Cellular pattern formation, establishment of polarity and segregation of colored cytoplasm in embryos of the nematode Romanomermis culicivorax

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Developmental Biology 315 (2008) 426 – 436 www.elsevier.com/developmentalbiology

Cellular pattern formation, establishment of polarity and segregation of colored cytoplasm in embryos of the nematode Romanomermis culicivorax Jens Schulze, Einhard Schierenberg ⁎ Zoological Institute, University of Cologne, Germany Received for publication 5 October 2007; revised 17 December 2007; accepted 31 December 2007 Available online 8 January 2008

Abstract We have begun to analyze the early embryogenesis of Romanomermis culicivorax, an insect-parasitic nematode phylogenetically distant to Caenorhabditis elegans. Development of R. culicivorax differs from C. elegans in many aspects including establishment of polarity, formation of embryonic axes and the pattern of asymmetric cleavages. Here, a polarity reversal in the germline takes place already in P1 rather than P2, the dorsal–ventral axis appears to be inverted and gut fate is derived from the AB rather than from the EMS blastomere. So far unique for nematodes is the presence of colored cytoplasm and its segregation into one specific founder cell. Normal development observed after experimentally induced abnormal partitioning of pigment indicates that it is not involved in cell specification. Another typical feature is prominent midbodies (MB). We investigated the role of the MB region in the establishment of asymmetry. After its irradiation the potential for unequal cleavage in somatic and germline cells as well as differential distribution of pigment are lost. This indicates a crucial involvement of this region for spindle orientation, positioning, and cytoplasmic segregation. A scenario is sketched suggesting why and how during evolution the observed differences between R. culicivorax and C. elegans may have evolved. © 2008 Elsevier Inc. All rights reserved. Keywords: Embryogenesis; Nematode; Polarity; Asymmetry; Midbody; Germline; Axis formation; Cytoplasmic segregation; Evolution; C. elegans

Introduction Establishment of cellular polarity, cell lineages, and cell specification have been analyzed in detail in the embryo of C. elegans (clade 9; Fig. 1). However, comparative studies in other nematodes have now revealed remarkable differences between species. For instance, in Acrobeloides nanus (clade 11, Fig. 1) early blastomeres are pluripotent and seem to compete for a primary fate. Here, eliminated cells can be replaced by neighbors, which adopt the developmental program of the missing blastomere (Wiegner and Schierenberg, 1998, 1999). This and other findings indicate that different paths can lead to essentially the same result. As nematodes belong to an ancient and diverse phylum (De Ley and Blaxter, 2002; Meldal et al., 2007) and many species

⁎ Corresponding author. Fax: +49 221 470 4987. E-mail address: [email protected] (E. Schierenberg). 0012-1606/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.12.043

can be cultured in the laboratory, they appear to be excellent candidates to study the evolution of developmental processes. Over the last decades various attempts have been made to better understand phylogenetic relationships among nematodes. Based mainly on DNA sequence data, a modern nematode phylogeny was suggested by Blaxter et al. (1998), extended and modified by De Ley and Blaxter (2002). Recently, drawing on a larger set of species, more than 300 nearly full-length SSU rDNA sequences were analyzed, revealing a backbone of twelve consecutive dichotomies that subdivide the phylum Nematoda into twelve clades (Holterman et al., 2006; Fig. 1). In the following we will rely on and refer to this work. A further extended phylogenetic tree with emphasis on marine taxa was presented by Meldal et al. (2007). Embryogenesis of representatives from clades 6 to 12 has been studied at least to some extent (Fig. 1; Sulston et al., 1983; Skiba and Schierenberg, 1992; Malakhov, 1994; Lahl et al., 2003; Houthoofd et al., 2003, 2006; Zhao et al., in press), including the classical model organism Ascaris (clade 8, Fig. 1; Boveri, 1899; Müller, 1903). They all show essentially the

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Materials and methods Nematode strains and culture

Fig. 1. Phylogenetic tree of nematodes. Simplified phylogenetic tree of nematodes based primarily on rDNA sequence data (Holterman et al., 2006). The tree is subdivided into twelve clades (1–12) and one unresolved branch (*). Branch lengths reflect substitution rates. Affiliations of selected representatives whose early embryogenesis has been studied are shown. For references, see Introduction.

same early cleavage pattern with a germline generating a small number of somatic founder cells. Also fate distribution seems to be similar among these taxa. We know much less in this respect about species belonging to clades 1–5 (Malakhov, 1994) positioned closer to the root of the nematode phylum. It was found that representatives of Enoplida (clade 1; Fig. 1) pass through early equal cleavages without a detectable germline (Malakhov, 1994), and the fate of most blastomeres appears not to be fixed during early development (Voronov and Panchin, 1998; Voronov, 1999). Gastrulation in the genus Tobrilus (clade 1, Fig. 1) differs from all other nematodes studied so far in that a large blastocoel forms, as is typical for most other animals (Schierenberg, 2005; our unpublished results). We chose to study Romanomermis culicivorax (clade 2) because its embryos are more transparent and develop faster than other members of this clade we have looked at (e.g., Dorylaimida and Mononchida) and because large numbers of fertilized eggs laid at the 1-cell stage are available. Another advantage of this species is that it can be cultured in large quantities in the laboratory. However, because of its partial parasitic life cycle in insects, refined procedures are required to pass the cultures from one generation to the next (Petersen, 1985; Platzer et al., 2005). Once specimens have emerged from the host, they develop to egg-laying adults without feeding. During recent decades Romanomermis and other mermithids have been used as biological control agents to fight insect-borne pests like malaria and dengue fever (Platzer, 1981; Petersen, 1985; Perez-Pacheco et al., 2005). In this paper we investigate to what extent major developmental differences to C. elegans are found during early embryogenesis of R. culicivorax, in particular with respect to the establishment of cellular polarity, asymmetric divisions, soma/germline separation, and cytoplasmic segregation, and gain some understanding of how these differences are generated.

Caenorhabditis elegans (N2) was cultured on agar plates with the uracilrequiring strain of Escherichia coli OP50 as a food source, essentially as described by Brenner (1974) except that, to reduce contamination with other bacteria, we used minimal medium plates (Lahl et al., 2003). Romanomermis culicivorax (Petersen et al., 1978), Romanomermis iyengari (Perez-Pacheco et al., 2005) and Strelkovimermis spiculatus (Poinar and Camino, 1986) were kindly provided by Dr. Edward Platzer, University of California, Riverside. Mermithids can be cultured in the laboratory using a variety of different mosquito genera as hosts (Perez-Pacheco et al., 2004). Insect larvae were hatched from eggs and infected as L3 stages by adding infective juvenile nematode worms to the liquid culture. Post-infective nematode larvae kill the host when they emerge and afterward can be cultured in distilled water without any food. More details are found in Petersen and Willis (1972) and Platzer and Stirling (1978). While nematodes which we had received from Dr. Platzer had been passaged through Culex pipiens as a host, we successfully initiated a culture using Aedes aegypti. Embryos of R. culicivorax generally develop inside the eggshell to the L2 stage, where they arrest. Hatching can be induced by osmotic shock (Perez-Pacheco et al., 2004).

Cell nomenclature For better comparison between R. culicivorax and C. elegans standard names for germline and somatic founder cells (Sulston et al., 1983) were given based on cell behavior (germline) and sequence of birth (somatic founder cells) as outlined in Results. This can lead to confusion if cells with the same name and origin make different contributions to the embryo. Depending on the outcome of our lineage studies, a different nomenclature may be considered at a later date.

Microscopy Development was studied with Nomarski optics and pigmented cytoplasm with brightfield illumination, both using a 100× objective. For cell lineage studies early stage embryos were collected from culture dishes with a drawn-out pasteur pipette. Specimens were mounted on slides carrying a thin 3% agarose layer as a mechanical cushion. The coverslip was sealed with petroleum jelly (Vaseline). Embryos were recorded at 23 °C, either on video tape (Lahl et al., 2003) or with a 4-D microscope (Schnabel et al., 1997). A Zeiss Axioskop 2 microscope with an internal focus drive was used to record Z-series (45 focal levels, increment 1.5 μm) through the embryo. Images from an analog camera (VC 45, PCO, Kelheim, Germany) were digitized with an Inspecta-2 frame grabber (Mikroton, Eching, Germany) and compressed 10x with a wavelet function (Lurawave, LuraTech, Berlin, Germany). 3-D tracing of cell behavior was software-supported (Biocell, Simi, Unterschleissheim, Germany). Laser scan microscopy was done with a Zeiss Axiovert 100M microscope coupled to an LSM 510 Meta laser unit.

Laser micromanipulation For irradiation an N2-pumped laser microbeam (Photonic Instruments, St. Charles, Ill.; coumarin dye, absorption maximum 440 nm) coupled to a Leica DMLB microscope via glassfiber optics was used. Each embryo was irradiated 5 × 1 min (at 5–10 Hz) with 2-min intervals in between.

Antibody staining To decrease the thickness of the eggshell, eggs were incubated for about 4 min in a NaOCl solution (0.75% NaOCl, 25 mM KOH) and afterward washed 3× in distilled water. Treated eggs were transferred to polylysine-coated microscope slides and covered with a coverslip. After freezing in liquid nitrogen, the coverslip was quickly pried off and slides were incubated in methanol followed by acetone (20 min each at –20 °C). Slides were washed 30 min in PBST (PBS, pH 7.4, 0.1% Tween 20) followed by 30 min in blocking solution (PBS, pH 7.4,

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2% BSA, 0.5% Tween 20, 0.02% sodium azide). Four monoclonal antibodies against C. elegans P granules (K76, L416, OICID4, PIF4) were used essentially as described in Strome and Wood, 1983. To visualize microtubules we used the monoclonal antibody T9026 (Sigma; dilution 1:400).

Centrifugation of embryos Eggs were incubated about 1 min in an NaOCl solution and subsequently washed in distilled water (as described above) to allow their reliable sticking to the microscope slide. Treated eggs were transferred onto polylysine-coated slides essentially following the procedure of Schlicht and Schierenberg (1991). As a standard protocol embryos were centrifuged for 20 min at 1500 g in a Hermle Z300 centrifuge with swing-out rotor. However, stratification can already be achieved with 500 g.

Results General similarities and considerable differences between R. culicivorax and C. elegans Comparing early embryogenesis of R. culicivorax to the standard C. elegans, we found basic similarities. In both species cleavage patterns are essentially invariant, although they differ considerably from each other. Extensive lineage studies in progress (to be published elsewhere) make clear that also in R. culicivorax a germline exists. Although the first cleavage is equal with respect to cell size (Figs. 2a, 3c), it is nevertheless a polar division as cytoplasmic components are segregated into one daughter cell (see below). Offspring of the posterior blastomere of the 2-cell stage cleave in a stem cell-like fashion with three consecutive asymmetric divisions (two are shown in Figs. 2b–d). The small daughter of the last asymmetric division executes a symmetric cleavage and both descendants follow the gut precursors into the center of the embryo without any further division as far as studied (data not shown). Thus, these cells show the typical germline behavior as found in C. elegans and therefore have been named P1–P4. Generally, the larger sisters resulting from the unequal divisions follow fixed cell lineages with rather synchronous, distinct cell cycle rhythms and thus behave like somatic founder cells. The supposed founder cells were given standard names AB, EMS and C (Fig. 2; Sulston et al., 1983) based on their consecutive generation from the germline. This, however, does not imply that cell fate distribution is the same as in C. elegans, e.g., the intestine has a different origin (see below). In addition, we found in R. culicivorax peculiarities with respect to the establishment of cell polarity, germline behavior, axis formation, early arrangement of blastomeres and segregation of cytoplasmic components, all as outlined in the following. Embryonic axes, early pattern formation and polarity reversal in R. culicivorax C. elegans and R. culicivorax form a diamond-shaped 4-cell stage where all three main axes can be defined (Fig. 2C, c). However, the way this is reached differs markedly between the two species Figs. 2A–C; a–c). Taking into account the area of a cell in the plane of maximal extension and cell shape, we determined that in R. culicivorax the first cleavage generates

Fig. 2. Scheme of early cleavage patterns in C. elegans and R. culicivorax. In comparison to C. elegans (A–E), R. culicivorax (a–e) shows several prominent differences. (a), first division generates two daughters of equal size forming cleavage spindles with linear orientation; (b, c), due to limited space the dividing cells reorient, forming a rhomboid 4-cell stage. In R. culicivorax positions of EMS (dorsal) and ABp (ventral) are switched relative to C. elegans; (d), ABp divides asymmetrically with a–p spindle orientation while ABa divides into left and right daughters; (e), reversal of cleavage polarity in the germline takes place already in P1 and not P2 as found in C. elegans (E). Cells are named according to lineage. Cell fates may differ between both species. Yellow lines, cleavage spindles; purple dot, stiff cell connection at the site where the first midbody had formed. Orientation: A–D, a–d: left lateral view; E, e: dorsal view.

two cells of essentially equal size (n = 5; Figs. 2a, 3c). Both blastomeres divide simultaneously (in some cases slightly asynchronously) with the same linear spindle orientation as during the first division (Figs. 2a, b, 3C) while in C. elegans the zygote P0 divides asymmetrically and P1 always divides after AB. Initially, both spindles are oriented transversely there, followed by a rotation of the P1 spindle (Figs. 2A, 3C, insert). In C. elegans the definitive embryonic a–p axis runs through the centers of AB and P1 (Fig. 2A) and the d–v axis becomes fixed with the skewing of the dividing AB cell (Figs. 2B, C). For recent reviews of early events in the C. elegans embryo, see Cowan and Hyman, 2004; Gönczy and Rose, 2005. In R. culicivorax there is no space for the linear stretching of the dividing AB and P1 within the round eggshell (Figs. 2a, b, 3d, e). In addition, the first two cells and later also P2 + ABp and

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Fig. 3. Early cleavage, midbodies and mitotic spindles in R. culicivorax. (A) early 2-cell stage, prominent midbody (MB) present between AB and P1. (B) Rotation of the reconstructed LSM image visualizes its peripheral position. (b) nuclei form in asymmetric positions. c, initially nuclei stay in close vicinity of MB region (see b, purple dot). (C) mitotic spindles in both cells form with longitudinal orientation, in contrast to C. elegans (c, insert). Note asymmetric position of cleavage spindle in P1. (D) both cells divide synchronously with strong asymmetric position of the P1 spindle. (d–f) in germline cells granule-free areas appear in the region where the first MB had formed (blue arrows). (E, F) in the emerging 4-cell and 7-cell stages (optical section showing only selected blastomeres) prominent MBs are found between sister cells. Note asymmetric position of cleavage spindle in P2. White arrows, midbodies; red arrows, asymmetrically positioned cleavage spindles in germline cells. A–F, anti-tubulin staining; a–f, corresponding stages except c (earlier stage), Nomarski optics. For orientation of 2–4 cell stages, see Fig. 2F, f, ventral view. Scale bar, 10 μm.

P2/P3 + ABpp never move relative to each other but form permanent stiff connections with each other (Fig. 2, purple circle) at the site where the prominent first midbody (MB) has formed (Figs. 3A, B), even when the MB itself cannot be detected anymore with an antibody against tubulin (Fig. 3D). This results in a skewing of both dividing blastomeres, AB and P1 (Figs. 2b, 3D,d), and consequently in a definitive embryonic a–p axis that is approximately perpendicular to its original orientation as well as the concomitant fixation of the d–v axis at right angles (Figs. 2c, 3e). AB and P1 divide asymmetrically. Based on cell diameter and shape we estimated that ABp has about 1.5× the volume of ABa and EMS has 4–5× the volume of P2 (n = 5; Figs. 2c, 3e). Our long-term recordings revealed that, different to C. elegans, in R. culicivorax the position of ABp marks the ventral side of the embryo and the position of EMS the dorsal side (Figs. 2C, c). While ABa and EMS divide with transverse spindle orientations into left and right daughters of equal size and similar subsequent cleavage behavior, ABp cleaves with longitudinal spindle orientation into unequal daughters (Fig. 2d) which express different cell cycle lengths and form different spatial patterns. In contrast, in C. elegans ABa and ABp divide with transverse and EMS with longitudinal spindle orientations (Fig. 2D) and the daughters of EMS acquire different fates (Sulston et al., 1983). With respect to their position and division pattern but also the origin of the intestine (see below), EMS in C. elegans resembles ABp in R. culicivorax and vice versa. This switch in cell fates can be described in formal terms as an inversion of the d–v axis polarity compared to C. elegans.

To investigate whether cell–cell interactions affect the characteristic cleavage pattern in R. culicivorax (Fig. 2), we performed laser irradiation experiments. We found that after ablation of AB (n = 24) or P1 (n = 13) the surviving cells execute the same sequence and orientation of early cleavages as in the intact egg. For example, ABa still divides into left and right and ABp into anterior and posterior daughters, and the normal pattern of cytoplasmic segregation (see below) occurs, supporting the view of autonomous early cell behavior in R. culicivorax. The special behavior of blastomeres in the 2–4 cell stages reveals another peculiarity. In C. elegans a reversal of cleavage polarity in the germline (i.e. a cleavage resulting in an anterior rather than a posterior position of the new germline cell relative to its somatic sister; Schierenberg, 1987), along with an anterior shift of the cleavage spindle, takes place in P2 (Fig. 2E; obvious only without the constraints of the eggshell). In R. culicivorax this polarity reversal is found already in P1 (Figs. 2b,e, 3C–E), i.e. P2 comes to lie anterior of EMS. This premature polarity reversal ascertains that a rhombic pattern with the germline adjacent to gut precursors is reached in the 4-cell stage as in C. elegans (Figs. 2c, 3e), despite the preceding massive cell rearrangements (Fig. 2a–c). Loss of cellular polarity after laser irradiation of the midbody region In contrast to C. elegans, we found prominent midbodies (MB) in the early embryo of R. culicivorax (Fig. 3). Our

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observation that nuclei in the 2-cell stage localize asymmetrically adjacent to the MB region (Figs. 3b, c) and that later the germline spindle is displaced toward this area (Figs. 3C, D) indicates a polarizing function. Our recordings reveal that during subsequent asymmetric cleavages of the germline the "region of the first midbody" (RFM) occupies a position at the junction between P2 and ABp (Figs. 2c, 3e), and later between P2/P3 and ABpp (Figs. 2d, 3f). This area is characterized by the absence of yolk granules (Figs. 3d–f), indicating an accumulation of cytoskeletal material. Anterior positions of the cleavage spindles of P2 (Fig. 3F) and P3 (not shown) suggest that the RFM continues to express a spindle-attracting function. Therefore, we irradiated the RFM in the 2-cell stage (n = 7). This resulted in minor retardations of the cell cycle but dramatic changes in cell behavior. In the 2-cell stage nuclei relocalized from peripheral to central positions (Fig. 4A) and spindles in AB and P1 either showed the normal linear orientation (Fig. 4B) or variably abnormal orientations up to completely transverse (Fig. 4b). In all cases subsequent cleavages produced cells of equal size (Fig. 4C,c) indicating that in these embryos polarity, i.e. the potential for correct spindle alignment and positioning, had been lost. In control experiments similar amounts of irradiation applied to P1 either to the cytoplasm or to the cortex outside the MB region (n = 14), did not affect normal cleavage. Thus, our results indicate a central role of the MB region for spindle orientation and asymmetric cleavages in the R. culicivorax embryo not only in the germline but also in the AB cell.

Bilateral symmetry In the 6-cell stage of C. elegans a shift of the two left AB descendants relative to those on the right side takes place (Schierenberg, 1984). This constitutes an early breaking of bilateral symmetry which is compensated by different cell fate assignments in contralateral lineage branches, particularly in ABa descendants (Sulston et al., 1983). In R. culicivorax we did not find such a shift, neither between ABal and ABar (ABp divides with a–p spindle orientation; Figs. 2d, 3F) nor between their daughters or granddaughters. Instead, these cells seem to form perfectly symmetric clones on the left and right side of the embryo (Figs. 5d–h;our unpublished data). This suggests that in R. culicivorax bilateral symmetry within the ABa lineage is established with its first division as found in MS, C and D of C. elegans (Sulston et al., 1983). Segregation of pigmented cytoplasm A prominent and thus far undescribed phenomenon in nematodes is the presence and segregation of colored cytoplasm in R. culicivorax. Initially, this marker is equally distributed all over the 1-cell stage. However, prior to first cleavage it is translocated toward the posterior pole (Fig. 5a), which is usually marked by the presence of polar bodies (98%, n N 200; Fig. 3b), while in C. elegans these products of meiosis are localized at the anterior pole. This demonstrates that an early cytoplasmic polarity is present despite an equal first cleavage. In the 2-cell stage the colored marker is found in P1 (Fig. 5b). With the division of P1 the brownish component is shunted into EMS (Fig. 5c). This segregation process is not necessarily fully exclusive and to a variable degree minor amounts may also be found in AB and P2. With the transverse division of EMS both daughters receive equal proportions of pigment (Fig. 5d). During the following divisions in the EMS lineage no further segregation takes place (Figs. 5e–h). The cells that receive the brown cytoplasm later extend over most of the embryo (Figs. 5g–i). Our studies in progress indicate that these cells contribute to ectodermal but not to endodermal tissue. Preliminary histological tests (Lillie and Fullmer, 1976) suggest that the brown pigment is melanin. Segregation of cytoplasmic pigment after irradiation and centrifugation

Fig. 4. Symmetric cleavage after ablation of the midbody region. (A) after ablation both nuclei shift to central positions. Two extreme variants of subsequent cell behavior are shown (B–D; b–d). (B) spindle positioning in the 2-cell stage is normal. (C) symmetric cleavage and normal cell rearrangements take place. (D) brown pigment (see Fig. 5) is differentially distributed to P1 daughters. (b) alternatively, spindle orientation is perpendicular to normal. (c) symmetric cleavage takes place. (d) cytoplasmic pigment is equally distributed to both blastomeres. Pink color of P1 descendants, loss of germline behavior; yellow lines, cleavage spindles; white connecting bars, sister cells.

We demonstrated that laser irradiation abolished asymmetric cleavage (see above). The segregation of cytoplasmic pigment allowed us to test whether under these experimental conditions cytoplasmic partitioning was affected. After laser treatment (n = 7) we found that concomitantly with symmetric division also the colored component was equally distributed to both daughter cells in five of seven embryos (Fig. 4d). However, in two cases the pigment was predominantly found in one of the sister cells (Fig. 4D). This observation could be explained by a late irradiation which prevented asymmetric positioning of the spindle but not the preceding cytoplasmic segregation. During

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Fig. 5. Segregation of pigmented cytoplasm in R. culicivorax. Prior to first cleavage colored cytoplasm accumulates at the posterior pole (a) and is then segregated into P1 (b); during the ensuing cleavages it is first shunted into EMS (c) and then into both daughters of EMS (d); consequently, all 4 (e), 8 (f), 16 (g) as well as the subsequent descendants of EMS (h, i) receive the brown component. Orientation (c, i), left lateral view; (d–h) dorsal view. Scale bar, 10 μm.

subsequent cleavages neither asymmetry nor (further) segregation of pigment was found. In order to test whether a cell-specifying potential is associated with the brownish cytoplasm and what effect its translocation has on further development, we centrifuged 1-cell embryos. This resulted in three distinct cytoplasmic layers similar to experiments in C. elegans (Schlicht and Schierenberg, 1991) with the brown pigment localized to the centripedal pole (Fig. 6a). Depending on the egg orientation relative to the centrifugal force, different abnormal patterns were obtained, with three variants most frequent (Figs. 7A-a′; B-b′; C-c′). If the first cleavage furrow bisects the layers, both daughter blastomeres receive the pigment (Figs. 6b, 7a; n = 36). Subsequently, it is usually segregated into ABp and EMS (Figs. 6c, 7a′). If the cleavage furrow is parallel to the stratification, the pigment is either shunted into AB (Fig. 7b) or into P1 (Fig. 7c). Subsequently, the pigment is usually exclusively segregated into ABp (Figs. 6d, 7c′; n = 11) or into EMS (Figs. 6e, 7b'; n = 7). In the former case the pigment is shunted into ABpp (not shown), in the latter case into both EMS daughters, as is found in the untreated embryo (Fig. 5d). In a few centrifugation experiments both AB descendants received the pigment (Fig. 6f; n = 4), probably because such embryos had been exposed to centrifugal force during a critical phase of the cell cycle, which prevented typical segregation. In these embryos only ABp segregated colored cytoplasm into its posterior daughter ABpp, while both ABa descendants received equal

amounts (not shown). Our observations support the view that differences in developmental potential are only partitioned along the a–p axis. We followed further development and terminal phenotypes of centrifuged embryos and found that all variants pass through the normal sequence of early cleavages (Fig. 2) and can develop into normal-shaped and moving juveniles furled in the eggshell. Hence, the prominent pigmented cytoplasm does not influence cell patterning and specification. The fact that in each experiment pigmented cytoplasm is confined to a specific cell cluster (not shown) indicates invariant cell pattern formation and that the pigment can be used as a marker for cell lineage studies. This is particularly obvious in embryos where it is restricted to ABp (Fig. 6d). Here, the pigment becomes concentrated in a group of cells in the center of the embryo which later gives rise to intestinal and pharyngeal tissue (Figs. 6g–i). Thus, in R. culicivorax gut is derived from ABp, in contrast to C. elegans, where it is generated by EMS descendants (Sulston et al., 1983). This conclusion is consistent with our ongoing lineage studies. Visualization of nuclear structures with antibodies against P granules We tested if in R. culicivorax germline-specific granules (“P granules”; Strome and Wood, 1983) can be visualized. For this, we applied several monoclonal antibodies which detect these structures in C. elegans. We found that none of them marked

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Fig. 6. Segregation of colored cytoplasm in centrifuged embryos of R. culicivorax. (a), formation of three distinct cytoplasmic layers after centrifugation of the uncleaved fertilized egg; (b), pigment is shunted into AB and P1 and (c) later into EMS and ABp. Alternatively, colored cytoplasm may become localized to ABp (d) and subsequently (arrow) to blastomeres giving rise to intestine and pharynx (g–i); (e) if localized to EMS, no further segregation takes place; (f) in rare cases both AB daughters are marked. Orientation (b–f, i), left lateral view, (g, h), dorsal view. Scale bar, 10 μm.

selectively germline structures, but two of them K76 and particularly L416 visualized nuclear spots in all blastomeres during all embryonic phases (Figs. 8A, B) The spots tend to be smaller and more numerous in early stages than in advanced embryos, where often only one or two per nucleus are found. Our preliminary data from fluorescent markers for DNA and RNA indicate that the regions of antibody binding mark sites of RNA synthesis (data not shown). However, like in C. elegans but different to some other nematodes (Laugsch and Schierenberg, 2004) we could not identify distinct nucleoli in early R. culicivorax embryos. Discussion

rather than a derived character as had been suggested earlier (Skiba and Schierenberg, 1992). The high variability provokes the question what its function may be (see below). In the R. culicivorax embryo polarity of the d–v axis appears to be inverted compared to C. elegans, such that EMS (generating epidermis) defines the dorsal and ABp (generating gut) the ventral side. However, in contrast to other systems like Drosophila (Moussian and Roth, 2005), in the nematode embryo specification according to position along the d–v axis appears not to play a role. We rather interpret this switch as a consequence of (a) a different fate distribution among somatic founder cells, (b) the early polarity reversal in the germline and (c) the characteristic cell rearrangements taking place in the 2–4 cell stages (Figs. 2 and 3).

Reversal of cleavage polarity and polarity of the d–v axis Region of the first midbody (RFM) and asymmetric cleavage The pattern of early embryogenesis in R. culicivorax differs considerably from all studied members of clades 3 to 12 (Fig. 1; Malakhov, 1994; Houthoofd et al., 2003; Schierenberg, 2006; Zhao et al., in press; our unpublished results). One of these differences is a reversal of cleavage polarity in the germline cell P1, which we also observed in other groups of clade 2 (i.e., Dorylaimida and Mononchida; Malakhov, 1994). In species of clades 9–11 polarity reversal occurs in P2, P3 or is even completely absent (Skiba and Schierenberg, 1992; Laugsch and Schierenberg, 2004). The fact that polarity reversal is found in various branches of nematodes suggests that it is a primitive

In P1 and later germline cells of C. elegans a bleb-like organelle associated with the anterior cell membrane forms which has been proposed to function as a cortical attachment site for astral microtubules (MT) required for centrosome rotation (Hyman and White, 1987; Hyman, 1989). Actin and capping protein transiently accumulate at this structure, which is thought to be a persistent remnant from prior cell divisions (Waddle et al., 1994). The observation in par-3 mutant embryos with a displaced midbody that the mitotic spindles are oriented toward it (Keating and White, 1998), suggests a central function

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appears not unlikely that a mes-1 homologue is also responsible for germline behavior in R. culicivorax, where it would be expected to be present already in P1. Similarities to how cellular asymmetry is generated in nematodes can be found in chordates. In ascidian embryos asymmetric divisions are induced by a cortical structure in the posterior vegetal region called CAB (“centrosome-attracting body”; Hibino et al., 1998) which is associated with an aPKC/ PAR-6/PAR-3 protein complex as found in the C. elegans zygote (Patalano et al., 2006). Loss of the CAB results in the abolishment of unequal cleavage and the ectopic formation of this organelle leads to ectopic asymmetric cleavages (Nishikata et al., 1999). A scenario for the evolution of germline behavior in nematodes

Fig. 7. Three most common patterns of cytoplasmic segregation after centrifugation. Centrifugation of 1-cell stages leads to pigment caps (black; A–C). Depending on the orientation of the cleavage spindle relative to this cap, either AB + P1 (a) or AB (b) or P1 (c) receive the cytoplasmic pigment at the next division. Due to subsequent segregation the brown component is found in either ABp + EMS (a′), ABp (c′) or EMS (b′). The latter is similar to the normal distribution (see Fig. 5c), but P2 is completely devoid of pigment. All variants are compatible with normal further development.

of this organelle or the adjacent region during cell division. However, an alternative model for positioning of the mitotic spindle in C. elegans has been suggested in which cell shape and a cortical band of LET-99 are crucial (Tsou et al., 2003; Wu and Rose, 2007). The unusual early cell rearrangements in R. culicivorax can be explained with the stiff connection between the dividing AB and P1 cells apparently caused by the prominent first midbody and the polarity reversal in P1 discussed above. The region of the first midbody (RFM) seems to play a crucial role in the asymmetric germline divisions since it attracts the mitotic spindle (Figs. 3C, D, F) and its ablation results in the loss of polarity (Fig. 4). The RFM shows a close resemblance to the area where MES-1 is expressed in C. elegans. MES-1 is a transmembrane protein which is only found on the surface of germline cells P2 (where a reversal of cleavage polarity takes place, see above) through P4 (where cleavage polarity is lost) adjacent to EMS, E and Ep, respectively (Berkowitz and Strome, 2000). Its position corresponds to the site where originally the midbody between P2 and EMS had formed. Elimination of MES-1 results in the loss of germline polarity after the 4-cell stage. It has been suggested that MES-1 pulls the nucleus–centrosome complex toward itself, resulting in proper alignment and asymmetric positioning of the spindle. Hence, it

Although Mermithida are positioned much closer to the base of the phylogenetic tree of nematodes than Rhabditida (Aleshin et al., 1998; Blaxter et al., 1998; Holterman et al., 2006), this does not necessarily imply that the cleavage pattern of R. culicivorax is more original than that of C. elegans. However, outgroup comparisons with Nematomorpha (Malakhov and Spiridonov, 1984) and Tardigrada (Hejnol and Schnabel, 2005; Gabriel et al., 2007) give some support to this assumption since in both taxa the first cleavage is symmetric like in R. culicivorax. In addition, other similarities in the early cleavage pattern to nematodes of clades 1 and 2 can be found (Malakhov, 1994; our unpublished results). Here, we hypothesize that it was changes in germline behavior that led to the shift from a R. culicivorax-like pattern to a C. elegans-like pattern and sketch the following simplified scenario incorporating our findings described above. In both species all germline cells divide with an a–p spindle orientation and thus disregard the “centriolic principle” (Costello, 1961) according to which a cell division occurs at right angles to the previous one if no additional forces act. Hence, a spindle-orienting mechanism must be postulated even during the symmetric division of P4. In R. culicivorax intestine is derived from the AB lineage and polarity reversal in P1 assures that germline and gut precursors become and remain neighbors (an association found in many organisms and thought to be essential for normal development; Wylie, 1999; Sulston et al., 1983). According to our model, this polarity reversal is induced by the region of the first midbody (RFM; see above), which functions as a spindleattracting site leading to asymmetric cleavages of P1, P2 and P3. In P0 asymmetry-inducing pulling forces (Cowan and Hyman, 2004) are not yet present, and in P4 not present any more, resulting in symmetric cleavages. During the course of evolution a shift in the developmental potential took place in an early ancestor of C. elegans, such that gut fate was now inherited by P1 and its daughter EMS instead of AB. To preserve under these conditions the germline/gut neighborhood, polarity reversal was postponed by one generation from P1 to P2 (polarity reversal was given up completely as an alternative in members of clade 11, thus requiring compensating germ cell migrations; Skiba and Schierenberg,

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Fig. 8. Visualization of nuclear components with antibody L416 against C. elegans P granules. (A) each nucleus of a 4-cell stage is marked with several granules; (B) each nucleus of a morphogenesis stage is marked with one or a few granules. (a, b) corresponding stages with Nomarski optics. Orientation, left lateral view. Scale bar, 10 μm.

1992). Asymmetric cleavages in P2 and P3 were now induced by the region of the second midbody (remnant of P1 division). To allow asymmetric cleavages in P0 and P1 (necessary to separate the additional developmental options carried by P1, e.g. ectodermal from endodermal potential), a new, temporarily acting cortical pulling force causing asymmetric mitotic spindle positioning was established at the posterior pole of the egg (Schierenberg, 1987; Grill et al., 2001; Grill and Hyman, 2005; Nguyen-Ngoc et al., 2007) in conjunction with fertilization (Wallenfang and Seydoux, 2000). Segregation of colored cytoplasm The most obvious difference between the R. culicivorax embryo and all other studied nematodes is the presence of colored cytoplasm which is segregated reproducibly into the EMS cell and its descendants. This segregation process starts during late interphase of the first mitosis with the movement of the pigment toward the posterior cell pole. This is reminiscent of the translocation of germline-specific P granules in C. elegans which takes place prior to mitosis and is microfilamentdependent (Strome and Wood, 1983). A comparable cytoplasmic segregation has been described in ascidian embryos where colored cytoplasm is shunted into future body muscle cells (Conklin, 1905; Jeffery and Swalla, 1997). The pattern of segregation is similar but not identical to that in R. culicivorax. In both species partitioning takes place during the very early cleavages only in divisions with an a–p spindle orientation, such that the pigment is found in only two blastomeres of the 8-cell stage. In ascidians the colored cytoplasm contains morphogenetic determinants. If this "myoplasm" is experimentally distributed to more than the normal number of blastomeres, additional muscle cells form (Whittaker, 1983; Nishida, 1992). Our centrifugation experiments

(Figs. 6, 7). did not give any hints for a cell-specification potential of the pigmented cytoplasm. If the colored component does not play a role in cell specification, what other function could it have? It is known that pigments are widely used in the animal kingdom to control the exposure to light. As in R. culicivorax it is shunted into cells that contribute to epidermis and is absent or reduced in the head region of the advanced embryo (Fig. 5i) it could be involved in guiding the juvenile from the bottom of the body of water to its mosquito host on the surface. Alternatively, the pigment could protect the nematodes from excessive UV radiation while moving through the water in search of a suitable host. The melanin pigment in the animal hemisphere of amphibian oocytes has been suggested to function as a sunscreen (Duellman and Trueb, 1986). The production of melanin as an innate immune response has been reported for arthropods (Christensen et al., 2005). However, we do not have any indication that R. culicivorax makes use of the brown pigment in a similar way nor that it is of mosquito origin. To determine, if segregation of pigment is a phenomenon restricted to R. culicivorax, we initiated studies in two close relatives, Romanomermis iyengari and Strelkovimermis spiculatus. We found an essentially identical situation in all three species including the characteristic reorientation of blastomeres in the 2–4 cell stages and the early segregation of brown cytoplasm into the dorsal EMS cell. Thus, our observations reported in this paper may apply to the taxon Mermithida in general. Our ongoing analysis of several species of the genera Mononchida and Dorylaimida (both also members of clade 2 like R. culicivorax) revealed the absence of colored cytoplasm in these taxa. On the other hand we found some obvious similarities to mermithids during early cleavage, including a polarity reversal in P1 and gut derived from the AB lineage (Malakhov and Spiridonov, 1984) which fits to the scenario sketched above. Acknowledgments We are very grateful to Dr. Edward Platzer, University of California, Riverside, for generously supplying us with mermithid nematodes, for sharing unpublished results and for advice. We thank Randall Cassada for critical reading of the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 572) to E.S. References Aleshin, V.V., Kedrova, O.S., Milyutina, I.A., Vladychenskaya, N.S., Petrov, N.B., 1998. Relationships among nematodes based on the analysis of 18S rRNA gene sequences: molecular evidence for monophyly of Chromadorian and Secernentean nematodes. Russ. J. Nematol. 6, 175–184. Berkowitz, L.A., Strome, S., 2000. MES-1, a protein required for unequal divisions of the germline in early C. elegans embryos, resembles receptor tyrosine kinases and is localized to the boundary between the germline and gut cells. Development 127, 4419–4431. Blaxter, M.L., De Ley, P., Garey, J.R., Liu, L.X., Scheldeman, P., Vierstraete, A., Vanfleteren, J.R., Mackey, L.Y., Dorris, M., Frisse, L.M., et al., 1998. A molecular evolutionary framework for the phylum Nematoda. Nature 392, 71–75.

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