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1 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
2 Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2559 Puesta del Sol Road, Santa Barbara, California 93105-2936
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Abstract |
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Dicyemids produce two distinct types of embryos: vermiform embryos from an asexual agamete and infusoriform embryos from fertilized eggs (Furuya et al., 1996). From the standpoint of morphological evolution, the vermiform embryo is the more pertinent target for study because its shape is similar to that of an adult. The cell lineage of vermiform embryos has been fully documented in only two dicyemids, Dicyema acuticephalum and D. japonicum (Furuya et al., 1994). Among other species, cell lineages have been described only to a limited extent in Microcyema vespa and Pseudicyema truncatum (Lameere, 1919; McConnaughey, 1938; Schartau, 1940; Nouvel, 1947; Bogomolov, 1970; Lapan and Morowitz, 1975). Details of cell lineage in the phylum as a whole remain to be determined.
In this paper we describe the pattern of cell divisions and cell lineages in the embryogenesis of vermiform embryos in dicyemids belonging to four genera: Conocyema polymorpha, Dicyema apalachiensis, Microcyema vespa, and Pseudicyema nakaoi. Our data reveal that cell lineages in vermiform embryos are highly conserved among species; but species-characteristic features appear in the later embryogenesis, and these are related to morphological evolution and speciation.
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Materials and Methods |
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Specimens of Pseudicyema nakaoi Furuya, 1999, were prepared for this study. A total of 57 host cuttlefishes, Sepia esculenta, were purchased in the western part of Japan. When dicyemids were detected in the kidney of the host cuttlefish, small pieces of renal appendages with attached dicyemids were removed and smeared on slide glasses. The smears were fixed immediately in Bouin’s fluid for 24 h and then stored in 70% ethyl alcohol. The fixed smears were stained in Ehrlich’s hematoxylin and counterstained in eosin. Stained smears were mounted using Entellan (Merck).
Embryos within the axial cell of parent nematogens were observed with the aid of a light microscope under an oil-immersion objective at a magnification of 2000 diameters. Cells were identified by their position within the embryo, their size, and the intensity of stain taken up by the nucleus and cytoplasm. By careful examination, we were able to identify each swollen nucleus that was about to divide and each metaphase figure in terms of the cell that was about to divide into two daughter cells. Each developing embryo was sketched at three optical depths, and three-dimensional diagrams were reconstructed from these sketches. Measurements and drawings were made with the aid of an ocular micrometer and a drawing tube (Olympus U-DA), respectively. Fully formed embryos consisted at most of 23 cells, and special techniques such as injection of a tracer and videoscopy were not required for determination of the cell lineage. Early divisions of the vermiform embryos examined in this study were the same as those of Dicyema acuticephalum and D. japonicum (see Furuya et al., 1994). The terminology of cells used by Furuya et al. (1994) was adopted in designating the cells in the present paper.
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Results |
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The sixth division is extremely unequal. Both the 3B and 3B cells divide, and they produce a pair of large cells and a pair of much smaller daughter cells. The smaller cells degenerate and eventually disappear. At around the 5-cell stage, cell 2a, the prospective axial cell, undergoes an extremely unequal division. The resultant smaller cell degenerates and eventually disappears. The seventh division is equal, and results in the 7-cell embryo. Cells 4B and 4B divide and produce two pairs of daughter cells, 5B1 and 5B2 plus 5B1 and 5B2, respectively. The future anterior-posterior axis of the embryo corresponds almost exactly to the 5B1-3A axis at the 7-cell stage. About the 7-cell stage, cell 3a, the prospective axial cell, undergoes an extremely unequal division. The resulting smaller cells degenerate and eventually disappear.
After the seventh division, the order of division is not necessarily identical among developing embryos. The 5B1 cell pair divides equally and produces two pairs of daughter cells, 6B11 and 6B12 plus 6B11 and 6B12. The 5B2 cell pair divides equally and produces two pairs of daughter cells, 6B21 and 6B22 plus 6B21 and 6B22. Neither pair divides further, and they form the anterior part of the embryo (Fig. 2a, b). The 6B22 cell pair develops into the parapolar cells, while the 6B11, 6B12, and 6B21 cell pairs eventually form a syncytium, which is more conspicuously stained with hematoxylin than the other cells (Fig. 1b). The two parapolar cells cover more than half of the syncytium (Fig. 2c). As peripheral cells are formed, the prospective axial cell, 4a, divides unequally. The large anterior cell, 5a1, undergoes no further divisions and becomes the axial cell, while the smaller posterior cell, 5a2, is incorporated into the axial cell and becomes an agamete (Fig. 2a). At this stage, cilia are first evident on the peripheral cells. Cilia on the external surface of the syncytium are directed anteriorly and are more densely distributed than on other peripheral cells.
The fully formed embryo consists of three types of cells: peripheral cells, one syncytial cell, and an axial cell, which contains two agametes (Figs. 1b, 2c). The peripheral cells are composed of two parapolar cells and two uropolar cells. The swollen cephalic head region consists of a calotte and two parapolar cells. The calotte is a syncytium. The trunk is composed of two uropolar cells. Further development involves growth and enlargement of the syncytium and branching of the axial cell (Fig. 1a). Total length, excluding cilia, of the fully formed vermiform embryo is about 50 µm, and the width is about 20 µm. The cell lineage of the vermiform embryo is summarized in Figure 3a. No variations in cell lineage were found in more than 50 embryos examined.
Conocyema polymorpha
Agamete diameter is about 7 µm. The first division is meridional and unequal, producing two daughter cells that are slightly different in size, A and B. The larger cell B becomes the mother cell of the peripheral cells of the embryo’s head (propolar and parapolar cells). The second division involves only the smaller cell A. This division is latitudinal and equal, producing two daughter cells, 2A and 2a. Cell 2A is the mother cell of the peripheral cells of the posterior trunk and tail, and cell 2a is the prospective axial cell (Fig. 3b). In the 2a lineage, extremely unequal divisions occur at around the 5-, 11-, and 13-cell stages (Fig. 3b), and the resultant much smaller daughter cells remain attached to the larger daughter cells until they ultimately degenerate without contributing to embryogenesis. The third division, involving cell B, is meridional and equal, producing two daughter cells, 2B and 2B. At this 4-cell stage, two pairs of cells, 2A-2a and 2B-2B, are arranged crosswise with respect to one another. The furrow of the third division coincides with the plane of bilateral symmetry of the embryo. The pattern of division and the cell lineage are the same for the descendants of cell 2B and those of cell 2B (Fig. 3b). The fourth division, involving cell 2A, is meridional and equal, resulting in the 5-cell embryo. The plane of cell division again coincides with the plane of bilateral symmetry, and it separates the right cell 3A from the left cell 3A. The pattern of cell division and the cell lineage are the same for descendants of cell 3A and for those of cell 3A (Fig. 3b).
The pattern of cell division beyond the 5-cell stage changes from spiral to bilateral. Beyond the 5-cell stage, divisions occur not one by one but in pairs, and they become almost synchronized. Subsequent developmental stages thus proceed with odd numbers of cells.
At around the 5-cell stage, cell 2a, the prospective axial cell, undergoes an extremely unequal division. The resultant smaller cell degenerates and finally disappears. The fifth cell division is equal and results in the 7-cell embryo. Thus, cells 2B and 2B divide and produce two pairs of daughter cells, 3B1 and 3B2 plus 3B1 and 3B2, respectively. The future anterior-posterior axis of the embryo corresponds almost exactly to the 3B1-3A axis at the 7-cell stage. The sixth division is slightly unequal. Cells 3A and 3A divide into two pairs of daughter cells, 4A1 and 4A2 plus 4A1 and 4A2. Cells 4A2 and 4A2 are the smallest cells at this stage. In addition to the 2a lineage, cells 3B2 and 3B2 undergo unequal divisions, each generating a pair of cells, one large and one much smaller. The smaller cells degenerate and finally disappear, although they remain in place on the developing embryo until later stages.
The 3B1 pair divide equally into 4B11 and 4B12 pairs. These cells undergo no further divisions. The 4B11 pair become the propolar cells, and the 4B12 pair become the parapolar cells (Figs. 2e, f). The cell in the 2a lineage (cell 3a) is incorporated into the inside of the embryo, and the 3B1 pair divide and rearrange their descendants. At around the 11-cell stage, the 3a cell undergoes an extremely unequal division.
The 13-cell stage is achieved by equal divisions of cells 4A2 and 4A2. The resultant 5A21 and 5A22 pairs undergo no further divisions and become diapolar cells and uropolar cells, respectively. Soon after these divisions, the 4B2 pair divide equally into two pairs of daughter cells, 5B21 and 5B22 plus 5B21 and 5B22. The 5B21 and 5B22 pair undergo no further divisions and become the propolar cells and parapolar cells, respectively (Fig. 2e, f). At around the 13-cell stage, the prospective axial cell 4a undergoes an extremely unequal division.
At the final stage of embryogenesis, the prospective axial cell, 5a, divides unequally. The large anterior cell, 6a1, undergoes no further divisions and becomes the axial cell, while the smaller posterior cell, 6a2, is incorporated into the axial cell and becomes an agamete (Fig. 2d).
The fully formed vermiform embryo of Conocyema polymorpha consists of 14 peripheral cells and one axial cell, which contains one or two agametes (Fig. 2f). The head peripheral cells are composed of four propolar cells and parapolar cells. The propolar cells have short, dense cilia and form the calotte, which is more conspicuously stained with hematoxylin than the other cells. Four diapolar cells make up the trunk peripheral cells. The caudal peripheral cells are uropolar cells. The length, excluding cilia, of the fully formed embryo is about 25 µm, and the width is about 10 µm. The cell lineage of the vermiform embryo is summarized in Figure 3b. No variations in cell lineage were found in more than 80 embryos examined.
Dicyema apalachiensis
Agamete diameter is about 5.5 µm. The first division is meridional and equal, producing two daughter cells of equal size. The subsequent patterns of development and cell lineage up to the 7-cell stage are the same as those described for Conocyema polymorpha.
At the 7-cell stage, cells 3B2 and 3B2 undergo unequal divisions, each generating a pair of cells, one large and one much smaller cell. The 9-cell stage is achieved by unequal divisions of cells 3B1 and 3B1. The resultant small cells, 4B11 and 4B11, divide again into two pairs of daughter cells, 5B111 and 5B112 plus 5B111 and 5B112, in the anterior part of the embryo (Fig. 2g-i). Almost simultaneously, the resultant large cells, 4B12 and 4B12, divide again into two pairs of daughter cells, 5B121 and 5B122 plus 5B121 and 5B122, in the anterior part of the embryo (Fig. 2h, i). These cells undergo no further divisions, and the 5B111 and 5B121 pairs become the propolar cells, while the 5B112 and 5B122 pairs become the metapolar cells. The cell in the 2a lineage (cell 4a) is incorporated into the inside of the embryo, and the other cells, 4B11 and 4B11, divide and rearrange their descendants.
At the 9-cell stage, cells 3A and 3A divide equally into two pairs of daughter cells, 4A1 and 4A2 plus 4A1 and 4A2. They do not divide further; the 4A1 pair become the uropolar cells, and the 4A2 pair become the diapolar cells (Fig. 3c). At the final stage of embryogenesis, the prospective axial cell, 4a, divides unequally. The large anterior cell, 5a1, becomes the axial cell, and the smaller posterior cell, 5a2, is incorporated into the axial cell and becomes an agamete.
The vermiform embryo of Dicyema apalachiensis consists of 14 peripheral cells and one axial cell, which contains one or two agametes. The peripheral cells of the head region are composed of four propolar cells, four metapolar cells, and two parapolar cells. The propolar and metapolar cells have short, dense cilia and form the calotte. Two diapolar cells make up the trunk peripheral cells. Two caudal peripheral cells are uropolar cells. The length, excluding cilia, of the fully formed embryo is about 30 µm, and the width is about 10 µm. The cell lineage of the vermiform embryo is summarized in Figure 3c. No variations in cell lineage were found in more than 50 embryos examined.
Pseudicyema nakaoi
Agamete diameter is about 6.5 µm. The first division is equal, producing two daughter cells of equal size. The subsequent patterns of development and cell lineage up to the 9-cell stage are the same as those described for Dicyema apalachiensis (see Fig. 3c).
At the 9-cell stage, cells 3B2 and 3B2 undergo extremely unequal divisions, each generating a pair of cells, one large and one much smaller cell. The smaller cells resulting from this division degenerate and finally disappear. In the 2a line, unequal divisions occur at around the 5-, 9-, and 11-cell stages (Fig. 3d). The pattern of development and cell lineages up to the 9-cell stage are the same in both P. truncatum and P. nakaoi. Further development was not studied in P. truncatum, because adequate material was not available.
After the unequal divisions of the 3B2 pair, cell pairs 4A1 and 3B1 undergo equal divisions almost simultaneously and produce two pairs of daughter cells, 5A11 and 5A12 plus 4B11 and 4B12, to form the 13-cell-stage embryo. At around the 13-cell stage, the prospective axial cell, 4a, undergoes an unequal cell division. The 5A11 pair divide equally and produce two pairs of daughter cells, 6A111 and 6A112 plus 6A111 and 6A112. The plane of this division is parallel to the anterior-posterior axis, in contrast to the previous division, which occurs parallel to the perpendicular axis. As a result, cells 6A111 and 6A111 are situated on the left and right sides of the embryo, respectively.
At the 15-cell stage, cell pairs 4B2 and 5A12 undergo equal divisions almost simultaneously, and produce two pairs of daughter cells, 5B21 and 5B22 plus 6A121 and 6A122, to form the 19-cell-stage embryo. These pairs undergo no further divisions. Cell pair 5B21 become the parapolar cells, and the 5B22 pair become the anterior diapolar cells (Fig. 3d). Cell pair 6A121 become the uropolar cells, and the 6A122 pair become the posterior diapolar cells (Fig. 3d).
At the 19-cell stage, the 4B11 and 4B12 pairs divide equally into two pairs of daughter cells, 5B111 and 5B112 plus 5B121 and 5B122, in the anterior part of the embryo (Fig. 2k, l). These divisions proceed cell by cell. The daughter cells undergo no further divisions, and the 5B111 and 5B121 pairs become the propolar cells, while the 5B112 and 5B122 pairs become the metapolar cells (Fig. 2j). The planes of these divisions, in contrast to the previous division, are oblique to the anterior-posterior axis. As the result, cells of the propolar tier alternate with cells of the metapolar tier (Fig. 2k, l).
At the final stage of embryogenesis, the prospective axial cell, 5a, divides unequally. The large anterior cell, 6a1, becomes the axial cell, and the smaller posterior cell, 6a2, is incorporated into the axial cell and becomes an agamete.
The fully formed vermiform embryo of P. nakaoi consists of 22 peripheral cells and one axial cell, which contains one agamete. The peripheral cells of the head region are composed of four propolar cells, four metapolar cells, and two parapolar cells. The propolar and metapolar cells have dense short cilia and form the calotte. Ten diapolar cells make up the trunk peripheral cells. Two caudal peripheral cells are uropolar cells. The body length, excluding cilia, of the fully formed embryo is about 70 µm, and the body width is about 16 µm. The cell lineage of the vermiform embryo is summarized in Figure 3d. No variations in cell lineage were found in more than 50 embryos examined.
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Discussion |
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Early developmental pattern and cell lineage
Comparisons of developmental processes and cell lineages among various species of dicyemids reveal conservative features in the early development. Although dicyemids from different host species and geographically different distributions were compared, the developmental processes and cell lineages are almost identical from an agamete to the 7-cell stage (Fig. 5). Cell-fate segregation appears in the very early stages of embryogenesis. Three types of prospective cells that form the body of embryos, such as the agamete, the axial cell, and the peripheral cells, can be identified as early as the 3-cell stage. In the development of vermiform embryos, cell fates may be initially segregated. This conserved feature among species may represent the basic plan in forming bodies of vermiform embryos (Fig. 6). These features in cell lineage suggest that the early developmental processes have persisted through the evolution of dicyemids. Vermiform embryos develop in the confined space of an axial cell located within the parent nematogen. This peculiar habitat thus may constrain the developmental process, as well as limit the size and number of cells that compose the body. As a result, development may appear to be conserved.
Variations of terminal divisions in cell lineages
Species-specific patterns of development and cell lineages appear in the later stages of embryogenesis. The most striking difference is seen in terminal divisions in the cell lineage that give rise to variations in peripheral cell numbers. For instance, species-specific differences in the peripheral cell number between Dicyema acuticephalum and D. japonicum can be attributed to the number of divisions of the 4A1 pair (Fig. 4; Furuya et al., 1994). In other species, additional terminal divisions occur toward the end of the establishment of another cell lineage as well. The various numbers of terminal divisions, which are genetically determined, clearly play a significant role in the morphogenesis of vermiform embryos and may be correlated with speciation in the dicyemids.
In most species of dicyemids, vermiform embryos have a constant number of peripheral cells. However, some species of dicyemids, such as Dicyema acuticephalum, D. bilobum, D. benthoctopi, D. erythrum, D. lycidoceum, and D. rhadinum, have a variable number of peripheral cells (Nouvel, 1947; Couch and Short, 1964; Hochberg and Short, 1970; Furuya et al., 1992a; 1994; Furuya, 1999). Such intraspecific variation in peripheral cell numbers could be attributed to minor differences in numbers of terminal divisions in certain cell lineages (compare Figs. 4a and b).
In the developmental patterns of vermiform embryos, the cell lineages do not vary, and the terminal divisions usually occur bilaterally. Thus, several even numbers of peripheral cells are formed as the result of a pair of terminal divisions in both the 2A- and B-cell lineages. In species that have a variable number of peripheral cells, such as Dicyema erythrum, D. lycidoceum, and D. rhadinum, some peaks are evident in even numbers of peripheral cells (see tables in Furuya, 1999). The number of terminal divisions may not be strictly programmed in these exceptional species.
Later development and larval morphology
In the evolution of dicyemids, various types of vermiform embryos must have been produced as deviations from a common developmental pattern. Unusual species, such as Microcyema vespa and Conocyema polymorpha, not only differ morphologically from other dicyemids but are distinct in the later stages of development. As shown in the cladogram (Fig. 7), these two species of dicyemids are clearly distinct and separate when compared with the clade composed of the genera Dicyema and Pseudicyema. Some changes that occur in cell lineages certainly are reflected in morphological features.
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In recent years, it has been argued that the evolution of morphological features requires alterations in developmental processes. In dicyemids, the cell lineage of Microcyema vespa is closer to a conservative lineage than in other genera in the phylum, but vermiform embryos of M. vespa show a distinctive form not seen in other genera. It is possible that in M. vespa the developmental process may be truncated, resulting in a simple cell lineage and a body organization with a very small number of peripheral cells. However, changes in cell lineage may not always contribute to morphological characters. For example, there are some differences in the later cell lineage between Dicyema acuticephalum and D. apalachiensis, but these dicyemids are very similar in general body shape.
Cell death
McConnaughey (1951) described chromatin elimination from the prospective axial cell. In Dicyema acuticephalum and D. japonicum, what appears to be a mass of eliminated chromatin is actually a small cell that is produced as the result of an extremely unequal division (Furuya et al., 1994). In Pseudicyema truncatum and Microcyema vespa, Lameere (1919) noted that the prospective axial cell underwent an unequal division and that the smaller daughter cell itself divided once or twice to produce two or four small cells that do not degenerate. We were able to examine the details of these small cells in several dicyemids, including the species studied by Lameere. In contrast to Lameere’s observation, the small cell does not undergo further divisions. In his report, the small cells were exclusively derived from a prospective axial cell (a-cell lineage), but we recognized they are formed in both the a-cell and B-cell lineages, as recognized in the previous study of D. acuticephalum and D. japonicum.
The small cells eventually die and are eliminated without contributing to the embryogenesis. This is considered to be a programmed cell death as described in the development of infusoriform embryos (Furuya et al., 1992b). In the dicyemids examined, extremely unequal divisions take place four to eight times during embryogenesis (Table 1). The number of such divisions is as definite according to species as the number of peripheral cells. In the a-cell lineage, much programmed cell death appears frequently in dicyemids that consist of a large number of peripheral cells. It seems possible that successive, extremely unequal divisions in the a-cell lineage may be required to maintain an increased amount of cytoplasm in the large axial cell. The axial cell retains most of the cytoplasm of the mother cell and enlarges after each cell division. In most dicyemids, the axial cell elongates as peripheral cell numbers increase. Thus, peripheral cell number appears to be correlated to the number of programmed cell deaths.
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Several features in developmental pattern and cell lineages among species
The early development of dicyemids is conservative and may be summarized as follows: (1) the first cell division produces prospective cells that generate the anterior peripheral region of the embryo; (2) the second cell division produces prospective cells that generate the posterior peripheral region plus the internal cells within the embryo; (3) in the lineage of prospective internal cells, several divisions ultimately result in the death of one of the daughter cells. Developmental processes to the 7-cell stage are almost identical in the vermiform embryos of the four genera examined (Figs. 5, 6).
In contrast, distinct species-specific differences appear in the order and number of terminal divisions of peripheral cells. Most of the changes in terminal divisions can be correlated with individual body length. Generic differences appear in the number of cells that contribute to the calotte during the final stage of embryogenesis. Distinct morphological features typically emerge following a final cell division or after the embryo escapes from the axial cell of the adult. Subsequent processes, proceeding without cell divisions, are cell differentiation in the head region and cell elongation in the trunk region.
On the basis of cell lineage, a simple cladogram was constructed (Fig. 7). Cell lineages from an agamete to the 7-cell stage were almost identical among species (bar 1). The terminal of B-cell lineage indicates some variation among species. In the family Conocyemidae, a calotte is formed with a tier of polar cells (bar 2A), whereas in the Dicyemidae a calotte consists of two tiers of polar cells, propolars and metapolars (bar 2B). Thus, the tree indicates that two clusters initially separate to form two families. In Microcyema, a calotte and peripheral cells form a syncytium (bar 3A), but in Conocyema a calotte is cellular and diapolars are present (bar 3B). In Dicyema japonicum, the calotte is formed in 3B1- and 3B2-cell lineages (bar 4A), but in D. acuticephalum, D. apalachiensis, and Pseudicyema nakaoi the calotte is formed only in 3B1-cell lineage (bar 4B). The orientation of propolars to metapolars separates Pseudicyema from Dicyema. In Pseudicyema, propolars are obliquely oriented to metapolars (bar 5B). In Dicyema, propolars are located perpendicularly above metapolars (bar 5A). In D. acuticephalum, cell death occurs both in 3B1- and in 3B2-cell lineages (bar 6A), but in D. apalachiensis it occurs only in 3B2-cell lineage (bar 6B). Based on the above criteria, separation of the dicyemids into two families may be justified; however, the generic state of Pseudicyema apparently warrants further study.
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Acknowledgments |
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