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Evolution of nematode development Ralf J Sommer Multiple evolutionary variations occur in the cellular and genetic programming of nematode development. Many changes involve alterations of inductive interactions. Surprisingly, inductive processes vary during evolution, irrespective of changes in the final cell lineages and morphological structures. Genetic studies in some nematodes also shed light on the underlying mechanisms of evolutionary change. Addresses Max-Planck Institute for Developmental Biology, Department for Evolutionary Biology, Spemannstrasse 35, 72076 Tübingen, Germany; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:443–448 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AC anchor cell EGF epidermal growth factor MAPK mitogen-activated protein kinase

Introduction Principles of development and pattern formation have been studied comprehensively in the free-living nematode Caenorhabditis elegans [1,2]. The invariant cell lineage has allowed developmental processes to be studied at a cellular level. In addition, intensive genetic and molecular studies provide a detailed picture of the molecular and biochemical interactions during cell-fate specification. With the sequencing of the C. elegans genome and the introduction of RNA-interference as a gene-knockout strategy, C. elegans is now heading for a ‘new career’ in applied sciences [3,4]. At the same time, however, the wealth of biological information from C. elegans makes it an excellent reference system for evolutionary studies [5,6]. Comparative cell-lineage analyses indicate that embryonic and postembryonic developmental processes vary substantially among nematodes. In some species, development can be studied using genetic tools so that the mechanisms of evolutionary change can be elucidated. Here, I discuss recent advances concerning the evolution of cell–cell interactions and inductive processes during nematode cell-fate specification.

Nematode phylogeny and diversity Nematodes were classically viewed as pseudocoelomates and many textbooks treated them as a minor phylum [7]. This picture has changed dramatically in recent years. Molecular studies indicate a close phylogenetic relationship of nematodes to arthropods and other phyla which undergo molting [8,9]. As a result, a new monophyletic taxon, the Ecdysozoa, has been proposed and exciting

discussions are underway between proponents of the competing phylogenetic hypotheses [10]. Nematodes inhabit nearly all ecosystems and are prolific [11,12]. Species can either be free-living or parasitic, infecting both plants and animals. Recent estimates suggest that the nematode phylum is the biggest in the animal kingdom, superseding the arthropods [13]: in view of the enormous nematode fauna in the marine benthic system and the number of nematode parasites in arthropods, up to 100 million species of nematodes have been estimated [14].

Embryogenesis Nematode embryogenesis has attracted researchers as early as the 19th century. Boveri identified the presence of a germline while studying the early cleavage in Parascaris (formerly Ascaris megalocephala) [15]. C. elegans, like Parascaris, undergoes a series of differential cleavages giving rise to distinct blastomere founder cells which develop in an invariant stereotyped pattern (Figure 1a,b) [16]. In C. elegans, it is known that the five somatic founder cells AB, MS, E, C and D develop by highly reproducible intercellular signaling processes [17]. For example, the founder cell E, which is the posterior daughter of the 4-cell blastomere EMS, forms the complete gut of the worm (Figure 1a). Furthermore, E is the only blastomere that is capable of forming gut. Elegant experiments by Goldstein [18] indicated that gut does not differentiate if EMS is isolated from its neighbors in the early 4-cell stage (Figure 1c). If EMS is recombined with its posterior neighbor P2, however, gut differentiation occurs (Figure 1d). This effect was not seen when EMS was recombined with its anterior neighbors ABa and ABp. Therefore, P2 induces the fate of the E cell in C. elegans. Subsequent molecular analysis has revealed that Wnt signaling is involved in the EMS–P2 interaction. Mutations in Wnt-signaling components resulted in a more-of-mesoderm (mom) phenotype, and both MS and E produced mesoderm [19–22]. In contrast, mutations in the gene for the downstream transcription factor pop-1 produced an opposite phenotype, with MS and E forming endoderm [22]. Comparative cell lineage and cell ablation studies indicated that the mode of founder cell specification differs among nematodes. In Acrobeloides nanus of the Cephalobidae family, the posterior part of the egg develops much faster than the anterior part so that the primordial germ cell P4 is already present at the 5-cell stage (Figure 1e) [23]. In contrast, in C. elegans, P4 is only generated at the 24-cell stage (Figure 1a). As a result, the spatial arrangement of the blastomeres differs substantially between A. nanus and C. elegans (Figure 1b,f). Cell-ablation studies in A. nanus indicated that all cells of the 3-cell embryo, AB–EMS–P2, are capable of forming gut

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(Figure 1g) [24]. Therefore, inhibitory interactions among these three founder cells are required to restrict gut fate to a single blastomere. Moreover, additional cell-ablation studies have indicated a surprising regulative potential: if blastomere AB is ablated, it is replaced by EMS, and EMS is itself replaced by its posterior neighbor C (Figure 1h) [25••]. Thus, hierarchical cell fate transformations are observed in A. nanus, a regulative phenomenon not seen in C. elegans. This pattern is, however, reminiscent of the sea urchin embryo — the paradigm example of regulative development. It would be interesting to know if this type of cell–cell interaction during gut specification also requires Wnt signaling.

division pattern varies between embryos [26,27]. At the 8cell stage, one blastomere is the founder cell for gut, whereas all other blastomeres form multiple cell types in various proportions. Thus, the E. brevis cell lineage is not invariant and the blastomeres are not determined. One might speculate, therefore, that the development of ancestral nematodes differs substantially from C. elegans. Additional support for such an hypothesis comes from the recent finding that some nematodes have variable cell numbers mostly as a result of individual-specific cell division patterns in the lateral hypodermis [28•].

Postembryogenesis Although many nematodes, like C. elegans, A. nanus or Parascaris, exhibit invariant cell lineages during embryogenesis, this pattern seems to be a derived character within nematodes. Free-living marine species, which are believed to represent the ancestral form [11], show variable cleavage patterns. Cell-lineage analysis and intracellular injection of fluorescent dyes in one such species, Enoplus brevis, indicated that blastomeres are indistinguishable and that the

Many sex- and position-specific structures are formed during postembryogenesis. Several of these characters — like the male rays [29], the female gonad [6], the buccal capsule [30], and general body size [31••] — have been compared among species and multiple evolutionary variations were observed. For instance, the study of nematode body size indicated that the size of the hypodermal syncytium can evolve repeatedly within nematodes and two

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Figure 2 (a) Cell-fate specification in the ventral epidermis of C. elegans. The 12 Pn.p cells are distributed equally between the pharynx and the rectum. P(1,2,9–11).p have an epidermal fate and fuse with the hypodermal syncytium hyp7 (white ovals). P(3–8).p form the vulva equivalence group and have the spatial pattern 3°-3°-2°-1°-2°-3°. 3° cells (dotted ovals) divide once and fuse with hyp7. 2° cells (grey ovals) generate seven progeny and form the anterior and posterior part of the vulva. The 1° cell (black oval) generates eight progeny, some of which connect the vulva to the uterus. The anchor cell is shown as a small grey circle. P12.p (hyp12) has a special fate and forms part of the rectum. (b) Modes of vulva induction in species of three different families of nematodes. Oscheius, two-step induction with both steps provided by the AC (small white circle). C. elegans, one-step induction. P. pacificus, continuous induction from early gonadal stages until the birth of the AC. Teratorhabditis palmarum and Brevibucca sp. are species with a posterior vulva not requiring a gonadal induction. Panagrolaimus sp. and Halocephalobus sp. with a two-step and threestep induction, respectively. Arrows indicate cell–cell interactions. The gonad is shown as a long white oval. Outer (2°) vulval fates are shown in grey, inner (1°) vulval fates are shown in black. Phylogeny is based on work described in [40••,42]. (See main text for details.)

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different cellular mechanisms, changes in cell number and the extent of acellular growth of the syncytium, can account for these variations [31••]. Surprisingly, free-living nematodes with a large body size grow predominantly in the final adult stage. Therefore, body size increase occurs mainly by changing the extent of acellular growth which requires somatic polyploidization. A comprehensive evolutionary study has been conducted on the development of the vulva, the egg-laying system of nematodes. In all species studied to date, the vulva develops from the ventral epidermis which consists of 12 epidermal precursor cells denoted as P(1–12).p from anterior to posterior (Figure 2a). In C. elegans, a total of six cells (P3.p–P8.p) have the competence to participate in vulva formation. The gonadal anchor cell (AC) induces three of these six cells, usually P5.p–P7.p, to form vulval tissue by EGF/RAS/MAPK signaling [32]. Recent findings reveal that Wnt signaling is also involved in the regulation of downstream transcription factors [33].

Previously, vulva development has been compared among many species of the Rhabditidae, the Diplogastridae and some Panagrolaimidae [34–38]. Félix et al. [39••] have extended this comparison to the Cephalobidae, Strongylidae, Brevibuccidae and the Myolaimidae. The most striking aspect of vulval variation is the diversity in vulval induction. In contrast to C. elegans, where the AC as a single cell induces vulva formation, induction can occur as a two-step process or as a continuous interaction between the gonad and the underlying epidermis (Figure 2b). In Pristionchus pacificus of the Diplogastridae, the gonad provides a continuous signal for vulva development which starts in the first juvenile stage and lasts until the birth of the AC [40•]. In several species of the Panagrolaimidae, the Cephalobidae and Oscheius sp. CEW1 of the Rhabditidae, a two-step induction occurs. In the latter, both signals come from the AC, whereas in some species of the Panagrolaimidae and Cephalobidae, the first signal already stems from the gonad (Figure 2b) [37,39••].

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Schematic summary of vulva induction in C. elegans and P. pacificus. (a) In C. elegans, the AC induces vulva formation in P(5–7).p by providing an EGF-related signal encoded by the gene lin-3. An EGFR/RAS/MAPK pathway transmits this signal and regulates the activity of several downstream transcription factors. One of these transcription factors is LIN-39, which provides positional information earlier in development for the formation of the vulva equivalence group. (b) In Cel-lin-39 mutants, the vulva equivalence group is not formed and P(3–8).p fuse with hyp7. (c) If Cel-lin-39 is provided early in development but not during vulva induction, P(3–8).p have an uninduced fate (3° fate), indicating that Cel-lin-39 is required during vulva induction. Genetic experiments have indicated that this function occurs in response to EGF/RAS/MAPK signaling. (d) In

P. pacificus, ventral epidermal cells in the anterior and posterior body region die of apoptosis. P(5–7).p form the vulva, as in C. elegans. The somatic gonad induces vulva formation in a continuous interaction with the underlying Pn.p cells. It is unknown if the EGF/RAS/MAPK pathway is involved in transmitting this signal. (e) In a Ppa-lin-39 mutant, P(5–8).p die of apoptosis like their anterior and posterior counterparts, indicating that Ppa-LIN-39 specifies the vulva equivalence group. (f) In the double mutant Ppa-lin-39 Ppa-ced-3, in which cell death cannot be executed because of a mutation in the cell death regulator Ppa-ced-3, a normal vulva is formed. Thus, Ppa-LIN-39 provides positional information for the formation of the vulva equivalence group but is not used to transmit gonadal signaling. (Cel, C. elegans; Ppa, P. pacificus.)

In Halicephalobus sp. JB128, three separate interactions occur between gonad and epidermis (Figure 2b) [38]. The gonad provides a survival signal for the vulva precursor cells. If the gonad is ablated early in larval development, the vulva precursor cells will die of programmed cell death. If the gonad is ablated before the AC is born, the vulva precursor cells will only form one of two alternative cell fates, indicating that a gonadal signal is involved in a first induction. Later, the AC contributes an additional signal for the specification of the second vulva cell fate (Figure 2b).

of nematodes (Figure 2b) [41]. This projection does not allow one to infer the ancestral pattern of vulva induction as too many different modes of induction exist but two important conclusions can be drawn. First, gonad-independent vulva formation in Mesorhabditis/Teratorhabditis on one hand and Brevibucca on the other evolved independently from one another and thus indicate a case of convergent evolution. Second, the one-step induction observed in C. elegans is specific for this genus and therefore represents a derived character. C. elegans vulva induction by the AC, a textbook paradigm for pattern formation and EGF/RAS/MAPK signaling, is a highly evolved feature. Like the syncytial blastoderm of the Drosophila egg, which differs substantially from the mode of early development in other insects, vulva induction by a one-step mechanism in Caenorhabditis is not representative for other nematodes.

In other nematodes, vulva differentiation is gonad-independent. Such a pattern was observed in Brevibucca of the Brevibuccidae [39••] and previously in Mesorhabditis sp. PS1179 and Teratorhabditis palmarum of the Rhabditidae (Figure 2b) [34]. The different modes of vulva induction — one-step, twostep, continuous induction and no induction by the gonad — can be projected onto a phylogenetic classification

A satellite organism: Pristionchus pacificus Given these variations at the cellular level, a question arises whether the underlying molecular processes have also

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changed. In Pristionchus pacificus, several genes involved in vulva formation were identified [42,43]. One such gene is the homeotic transcription factor lin-39. Cel-lin-39 is used twice during vulva formation: early in development, Cel-lin-39 specifies the vulva equivalence group, whereas later on it provides specificity to the EGF/RAS/MAPK signaling pathway (Figure 3a–c) [44,45]. Ppa-lin-39 also specifies the vulva equivalence group and the vulva precursor cells die of apoptosis in Ppa-lin-39 mutants (Figure 3d,e) [46]. Double mutants between Ppa-lin-39 and the cell death regulator Ppa-ced-3 form a normal vulva, however, indicating that in contrast to Cel-lin-39, Ppa-lin-39 is not re-used during vulva induction (Figure 3f) [47]. Thus, cellular and molecular differences do exist in vulva induction between C. elegans and P. pacificus. It is unknown, however, if only lin-39 or the complete EGF/RAS/MAPK signaling cascade changed its function during nematode evolution (Figure 3d).

There is no obvious reason why this diversity in mechanisms should be unique to nematodes. Comparative studies among arthropods or vertebrates point in a similar direction [53]. At first sight, these findings are in contrast to the general notion of the conservation of developmental control genes but no matter how similar the primary sequences of developmental control genes are, what they do and how they interact with each other changes greatly during evolution. Similarity or divergence is, thus, just a matter of consideration. Therefore, the real question ahead of us is to understand by what type of mechanisms genes change their function to create the diversity of life.

The case of lin-39 does not seem to be unique — other genes, like mab-5, also changed their functional specificity during nematode evolution. The comparison of the mab-5 mutant phenotypes between C. elegans and P. pacificus has revealed several functional differences with regard to vulva formation [48]. Genetic analysis in selected nematodes thus provides a tool for the analysis of the molecular evolution of developmental processes. Other than P. pacificus, the rhabditid Oscheius sp. CEW1 is also amenable to genetic analysis [49].

References and recommended reading

In summary, multiple mechanisms are used to build a perfect vulva in a species-specific way. Although no molecular picture as clear as in C. elegans is yet available in any other nematode, available data indicate that the relative importance of signaling systems and transcription factors changed during evolution. Nonetheless, the organ structure itself and the cells contributing to it are homologous in all nematodes studied [50].

A genomic approach The completed sequence of the C. elegans genome provides a platform for the systematic analysis of gene function. For example, DNA arrays and two-hybrid analyses are used to study the biochemistry of developmental processes [51,52••]. Taking these large-scale approaches in C. elegans as a reference, future work on selected evolutionary problems should also include genomic approaches. As a starting point in P. pacificus, an expressed sequence tag sequencing project has been initiated which will provide some of the resources required for a detailed analysis at a genomic level (RJ Sommer, J McCarter, unpublished observations).

Conclusions Evolutionary developmental biology in worms provides two major findings. Strikingly, developmental processes vary during evolution whether or not the final morphological structures differ between species. Furthermore, molecular and cellular aspects underlying developmental processes change rapidly during evolution and multiple mechanisms can be involved in cell-fate specification.

Acknowledgements I would like to thank J Srinivasan for critically reading the manuscript and the present and former lab members for discussions. I would also like to thank my many colleagues in ‘nematode evolution’ for fruitful and always open interactions.

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38. Félix MA, Sternberg PW: A gonad-derived survival signal for vulval precursor cells in two nematode species. Curr Biol 1998, 8:287-290. 39. Félix MA, De Ley P, Sommer RJ, Frisse L, Nadler S, Thomas KW, ·· Vanfleteren J, Sternberg PW: Evolution of vulva development in the Cephalobina (Nematoda). Dev Biol 2000, 221:68-86. Variations in the inductive interactions giving rise to the vulva are observed in species of the Cephalobina in the absence of major cell lineage alterations. This work, for the first time, includes a character analysis, indicating the direction of change during nematode vulval evolution. 40. Sigrist CB, Sommer RJ: Vulva formation in Pristionchus pacificus · relies on continuous gonadal induction. Dev Genes Evol 1999, 209:451-459. In contrast to various modes of 2-step induction, a continuous interaction between the gonad and the epidermis exists in P. pacificus involving multiple cells of the gonad in a time interval of ~20 hours. Multiple signaling pathways might be involved in vulva induction in P. pacificus, which can be analyzed by studying mutants defective in vulva induction. 41. Blaxter ML, De Ley P, Garey JR, Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR et al.: A molecular evolutionary framework for the phylum Nematoda. Nature 1998, 392:71-75. 42. Sommer RJ, Sternberg PW: Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr Biol 1996, 6:52-59. 43. Eizinger A, Jungblut B, Sommer RJ: Evolutionary change in the functional specificity of genes. Trends Genet 1999, 15:197-202. 44. Maloof JN, Kenyon C: The Hox gene lin-39 is required during C. elegans vulval induction to select the outcome of Ras signaling. Development 1998, 125:181-190. 45. Clandinin TR, Katz WS, Sternberg PW: Caenorhabditis elegans HOM-C genes regulate the response of vulval precursor cells to inductive signal. Dev Biol 1997, 182:150-161. 46. Eizinger A, Sommer RJ: The homeotic gene lin-39 and the evolution of nematode epidermal cell fates. Science 1997, 278:452-455. 47.

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