already given rise to multiple mesodermal cells. The two rows are still connected by cells of mc4 in the lateral part (arrow). The median cell of m(2) has become separated from the remaining cells. Cells of the more posterior rows (m(3) and following) undergo a stereotyped division pattern (sister cell relationship is indicated by white bars). See text for details. After the founders of mc1, mc2, and mc3 have divided at least once, cells originating from these columns move closer to each other (here seen in m(3)); finally they will form one cluster with only the cell of mc1 one being transiently detached from it. This pattern is not seen in m(1) but in m(2). B: Detail of a sequence of four mesodermal rows and the relative position of the mesodermal cells toward the ectoderm. The brackets on top and bottom of the figure indicate the area from where the limb bud will initially grow out (cells originating from ectodermal columns E(3-6) (see Wolff and Scholtz, 2008). Beginning segment differentiation becomes evident by the pattern of differential cleavages within the ectoderm and by the appearance of segmental furrows anterior and posterior to one mesodermal row (position marked by yellow arrows). See text for description of the exact positions of mesodermal cells in relation to the ectoderm at this time point. C: Dorsal view, anterior part of the germ band of a slightly advanced developmental stage when compared with A and B. m(1) and m(2) are still connected with one another by means of cells of mc4 (arrowhead). The median cell in m(2) has produced a cluster of three cells (double headed arrow). In the more posterior rows, cells of mc4, mc3, and mc2 form clusters, with the cells being closely connected to each other (here seen in m(3) and m(5)). These assemblies are preceded by the formation of broad cytoplasmic bridges that stretch toward the cells of the neighboring column (m(5), m(4), arrows). Cells of mc1 remain in a median position at some distance to the other ones. Anterior is up, sketches indicate labeled blastomeres. A: Epifluorescence image, labeled mesodermal cells in live embryo. B,C: Dorsal view at dissected germ bands, reconstructions of confocal image stacks. en, endodermal cells; m(1), mesodermal row 1 (following named accordingly); ml, midline; red: DiI, turquoise: nuclear stain.

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The Naupliar Mesoderm at Early Germ Band and Beginning Segmentation Stage

The somatic mesoderm is produced by the mesoteloblasts and shows a highly stereotypical pattern (see above). The spatial starting point of this system is marked by a clear line: the daughters of the mesoteloblasts are exclusively found in the second maxilla and posterior-following segments. The remaining naupliar (head-) mesoderm has a different ontogenetic origin than the post-naupliar (somatic) mesoderm (Wolff and Scholtz, 2002).

Macromere A of the eight-cell stage is responsible for the formation of the whole naupliar mesoderm and median parts of the endoderm. This means that the descendants of macromere A are responsible for the mesoderm in segments of the first and second antenna, the mandible and first maxilla (Fig. 3E,F).

It is notable that at early germ band formation this arrangement is never completely symmetric (Fig. 3A). In contrast, the cell lineage of the early somatic mesoderm is separated from the endodermal line by differentiation of the mesoteloblasts and therefore easier to identify. Based on morphological characteristics it is much harder to distinguish the naupliar mesoderm from endodermal cells within the early germ band (Fig. 3A–C,E). This is the reason for using the term mesendoderm at this developmental stage. During ongoing development endodermal cells form massive cell sheets laterally within the embryonic midgut gland anlagen (white dashed lines in Fig. 3E,F). The mesodermal cells of the head arrange in units that are assigned to single segments (Fig. 3F), but not as clearly distinct from one another as realized in the post-naupliar mesoderm.

Additionally, A-clones are together with ba- and da- clones responsible for the formation of the midgut glands (endoderm) (e.g., Fig. 3A,E,F). In some labeling experiments, we found A-clones posterior to the segment of the second maxilla but we could not connect these cells to mesodermal tissues (see e.g., Fig. 3F).

Another interesting aspect is that within the patterning of the head mesoderm (derived from macromere A) there is a less strict separation of the left and right parts of the later germ layer with respect to the two progenitor cells at 16-cell stage (see Fig. 1A). Whereas in somatic mesodermal patterning the right precursor ba always exclusively produces right mesoderm and the left precursor da accordingly only left mesoderm, the separation of the macromere A into the right Ar and left Al daughters in the 16-cell stage is less strict. They do mainly contribute to their corresponding body half, but often clones originating from Ar are also found beneath the left part of the germ band and daughters of Al under the right part (e.g., Fig. 3F). This might be related to the fact that Ar and Al together form a common cell cluster which remains undivided throughout the germ disc development (see Fig. 1) and only starts to split when the naupliar segments start to differentiate. Instead, the precursors for the post-naupliar mesoderm, bae and dae, are already spatially separated before germ disc formation (see Fig. 1). In summary, the patterning mode of the naupliar mesoderm is less stereotyped at the cell lineage level and is characterized by cell migration and a final partitioning of a primarily uniform mesendoderm.

Post-naupliar Mesodermal Patterning During Late Germ Band Stage and Beginning Segment Differentiation (Stage 1 and 2)

With a developmental gradient from anterior (more developed) to posterior (less developed), the mesodermal cells in the anterior part of the germ band start to divide differentially, thus enlarging the number of mesodermal cells in each segment (Fig. 4).

The pattern seen in m(1) and m(2) differs from the one seen in more posterior segments, because the initial set of founders has also been different. In m(1), which initially consisted of two founders, one smaller cell is produced by cell m(1)2 laterally, lying in one row and being connected to the two cells produced by mesoteloblast four in m(2) (Fig. 2G,I). As described above, cell m(1)1 produces one posterior daughter that initially stays connected to it. Through these two bridges the two anterior rows stay connected for a while. With the movement of the posterior daughter cell (m1p) of row m(1) beneath the ectodermal unit forming the maxilliped, the rows are separated medially (Fig. 2H, arrow), but the connection of the cells in the lateral part is maintained until late germ band stage (Figs. 2H, 4A,C). Cells of both rows are dividing, they form units of a more compact shape compared with the posterior-following rows. The median cell of row m(2) gives rise to two more daughters, this cell cluster then consists of three cells and is detached from the other cells of the row (Fig. 4C, double headed arrow).

The more posterior mesodermal row m(3) and the following rows are produced by regular iterated divisions of all four mesoteloblasts. The cleavage pattern is more uniform than in the anterior rows. There is a fixed sequence in the divisions of cells belonging to one row and a typical orientation within the division of each segmental founder. Due to the developmental gradient from anterior (more developed) to posterior (less developed) the sequence of divisions can be studied by comparing one mesodermal row with the anterior-following ones. In most cases, the first round of mitosis starts with the division of the cell of mc3 immediately followed by the cell of mc2. In some cases, the mc2 cell can be the first to divide (Fig. 4A,B). The next cell that divides is the most lateral cell of mc4 (Fig. 4A–C). After some delay, the most median cell of mc1 divides. The divisions of cells of the columns mc2, mc3, and mc4 are generally oriented in the anterior–posterior direction but oblique to the longitudinal axis, whereby one daughter is produced toward anterior-lateral. Despite the oblique orientations of the divisions, in most cases the two daughter cells will be arranged in front of each other, parallel to the anteroposterior axis later on (Fig. 4B,C). This requires that the cells rearrange with respect to the longitudinal body axis after the division.

Initially, the lateral-most cells of mc4 are situated some distance from cells of the adjacent mc3. This arrangement changes with proceeding segment differentiation; the cells of mc2, mc3, and mc4 cluster together, and instead the innermost cells of mc1 are now positioned some distance from the remaining columns. An exception is m(1) where all cells form one compact unit (see Figs. 4A,B, 5A–D).

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Figure 5. Development of the mesodermal rows at stage 2 and 3. A: Overview of a stage 2 embryo with 15 of 16 mesodermal rows formed. The germ band split (gbs, indicated by white dashed lines) affects the segments mxp to pl1. Dye has become free in the central part of the embryo, especially in the area of the posterior conjunction of the two germ band halves (asterisk). B: The sequence of posterior rows of a stage 3 embryo illustrates the ongoing cleavage of the mesodermal cells. The rows in the posterior part show the same pattern of cell clustering of mc2, mc3, and mc4 and the separation of the median cells. Cytoplasmic connections are evident; they bridge the greater distance between mc1 and mc2 cells (arrow). C: Overview of mesodermal units as seen in context of the surrounding ectoderm. D: Reconstructed sections through the segmental mesodermal units (developmental sequence of m(8) to m(11) as seen in C), posterior view. Segment m(11): cells of mc2-mc4 have not yet clustered together, they lie in a flat line above the ectoderm. The ectoderm itself shows only a slight bending ventrally, indicating the beginning of limb bud formation. This is separated from the median cells surrounding the mc1 cell by a slight inward vaulting (arrow). This marks the later border between lateral limb bud and more median tissues. Segment m(10): cells of mc2-mc4 are connected. The bending of the ectoderm and the bordering furrow (arrow) become more pronounced but the outgrowth of the bud has not yet started. Segment m(9): The limb (lb) starts budding out and cells of mc2 and mc3 start to extend into it (white arrow). The mc1 cells stay medially outside the limb bud and cells of mc4 flank the limb bud laterally. Segment m(11): Mesoderm cells proceed into the outgrowing limb bud. The mesoderm does not fill the most distal part of the limb bud (double headed white arrow). Anterior is up, sketches indicate labeled blastomeres. A: Fluorescence signal of DiI labeling, superimposed on a light microscopy image of live embryo. B: Epifluorescence image of DiI signal in posterior mesodermal rows in live embryo. C,D: Confocal microscopy image stack reconstructions of dissected embryos. ml, midline; gbs, germ band split; mx2, second maxilla; mxp, maxilliped; pe1-7, pereomere 1-7; pl1-6, pleomere 1-6; mc1-4, mesodermal column 1-4; lb, limb bud; red: DiI, turquoise: nuclei stain.

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As shown in Figure 5A, the left and right half of the germ band split during development (see Ungerer and Wolff, 2005). After this process we often found a weak signal of the vital marker DiI in the centre of the embryo. This phenomenon is especially observable in the posterior region where the embryo starts its ventral closure after the split (Fig. 5A). We interpret this acellular signal as a result of decrease of volume of some cells within the visceral or somatic mesoderm, which may be related to the unknown mechanism of germ band split or closure. At this stage an anterior–posterior developmental gradient still characterizes the embryo (Fig. 5A). The sequence of mesodermal rows presented in Figure 5B shows the proceeding proliferation of the mesodermal cells. mc2 and mc3 each comprise three cells, then the clusters establish closer contact to the neighboring columns until mc2, mc3, and mc4 cells have formed one unit. This group of cells gains a prominent appearance by consisting of at least two cell rows in the longitudinal direction (Fig. 5B,C) and also by enlarging along the dorsoventral axis (Fig. 5D). At this developmental stage limb buds start to bulge out from the ectodermal germ layer. During this process the segmental mesoderm coming from mc2 and mc3 fills the limb anlagen with muscle progenitor cells (Fig. 5D). Cells originating from mc2 directly underlie the ectodermal columns that are first involved in limb development (compare with Fig. 4B) and contribute to distal parts of the appendage (Wolff and Scholtz, 2008), most likely contribute to the growing tip and distal parts of the limb mesoderm. But there is clearly a contribution of cells from mc3 (Fig. 5D) that underlie parts of the ectoderm which later become involved in limb development. These cells mainly contribute to basal lateral parts of the leg (see Wolff and Scholtz, 2008). To conclude from this, cells originating from mc3 also contribute mainly to the more proximal parts, and the whole limb mesoderm is a combined product of descendants of mc2 and mc3 (Fig. 5D). Cells of mc4 are also part of a major mesodermal cell group but they are situated more laterally and will most likely contribute to dorsal muscle groups and the heart. Cells of mc1 are clearly separated from the remaining cells and are not involved in limb mesoderm formation. At this stage they form a distinct median population of mesodermal cells (Fig. 5A–D).

With the exception of the two anterior-most rows, the mesodermal trunk units follow a very stereotypical patterning process. The first divisions of the segmental cells and the following dynamics of the segmental units are summarized in Figure 9B I-III.

Mesodermal Germ Layer at Limb Bud Stage and During Limb Growth (Stage 3 and 4)

The ensuing stage 3 is still characterized by the split of the left and right germ band halves. The limb buds are elongated but are not yet subdivided into podomeres (see Figs. 6A,C,D). The median mesodermal cell group coming from mc1 that was formerly separated has now moved toward the remaining cells and is attached to them (Fig. 6D). Thus the whole segmental mesoderm is in contact. Nevertheless, the mc1 cells are still flanking the median side of the limb anlagen, being situated ventrally beneath the sternites (Fig. 6D). The central cells, deriving from the mc2 and mc3, have further proliferated and extend into the elongated limb anlage. The largest number of cells is thereby found in the proximal part of the limb primordium (Fig. 6E, bracket), with only a few cells protruding distally (Fig. 6E, arrow). There is one characteristic mesoderm protrusion (Fig. 6D, double arrowheads) that fills the inner side of the coxa, which at this stage is marked by a vaulting of the ectoderm (Fig. 6D). Another less pronounced mesodermal protrusion is found beneath the developing coxal plate, a derivative of the coxa (Fig. 6D, cxp). Dorsolaterally to the growing limb the mesoderm reaches the developing tergites. These dorsal extensions are pronounced in the segment of the second maxilla and the pereopods, but not in the maxilliped (Fig. 6A, arrows) and also not yet in the posterior segments of the pleon (white arrows in Fig. 6A). The dorsolateral cells originate from the lateral mc4, while the cells closer to the limb bud must have their origin in mc3. A clear discrimination between cells from different columns is no longer possible as all somatic mesodermal cells of one segment are now closely connected. However, we consider it unlikely that cells within one segmental mesodermal unit intercalate and change their relative positions toward one another.

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Figure 6. Development of the mesodermal units at stage 3 and 4. A: Overview of stage 3 embryo. Anterior segments are at the stage of limb elongation whereas pleon segments are only at the stage of beginning limb bud formation. B: Dorsal view of a stage 4 embryo, overview of the pereon and first pleonal segments. The germ band halves are united again, the ganglia anlagen become evident along the midline (dashed white line). All mesodermal segmental units are now connected, forming a mesodermal band along the trunk (white arrows) and the pleonal segments have also reached the stage of limb outgrowth. C: Detail of the first three limbs containing trunk mesoderm (mx2, mxp, and pe1) from same embryo as shown in A. Insight into limbs from ventral. Arrows point to intersegmental connections. The first maxilla (mx1) is of a more compact shape than the more posterior limbs and is completely filled by mesodermal cells. In the mxp and pe1, the mesoderm does not reach into the distal parts but numerous cells are found in the basal parts. D: Transversal section through pereopod 5 of embryo shown in A. Black arrowheads indicate the origin from the different mesodermal columns of the mesodermal cells along the trunk and in the ventral limb outgrowth. Cells of mc1 are now reconnected with the remaining mesodermal unit forming a ventromedian protrusion from it (arrow). The basal parts of the limb are filled by a prominent mesodermal cell group. This fills the pronounced vaulting of the developing coxa (double arrowhead) and is also slightly protruding toward the anlage of the coxal plate (cxp). See text for details. E: Ventral view of pereopod 4 and 5 (pe4-5) of the same embryo as shown in B. The limbs have elongated and all podomeres are present. Gills (gi) differentiate medially at the proximal limb insertion (here shown for pe4). See text for details. F: Transection through a pleopod (pl3). The protopodit (pp, area marked by bracket) is filled by mesodermal cells. It is broadly connected to the mesodermal band of the trunk (white arrowhead). At this stage only the endopodit (enp) is filled by a single mesodermal cell (arrow) that comes from the major proximal population and is still connected to it. Anterior is to the left, sketches indicate labeled blastomeres. Reconstructions of confocal image stacks of fixed embryos. a1, segment of antenna 1; a2, segment of antenna 2; md, mandibular segment; mx1, segment of first maxilla; mx2, segment of second maxilla; mxp, segment of the maxilliped; pe1-7, segments of pereopods 1-7; pl, pleonal segments; gbs, germ band split; ml, midline; ter, tergite; mc1-4, mesodermal columns 1-4; cxp, coxal plate; cx, coxa; ba, basis; is, ischium; me, merus; ca, carpus; pr, propodus; da, dactylus; gi, gill; pl3, pleopod 3; pp, protopodit; exp, exopodit; enp, endopodit; red: DiI, blue/turquoise: blue/turquoise: nuclei stain.

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At this developmental stage a connection between the mesodermal units of adjacent segments is formed (Fig. 6C, arrows). These connections are established between the ventral parts of the units. In the subsequent stage 4, all segmental mesodermal units are connected through a continuous band along the trunk (white arrows in Fig. 6B,C). The limbs have now elongated and the pereopods are subdivided into podomeres. The limb mesoderm stays connected to the segmentally iterated trunk mesoderm (Fig. 6B,E). Most of the mesodermal cells of the limb are found in the proximal podomeres. The basis, ischium, merus, and carpus (Fig. 6E, brackets) in particular contain a large number of mesodermal cells. Out of this prominent group, single cells protrude further into the more distal segment of the propodus (Fig. 6E, arrow). The terminal dactylus remains free of mesodermal cells, as later it will not contain intrinsic musculature. Within the posterior biramous pleopods we find a pattern resembling that of the distribution of mesodermal cells. The proximal segment (Fig. 6F, pp/bracket) contains the major part of the mesodermal cells. Single cells extend into the distally bifurcated part of the limb, first into the endopodit (Fig. 6F, arrow). Here it becomes evident that the distal cells remain connected to the more proximal situated mesodermal cells through cytoplasmic bridges. This is probably also the case for the distal cells of the pereopod but is due to the smaller size of the cells not visible in the images.

Summing up, at late germ band and beginning limb differentiation stage the mesodermal units form coherent blocks of a tripartite shape, comprising a ventromedian cell population, a central part that forms the limb mesoderm and a dorsal extension (see also Fig. 9B III-V).

The Muscular System of the Embryo Before Hatching (Stage 6)

The muscular system of the amphipod O. cavimana is generated in the embryo before hatching. The first actin-positive strands can be detected in stage 5 embryos in the ventral trunk. A complete muscular system is detected in stage 6 embryos shortly before hatching (Fig. 7A). The muscular system can generally be divided into two parts. First is the head musculature with prominent muscle groups related to the feeding apparatus and the muscles of the first and second antennae. The second and more complex part is the trunk musculature (Fig. 7A). Within the segments of the walking legs the musculature can be subdivided into several serial homologous muscle groups. The same is true for the posterior pleopods, only differing in the specification of the limb musculature. The musculature of a pereon segment consists of a ventral median muscle (Fig. 7B,B′, yellow), extrinsic limb muscles connecting the basal leg segments with the trunk and the intrinsic muscles of limbs that connect the exoskeleton of one podomere with that which is distally-following (Fig. 7B,B′, green). The dorsal muscles of the trunk (Fig. 7B,B′, blue) consist of several muscles with longitudinal orientation; some are parallel to the longitudinal axis of the embryo whilst some are obliquely orientated.

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Figure 7. Reconstruction of the muscular system of a stage 6 embryo. A: Red: muscles (phalloidin), overview of complete embryo. The musculature within the head consists of major groups connected to the feeding apparatus. Within the pereon the segmental muscular units are highly stereotyped comprising dorsal trunk muscles (dtm, bracket), the musculature connecting the limbs to the trunk (extrinsic limb musculature, elm), the musculature of the limb basis (lmb) and distally-following podomeres and a ventral median muscle (arrow). B,B′: Color coded model of the musculature of a pereon segment. Colors reflect the most likely origin of the distinct muscle groups in the respective mesodermal columns. Gray: exoskeleton, red (coming from mc4): heart musculature, blue (coming mostly from mc3): dorsolateral trunk musculature, green (coming mostly from mc2): extrinsic and intrinsic limb musculature, yellow (coming from mc1): ventromedian muscle. elm, extrinsic limb musculature; lmb, limb musculature of the basis; dtm, dorsal trunk musculature; cx, coxa; ba, basis; is, ischium; me, merus; ca, carpus; pro, propodus; da, dactylus; ter, tergite.

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The dorsal vessel of the adult O. cavimana inserts at the rear edge of the cephalothorax and stretches to the seventh thorax segment. Ostia are found in segments number three, four and five, arterial openings toward the ventral side in segments four, five and six (Wirkner and Richter, 2007). The heart of the embryo gains its functional shape at stage 6 when the more ventrally-situated muscle groups are already differentiated. Inspection of an early stage 6 embryo showed the heart musculature before closing of the heart tube (Fig. 8A). Here, it becomes clear that the circular heart musculature is generated by a contribution of fibers originating from both body halves of the embryo and that they fuse at the dorsal midline (arrows in Fig. 8A). The bilateral character of the heart is also reflected in the structure of the musculature of the readily differentiated heart of the late stage 6 embryo (Fig. 8B,C). The fine circular fibers do not span the whole circumference of the heart but show fusion sites along the dorsal midline (Fig. 8B) and along the ventral midline (Fig. 8C). Muscle fibers are arranged in groups separated by narrow gaps (arrows in Fig. 8B,C), giving the heart a metameric appearance.

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Figure 8. Heart musculature of stage 6 embryos. A: Dorsal view of an early stage 6 embryo. The embryonic heart tube is closing along the dorsal midline (arrows). B: Dorsal view of the fully differentiated heart tube consisting of fine circular muscle fibers arranged in metameric groups separated by narrow gaps (arrows in B,C). Fibers do not cross the dorsal midline (dml) but are attached to the equivalent heart tube fibers of the other body half here. C: Ventral view of heart tube (same specimen as B). The circular fibers also do not run over the ventral midline. A weaker area of staining possibly indicates an attachment site to a matrix along the ventral midline (vlm) of the heart. dtm, dorsal trunk musculature; dlm, dorsal midline; os, ostia; ao, arterial opening; vml, ventral midline; white: nuclei stain, red: musculature (phalloidin).

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In conclusion, the musculature of the embryo comprises four groups (ventromedian muscles, limb musculature, dorsal trunk musculature and the dorsal vessel). Figures 7B,B′ and 9B illustrate the derivation of the muscle groups from different parts of the mesodermal units and therefore from different mesodermal columns that go back to the single mesoteloblasts.

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Figure 9. A: Distribution of mesodermal cells of the O. cavimana germ band in relation to the genealogical units and later segments (based on Scholtz et al. (1994) and Wolff and Scholtz, (2006) and this work). Light red indicates the head mesoderm, producing the segmental units through cells protruding into the naupliar segments. Dark red symbolizes the post-naupliar mesoderm units formed by the activity of the mesoteloblasts. The boundary of ectodermal naupliar and post-naupliar patterning coincides with the E1/E2 border, crossing the segment of the first maxilla (arrow). While post-naupliar mesodermal cells respect this border, cells of the naupliar mesoderm might occasionally be found posterior to it. B: Summary of segmental mesodermal development. Arabic numbers indicate parental mesoteloblasts. I, II: view from top, III-V: transection through mesodermal unit (muscle group). I: First round of differential cleavages at germ band stage. The mc2 cell is the first to divide, followed by the column three cell shortly afterward. Then the lateral mc4 cell divides. The mc1 cell divides last. II: Clustering of cells, late germ band (stage 1). Cells originating from the mc2-4 cluster together in one group. The most median cell from mc1 stays separated at this stage. III: Beginning of the shaping of the mesodermal units. Stage 2: mesoderm originating from mc2 and mc3 migrates in growing limb buds. IV: Tripartite shape of segmental mesoderm, stage 3-4: The previously separated median cluster is now attached to the inner side of the mesodermal unit. Hence, one unit consists of a median arm, a branch reaching into limb and one protruding dorsolaterally. From this initial architecture of the units, differentiation processes like myogenesis start. V After the differentiation of the musculature (mostly taking place during stage 5) the muscular system of the embryo close to hatching (stage 6) can be subdivided into four major groups: the ventromedian muscles (yellow), extrinsic and intrinsic limb musculature (green), dorsolateral muscle groups (blue) and the heart musculature (red). The muscle group median to the limbs derives from the inner branch of the tripartite mesodermal unit, hence from the mc1. Limb musculature derives in large parts from mc2 cells with a contribution of mc3 to laterobasal parts. Most of the mc3 offspring are found in the dorsolateral branch of the mesoderm and will give rise to the prominent dorsolateral muscle groups. The most lateral cells deriving from mc4 are involved in the formation of the dorsolateral trunk musculature as well, but by the time the bilateral mesodermal portions meet at the dorsal midline the most lateral cells will give rise to the heart.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Gastrulation and the Origin of Mesoderm

The first three cleavages and the arrangement of the blastomeres during the four-cell and eight-cell stages are typical of amphipod crustaceans, and are suggested to be apomorphic characteristics for the amphipods (Scholtz and Wolff, 2002). This is not the case for the following fourth cleavage. While in O. cavimana a stereotypic blastomere arrangement at 16-cell stage (Scholtz and Wolff, 2002; this study) occurs, in the amphipod P. hawaiensis several different arrangements of blastomeres are possible (Alwes et al., 2011). Also, the further development of P. hawaiensis is more variable and a sickle-shaped cell arrangement like in O. cavimana is not formed (Alwes et al., 2011). However, there are several processes of cell behavior and gastrulation that are very similar in both amphipod species (Scholtz and Wolff, 2002; Wolff and Scholtz, 2002). In both amphipods, the descendants of the germ line (a in O. cavimana and g in P. hawaiensis) form the centre of the initial gastrulation. Comparable to the sickle formation in O. cavimana is the rosette stage in P. hawaiensis (Alwes et al., 2011). Remarkably, this is similar to the blastomere arrangement of future mesendodermal cells (ba, da, A, and a in O. cavimana and mra, mla, Mav, and g in P. hawaiensis) and the germ line progenitors (a in O. cavimana and g in P. hawaiensis). During gastrulation, these cells get internalized by epibolic migration by the macromere derivatives that give rise to the left and right ectoderm (B and D in O. cavimana and Er and El in P. hawaiensis). A plausible explanation of a lack of a sickle formation and a missing visible blastopore (open gastrulation centre) could be that eggs of O. cavimana are bigger and contain relatively more yolk. This provides more space for the embryonic tissue compared with P. hawaiensis where the early germ has relatively less space and the steric pressure is probably higher.

Teloblastic Mesodermal Row Formation and Subsequent Divisions of the Segmental Founders From a Comparative Phylogenetic Perspective

The generation of four mesoteloblasts through a characteristic series of divisions of the mother cells is conserved in all amphipods studied in this respect (Scholtz, 1990; Price and Patel, 2008; this work). Although studies in other malacostracan representatives are scarce, descriptions of two other peracarids, Leptochelia sp. (Tanaidacea, Dohle, 1972) and Diastylis rathkei (Cumacea, Dohle, 1970), and of the decapod Hepatacarpus rectirostris (Oishi, 1959) suggest a wide conservation of this formation within Malacostraca. There are other descriptions about the mesoderm formation in crustaceans but unfortunately they lack details about cell lineages for the mesoderm (e.g., Hemimysis lamornae: Manton, 1928, Gammarus pulex: Weygoldt, 1958).

The cellular composition of the post-naupliar germ band, patterned through the mitotic waves of the ectodermal rows (ectoteloblasts are absent in Amphipoda, see Scholtz, 1990) is highly stereotyped. The same is true for the early divisions of the direct offspring of the mesoteloblasts, the segmental mesodermal founder cells. An initial number of four founders per hemi-segment is the starting point for segmental mesodermal differentiation. This strict pattern of four cells in each hemi-segment begins with the segment of the first walking leg. From here onward, we observed a characteristic timely sequence of divisions, beginning with the cells of mc2 and mc3 and ending with the most median cells of mc1. The spindle axes of the divisions are obliquely oriented toward the anteroposterior axis. Only later in development the two daughter cells rearrange and become situated in front of each other, parallel to the anteroposterior axis. The two most anterior segments of the second maxilla and the maxilliped obtain their mesoderm differently. Division products of the early differentiation of the mesoteloblasts provide a distinct set of mesodermal cells for these segments. It is also notable that in these segments the mesoderm is delayed in development. The separation into different segmental domains starts at a time when the mesoteloblasts have already generated many mesodermal rows. The connection between the most lateral cells of the mesodermal units m(1) and m(2) is retained even up to segment differentiation. The early divisions of the founder cells in these segments seem to a large extent to also follow a fixed pattern, but this pattern differs from the stereotypic division sequence seen in the more posterior segments.

The cell division pattern of the post-naupliar mesoderm up to segment formation has been described in two other peracarids, allowing a comparison between closely related species. In Gammarus pulex, another amphipod, the first divisions of the mesodermal founders are also oriented oblique to the longitudinal axis, but the sequence of divisions is slightly different with the most lateral cell dividing last (Scholtz, 1990). In the tanaidacean Leptochelia, a similar timing of the divisions as in G. pulex is described (Dohle, 1972), suggesting that this is the original state and that it was slightly altered in O. cavimana. Unlike in amphipods, the orientations of the divisions in Leptochelia are oriented immediately parallel to the anteroposterior axis, a pattern that is seen in O. cavimana a little later after a rearrangement of the cells. Despite these subtle differences the outcome of the first divisions in all investigated peracarids are two cell-wide mesodermal rows beneath each ectodermal segment.

Apart from malacostracan crustaceans the only other animal group that has been shown to form the segmental mesoderm by a teloblastic mode of growth are clitellates (Annelida) (discussed in Scholtz, 2002). In contrast to malacostracans, only one pair of mesoteloblasts is specified, forming bilateral and one cell-wide mesodermal bands underlying the four ectodermal columns. Each founder that is produced in this way can be assigned to one segment, giving rise to the segmental mesoderm of its body half. These founders also undergo a stereotyped cell division pattern that leads to the formation of uniform segmental packets (Storey, 1989; Shankland, 1991; Goto et al., 1999a, b). Although not completely similar, the pattern seen in malacostracans comprises more shared features with the mode found in annelids than with other arthropods. However, due to the phylogenetic position of both lineages a common evolutionary origin is much less plausible than a convergent evolved concentration of a posterior growth mode to single, identifiable cells that give rise to segmental structures by a fixed cell lineage.

The Head/Trunk Separation During Early Mesodermal Patterning of Amphipod Development

The segmental head mesoderm originates from macromere A of the eight-cell stage (respectively from the Al and Ar of the 16-cell stage) (Scholtz and Wolff, 2002; Wolff and Scholtz, 2002) and not from the adjacent micromeres b and d. An origin of the naupliar mesoderm from the b and d micromeres was described for the amphipod P. hawaiensis (ml and mr cells (Gerberding et al., 2002) but was then also corrected by (Browne et al., 2005) so that the early separation of head and trunk mesodermal lineages in amphipod development is found to be similar in both amphipod species.

Descendants of A (Mav in P. hawaiensis) do not contribute to the trunk mesoderm, only to the head mesoderm. Cells from this lineage are also found in large numbers beneath the midgut gland primordia and form either visceral mesoderm or endoderm. Questions for a cell division pattern or the exact pattern through which the anterior segmental mesoderm is separated from the endodermal cell mass in P. hawaiensis and O. cavimana are still unanswered. Within the head ectoderm no stereotypic arrangement of the early cells can be found and segments are formed out of an apparently randomly organized cell assembly (Scholtz, 1990; Scholtz et al., 1994; Wolff and Scholtz, 2006). Stripes of engrailed expressing cells as markers for early segmentation are generated by different patterns in head and trunk and the head stripes do not appear regularly sequential as the stripes in the trunk do (Scholtz et al., 1994). Therefore the anterior mesodermal patterning mode is different from patterning in the trunk, as is true for the patterning of the ectoderm. Studies on the origin and organization of head mesoderm in other arthropods are rare. Nevertheless, a divergent patterning mode between head and trunk mesoderm seems to be characteristic in arthropods, as major differences have been described in the fruit fly Drosophila melanogaster. Here the formation of mesoderm in the head is also not as uniform as in the trunk. Only a small portion of the head mesoderm originates from the anterior ventral furrow and additional cells with mesodermal fate segregate from the surface epithelium after formation of the ventral furrow (Tepass et al., 1994; Seecoomar et al., 2000). Furthermore, cells from different locations along the head anteroposterior axis form very different cell types, unlike in the trunk (de Velasco et al., 2006).

The lineage-correlated head/trunk separation within the mesodermal germ layer takes places between the segments of the first maxilla and second maxilla. Previous descriptions of O. cavimana were incorrect about the origin of the mesodermal cells of the first maxilla in assuming a post-naupliar identity (Scholtz and Wolff, 2002; Wolff and Scholtz, 2002). The cut between naupliar and post-naupliar ectoderm is made with the transition from ectodermal unit E(1) to E(2) of the germ band, due to the parasegmental shift crossing the later segment of the first maxilla (Wolff and Scholtz, 2002). This does not, however, stand in contradiction to the head/trunk transition within the mesoderm where the first mesodermal cells can be found beneath the posterior part of E(2), contributing to the second maxilla. Mesodermal cells of E(1) have a naupliar origin, as is true for the ectoderm (Wolff and Scholtz, 2002), the seemingly more posterior beginning of the post-naupliar mesoderm is due only to the lack of a shift between genealogical units and later segments within the mesoderm. This corresponds to findings in the amphipod crustacean Parhyale hawaiensis where the post-naupliar mesoderm starts in the segment of the second maxilla (Price and Patel, 2008).

In summary one can say that the split between the naupliar and post-naupliar regions is between the first and second maxilla and that both regions underlie different patterning modes for their organization. The split of both body parts can be traced back to the 16-cell stage where major embryonic components have their origin in different blastomeres. Blastomeres Al/Ar (mesendoderm), Ba/Da (ectoderm), and al/ar (germ cells) form the naupliar part; the blastomeres Bp/Dp (ectoderm) and ba/da (mesendoderm) contribute exclusively to the post-naupliar part of the embryo.

That single blastomeres are responsible for the naupliar region may reflect a remnant of a nauplius stage. A nauplius stage (nauplius larva or head larva) is a unique feature of crustaceans, but amphipods as many other malacostracan crustaceans completely lack a larval stage and display a direct development (see Scholtz, 2000). A typical nauplius larva is built up by a pear-shaped body with three pairs of appendages (first and second antenna and mandible) and a posterior pre-anal growth zone responsible for post-naupliar segments (Scholtz, 2000). Interestingly, our data on mesodermal distribution together with data on the ectodermal patterning (see Wolff and Scholtz, 2002) show a clear border between the naupliar and post-naupliar parts of the body, between the first and the second maxilla. This could indicate that the limbless segment of the first maxilla belongs to a nauplius stage of recent crustaceans. Additional support is given by the view that a crustacean nauplius larva (head larva) comprises not only three limb-bearing segments, but also a fourth limbless segment (first maxilla). Additional support is given by the fact that representatives of the crustacean stem line have larval forms with an additional fourth limb-bearing segment (the first maxilla) posterior to the mandible (Walossek and Müller, 1997; Waloszek and Maas, 2005).

The first maxillae are feeding structures in all recent crustaceans and belong to the ground pattern of Crustacea (Walossek and Müller, 1997). In contrast to this, the second maxilla is used as a walking leg in basally branching crustaceans (like cephalocarids and branchiopods) and has been transformed into a feeding structure only in malacostracan crustaceans. This speaks in favor of a trunk identity for the second maxilla, but not for the first maxilla.

Gene expression data and experiments on misexpression of the Hox gene Ultrabithorax (Ubx) support this view. As a Hox gene, Ubx is involved in the specification of appendage identities in crustaceans and typically expressed in the thoracic segments of crustaceans which are specialized as walking legs (Averof and Patel, 1997). Ubx is excluded from anterior thoracic segments that are feeding appendages, called maxillipeds. Overexpression of Ubx in the amphipod Parhyale hawaiensis caused a transformation of the second maxilla and the maxilliped (first thoracic appendage) to walking legs. However, phenotypic transformations of mandibles or first maxilla transformations were not obtained (Pavlopoulos et al., 2009).

Mesodermal Patterning in the Post-naupliar Limbs

Recently, an increasing number of studies has dealt with differentiation processes in amphipods (e.g., Ungerer and Wolff, 2005; Wolff and Scholtz, 2006, 2008; Price and Patel, 2008; Ungerer and Scholtz, 2008), enabling a discussion of mesodermal development in the context of ectodermal patterning processes. Lineaging experiments in O. cavimana have shown how ectodermal cells of the grid like pattern of the germ band give rise to the limb in a well-defined temporal and spatial sequence (Wolff and Scholtz, 2008). The outgrowth of the limb bud starts from cells of the transversal row d, which is the most posterior of four ectodermal cell rows of a genealogical ectodermal unit (Wolff and Scholtz, 2008). Soon after, cells of the anterior-most row a from the posterior-following unit become involved. Thus one limb originates from two genealogical units, as is true for the whole ectodermal segment (Dohle and Scholtz, 1988; Wolff and Scholtz, 2008). At early germ band stage mesodermal cells of O. cavimana underlie the rows c and d of the ectoderm. In contrast to the ectoderm, the mesoderm shows no shifted or parasegmental pattern. The transversal rows get situated in the centre of the respective segments, with the segmental furrows being drawn in anterior and posterior to it, and one transversal row of mesodermal cells contributes to the limb of one segment. Connections between the segmental portions of the mesoderm are only formed after the growing limbs have been populated with mesodermal cells. In the case of the longitudinal ectodermal columns (ec) of the germ band, it has been shown that cells of ec2 until ec9 become involved in a progressive manner (see Wolff and Scholtz, 2008). Initially, cells of ec3-6 form the limb bud. They generate limb growth by means of a distally situated growth zone and cells originating from this lineage are found along the whole proximo–distal (PD) axis of the leg. Lateral cells of ec2 and cells of ec7-9 become incorporated into the process after initial limb outgrowth and contribute to proximal parts of the leg (Wolff and Scholtz, 2008). Our data show that mesodermal cells originating from the cluster formed by mc2-4 grow into the limb bud once it is formed ectodermally. There is clearly a contribution of mc2 cells that underlie the ectodermal cells forming the early limb bud at germ band stage, but also of mc3 cells that underlie the parts of the ectoderm that contribute to basal parts of the limbs later on. Although the cells originate from the same parts of the germ band there is no clear sign that the limb patterning of ecto- and mesoderm is corresponding one-to-one. At the time when limbs have grown out and their initial segmentation has emerged the majority of mesodermal cells of the uniramous walking leg is found in the podomeres basis, ischium, merus, and carpus, with only single cells protruding into the more distal propodus and no mesodermal cells being found in the dactylus.

The biramous pleopods of O. cavimana ectodermally develop by means of a secondary split of the main axis in the distal part of the leg into endopodit and exopodit (Wolff and Scholtz, 2008). Like in the pereopods, the majority of mesodermal cells of the pleopod are found in the proximal segment, the unbranched protopod. The fact that we initially only find one of the branches populated with single mesodermal cells shows that the mesoderm of the biramous legs is not produced by a secondary split of a main axis like in case of the ectoderm. This speaks in favor of a mode of mesodermal growth with a main proliferation zone in the proximal/central limb segments, with a movement of some cells toward the distal part. This model would, however, assume a mesodermal growth mode different from the one found in the ectoderm, where cells of a distally situated growth zone divide to elongate the limb along the PD axis (Wolff and Scholtz, 2008). The high number of mesodermal cells (more than 30 cells could be counted in a pereopod at stage 4) stands in contradiction to the few cells that are singled out and become myoblasts, as was shown in isopods by Kreissl et al. (2008) using a myosin heavy chain (MHC) antibody as a marker for early myogenic cells. Accordingly, in the O. cavimana leg few cells become MHC positive at late stage 5 (unpublished observation). The presence of a much larger number of mesodermal cells within the limb suggests that multinuclear muscle fibers are generated by means of the founder and fusion model as it was described in the grasshopper (Ho et al., 1983) and studied in Drosophila (reviewed in Paululat et al., 1999): single cells become specified as muscle founder cells and recruit fusion-competent surrounding mesodermal cells to form multinucleated myotubes, each giving rise to a single, individual muscle fiber. The current data support this model to be true in crustaceans as well, although no actual fusion events were captured with the methods applied here.

In future studies, it would be interesting to see how far and in which way ecto- and mesodermal growth and differentiation processes depend on each other. First data regarding the interaction of mesoderm and ectoderm are available. Ablation experiments in the amphipod P. hawaiensis show that mesodermal and ectodermal precursor cells at eight-cell stage display intra-germ layer compensation. Both germ layers have the ability to replace ablated cells. Ablation experiments at later developmental stages (after gastrulation) revealed that compensation no longer occurs (Price et al., 2010). A recent study by Hannibal et al. (2012) could show in addition that segment formation within the mesoderm is dependent on the presence of the developing ectoderm. Segmentation of the ectoderm does not, however, require the presence of mesodermal cells (Hannibal et al., 2012).

Mesodermal Patterning and Heart Formation

The dorsal vessel of the O. cavimana embryo is believed to originate mainly from the trunk segmental mesoderm. Weygoldt (1958), in his study on embryonic development of the amphipod Gammarus pulex, describes segmentally arranged mesodermal tissues in the trunk that grow toward the dorsal side of the body and there give rise to a pericardial septum and the heart anlage itself. Our data shows that the heart forms along the dorsal midline by a closure of myofibers originating from both body halves of the embryo, most likely progeny of the most dorsolateral edges of the mesodermal units (mc4) and thus going back to MT4. In P. hawaiensis an antibody against even skipped, which in Drosophila is a marker for progenitor cells of dorsal musculature and heart tissue (Frasch et al., 1987), stains the anterior daughter cells of the mc4 cells (m4a cells) (Vargas-Vila et al., 2010). This adds further support to an origin of the heart cells in the dorsal cells of the mesodermal units, deriving from MT4.

Accordingly, it was shown in the beetle Tribolium castaneum and in the spider Cupiennius salei that tinman, as a molecular marker for heart-forming cells, is expressed in the lateral edges of the mesodermal primordium. These cells then move dorsally where they fuse to form the heart tube (Janssen and Damen, 2008), a mode that most likely takes place in a very similar way in the O. cavimana embryo.

Mesoderm Development in Arthropods: Conserved and Diverged Aspects and Future Perspectives

In crustaceans, the formation of the segmental mesoderm during embryogenesis is characterized by the posterior addition of segmental rows. In malacostracans, this is achieved by the activity of mesoteloblasts, as it was shown in different peracarid species (Dohle, 1970, 1972) and also in decapods (Oishi, 1959; Hertzler and Clark, 1992; Hertzler, 2002, 2005; Jirikowski et al., 2010). Although true teloblasts are absent in branchiopods, the segmental mesoderm in Artemia salina also originates from a posterior growth zone. As seen in O. cavimana, the produced segmental cell units stretch in a dorsolateral direction before they finally give rise to the mesodermal organs (Fränsemeier, 1939). In contrast, in the fruit fly Drosophila melanogaster, which represents the best-studied arthropod model, the mesoderm is specified in the ventral part of the blastoderm and a mesodermal monolayer resulting from the migration of cells from the ventral part only secondarily becomes subdivided into segmental packets (Bate, 1990; Leptin and Grunewald, 1990; Leptin et al., 1992). This mode of mesoderm formation has probably evolved along with the transition to long germ development in the insect lineage leading to the fly. The beetle Tribolium castaneum represents an intermediate state between long and short germ development within insects. Here, the mesoderm of the anterior segments that are formed in the syncytial blastoderm develops by an invagination of ventral cells, as in Drosophila. But the mesoderm of the posterior segments is added sequentially from a posterior growth zone (Sommer and Tautz, 1994; Handel et al., 2005). Little is known about mesodermal development in myriapods and chelicerates, although it was shown in a spider that stripes of twist-expressing cells emerge from a posterior-situated growth zone (Yamazaki et al., 2005). Also, in one tardigrade representative, a phylum that can serve as an outgroup to the arthropods, it has been shown by a 4D microscopic analysis that the segmental mesoderm is produced by posterior elongation of mesodermal bands (Hejnol and Schnabel, 2005). Hence, the posterior addition of mesodermal precursors within the trunk is most likely an ancestral feature to the arthropod lineage, while within malacostracans it has been tied to a fixed number of teloblasts and thereby to a stereotypic cell lineage.

Although representing a derived state, most insights into arthropod mesodermal development do come from the insect model Drosophila melanogaster where the application of powerful genetic methods is possible. In Drosophila, the secondary subdivision of the mesoderm along the anteroposterior axis is promoted by the segment polarity genes even skipped and sloppy paired, dividing the mesoderm of one segment into anterior and posterior domains that give rise to different mesodermal organs. A further subdivision of the segmental mesoderm takes place along the DV axis. Domains along this axis give rise to the visceral muscle, the somatic musculature and to the heart in a well-defined manner (reviewed in Paululat et al., 1999). When compared with Drosophila the amphipod represents an extreme of development characterized by a stereotypic cellular composition with large and individually identifiable cells, especially during mesodermal development. By the teloblastic mode of row formation the germ layer is subdivided into segmental domains from its emergence onward, and a ventral /dorsal fate of the mesodermal cells is clearly predetermined in their origins from the different mesoteloblasts, hence their position in one of the four longitudinal columns of the germ band. A further question to answer in the study of amphipod mesodermal development would be how specific fate-determining factors act in this well-defined cellular environment. There is a first indication that the four mesodermal founders have different molecular characteristics. It was shown in Parhyale hawaiensis that at germ band stage the mesodermal factor twist is only expressed in progeny of mesoteloblast 2 (m2a cells) (Price and Patel, 2008), while even skipped as a marker for dorsal mesoderm is expressed in progeny of MT4 (m4a cells) (Vargas-Vila et al., 2010). Another interesting aspect within this context of prepatterning of the amphipod segmental mesodermal units are the described cytoplasmic bridges between progeny of different mesoteloblasts (best visible in Fig. 5B). They might be functionally related to cytonemes that were described in the spider cumulus (Akiyama-Oda and Oda, 2003; Chaw et al., 2007) and in the Drosophila imaginal discs, where they are correlated with dpp-signaling and are thought to be involved in cell–cell communication (Hsiung et al., 2005).

Future studies on the amphipod mesodermal cells will be able to combine the knowledge about the exact origin and history of the cells with their molecular characteristics and final fate. This will allow deep insights into mesodermal differentiation in a crustacean system and will complement the data from Drosophila, thus leading to a broader understanding of mesodermal patterning and its phylogenetic deviations in arthropods.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Embryo Handling and Microinjections

Stock keeping, embryo collection and microinjections of Orchestia cavimana (Heller 1865) embryos were described earlier in Wolff and Scholtz (2002). The vital dye DiI (1,10,di-octadecyl-3,3,30,30,-tetramethylindo-carbocyanine perchlorate) (2 mg/ml, Molecular Probes) was applied to cell da, ba, or both at 16-cell stage (see Fig. 1A). In some cases, cell d or b of the 8-cell stage was labeled (Fig. 1A), which led to an additional labeling of extra-embryonic areas of the egg. For labeling of the naupliar mesoderm, cell A of the 8-cell stage or cell Al/Ar of the 16-cell stage (Fig. 1A) was marked. See Table 1 for numbers of performed labeling reactions. Labeled embryos were cultured in amphipod saline (isopod saline [205 mM NaCl, 21.5 mM KCl, 14.4 mM CaCl2, 16.8 mM MgCl2, 2.4 mM NaHCO3 to 1,000 ml distilled water] and NaCl-frog saline [115 mM NaCl, 2.5 mM KCl, 2.15 mM Na2HPO4, 0.85 mM NaH2PO4, 1.8 mM CaCl2 to 1,000 ml distilled water] in ratio of 1:2) plus 1% antibiotics (Pen-Strep-Premix, Roth) at 22°C. Approximately 3 days after injection, mesoteloblasts appeared and documentation was started. For this, a Zeiss Axioskop 2 fluorescence compound microscope with an AxioCam HRc and a Zeiss Lumar V12 stereomicroscope with AxioCam MRm were used.

Table 1. Number of Performed Labeling Experimentsa
Labeled cellbdb+dbadada+daAArAlAl+ba
  • a

    Only embryos that survived the injection process and were subsequently analyzed are listed. See Fig. 1A for cell nomenclature.

Number of embryos232121464672

Fixation and Fluorescence Staining of Nuclei

DiI labeled embryos were raised to the desired stages (see Table 2) and were then put into 4% paraformaldehyde in amphipod saline for fixation. After 30 min, prefixation the egg membranes were removed manually, as well as the yolky extra-embryonic parts. Embryos were left for 60 min in the fixative. Subsequently, a Hoechst (Bisbenzimide H 33258) staining of nuclei was carried out. For this, embryos were put into the staining solution (100 mg ml−1 Hoechst in phosphate buffered saline [PBS; 1,86 mM, Na2H2PO4, 8,41 mM Na2HPO4, 175 mM NaCl in ddH2O, pH 7,4]) for 15 min. After several washes in PBS+0.1% Tween-20, embryos were embedded in Slow fade Gold (Invitrogen) for microscopic inspection.

Table 2. Overview of Developmental Stages of the Orchestia cavimana Modified from Ungerer and Wolff (2005)a
StageCharacteristics
  • a

    The lineage study presented here covers the early stages up to stage four. Additionally an overview of the differentiated muscular system of the late embryo (stage 6) is given.

Germ discStarting with a characteristic sickle-shaped arrangement the embryonic germ disc is formed and gastrulation proceeds
early germ bandEctodermal germ disc cells arrange in an orderly row pattern and start, contemporary with mesoteloblasts, their stereotyped division pattern
#1Formation of naupliar limb buds; post-naupliar segment formation
#2Formation of the post-naupliar limb buds; initiation of germ band split
#3Limb elongation and initial differentiation, maximum of germ band split
#4Ventral closure of the germ band after split; limbs readily segmented
#5Dorsal closure of embryo, characteristic tagmata are evident
#6Late embryo shortly before hatching, all organ systems like the heart are fully differentiated

Phalloidin Staining of Musculature

Untreated embryos of adequate stages (stage 5, 6, and hatchling) were fixed as described above. After several washes in PBS+0.1% Tween-20 embryos were transferred into a freshly prepared staining solution (3.3 mM Phalloidin-TRITC [Sigma] in PBS) and incubated for 60 min in the dark. The staining reaction was stopped by a few washes in PBS+0.1% Tween-20. The embryos were subsequently counterstained with Hoechst as described above.

Confocal Laser Scanning Microscopy

Digital image stacks of the specimens (DiI-labeled embryos as well as Phalloidin staining) were taken with a Leica DMIRE2 confocal laser scanning microscope. Excitation of DiI was achieved by using a Helium-Neon Laser (543 nm). For Hoechst excitation, a UV Diode Laser (405 nm) was used. Phalloidin TRITC was also excited with the 543-nm Laser. For multicolored picture stacks, the same plane thickness was applied for each channel to avoid optical shift. Reconstructions of the image stacks were generated with the software Imaris (Bitplane).

Live Imaging

For live-embryo imaging embryos were mounted on microscope slides and covered with amphipod saline. Embryonic development was recorded using a 4D-microscope (Zeiss Axioskop 2 equipped with a PCO camera). Data were analyzed with SIMI BioCell software and videos were created using Adobe Premiere software.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Stefan Richter and Gerhard Scholtz for constructive advice during the experiments and K. Lindemann for help with collecting specimens. We also thank Frederike Alwes and the anonymous reviewer for valuable comments on the manuscript. Furthermore, we thank Richard Claes for comments on the manuscript and for improving the English. C.W. was funded by a DFG grant.

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  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_23758_sm_SuppMovieS1.mov4642KSupporting Information Movie S1. This movie shows the embryonic development of an embryo at approximately 20°C; the A-quadrant of an egg at 16-cell stage is in the centre. Overlay of original imaging data and SimiBioCell analysis with highlighted cells which are involved in sickle-shape formation (color code: A: yellow spheres; a: yellow stars; B: green spheres, b: green cubes; D: red spheres, d: red cubes). The movie starts at 16-cell stage and ends with the sickle-shaped cell formation (indicated with a black dashed line at the end of the movie). Duration is 14 hours of development and time interval between each frame is 2 min.

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