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AC ablated

Vulvaless mutant

Figure 9.1 Schematic summary of vulva formation in C. elegans. A, During the L1 stage, the 12 ventral epidermal cells P(1-12).p are equally distributed between pharynx and rectum. B, P(1,2,9-11).p fuse with the hypodermal syncytium hyp7 (F, white ovals). P(3-8).p form the vulva equivalence group and adopt one of three alternative cell fates. P6.p has a 1° fate (black oval), and P(5,7).p have a 2° fate (grey ovals). P(3,4,8).p have a 3° fate and remain epidermal (dotted ovals). The anchor cell (AC, black circle) provides an inductive signal for vulva formation. C, Cell lineage pattern of the vulval precursor cells. P(3,4,8).p divide once and then fuse of the vulva, respectively. The fate of these two cells has been designated as 2° (secondary). Two features made C. elegans vulva formation of special interest to developmental biologists. First, there are three more cells in the ventral epidermis that have the developmental competence to participate in vulva formation (Figure 9.1E). P(3,4,8).p can form vulval tissue after P(5-7).p, or a subset of these cells, have been ablated by laser microbeam irradiation early in development. In wild-type animals, P(3,4,8).p remain epidermal and adopt the 3° (tertiary) fate. Second, vulva formation requires inductive input by the gonadal anchor cell (AC). If the AC is ablated at birth, P(5-7).p remain epidermal and have a 3° fate, like their neighbouring cells P(3,4,8).p (Figure 9.1E).

Genetic and molecular studies in the past two decades have revealed the mechanistic basis of vulva formation in C. elegans (for review see Sternberg 2005, Eisenmann 2005). The inductive signal is provided by the AC and encodes an EGF-like molecule. Within the vulval precursor cells (VPCs), this signal is transmitted by EGF/RAS/MAPK signalling. A Wnt signalling pathway plays a redundant role in vulva formation. Cross-talk among the VPCs requires a Notch-like signalling system. After more than 20 years of active research, C. elegans vulva formation represents a paradigm for the molecular understanding of the interaction of signalling pathways in animal development and provides a platform for evolutionary developmental biology and comparative studies of vulva formation between different nematode species.

a satellite organism in evolutionary developmental biology: the nematode pristionchus pacificus

Comparative studies on vulva formation were initiated more than a decade ago and evolutionary changes of nearly all aspects of vulva

Figure 9.1 (cont.) with hyp7 (S). P(5,7).p generate seven progeny each. The first two cell divisions occur along the antero-posterior axis, the third division can be longitudinal (L), or transversal (T), or can be absent (N). P6.p generates eight progeny. D, Schematic summary of signalling interactions during vulva formation in C. elegans. An inductive EGF-like signal originates from the AC (black arrows). P6.p signals its neighbours to adopt a 2° fate via 'lateral signalling' (dotted arrows). Negative signalling (bars) prevents inappropriate vulva differentiation. E, Summary of cell ablation experiments. After ablation of P5.p, P4.p adopts a 2° fate and forms part of the vulva. After ablation of P(5-7).p, P(3,4,8).p can form a functional vulva. After ablation of the AC, all precursor cells adopt a 3° fate. Vulvaless mutants have a similar phenotype as AC-ablated animals.

formation have been identified (Sommer and Sternberg 1994, 1995, 1996, Félix and Sternberg 1998, Félix et al. 2000). Basically, changes involve (1) the size of the vulva equivalence group, (2) the number of VPCs participating in vulva formation, (3) involvement of the gonad/ AC in vulva induction, (4) novel signalling activities and (5) cell lineage alterations (for review see Sommer 1997). For example, in species with a posterior vulva, such as Teratorhabditis palmarum and Mesorhabditis sp. PS1179, the vulva is made from P(5-7).p, as in C. elegans. However, these cells migrate towards the posterior prior to differentiation and form a vulva even in the absence of the gonad (Sommer and Sternberg 1994). Further ablation experiments revealed that the VPCs in these species have strong autonomous properties to form vulva tissue, but the genetic program involved in vulva formation has not been investigated.

As a result of comparative studies of more than 50 nematode species, a few of them were selected for more detailed comparisons. We have developed the diplogastrid Pristionchus pacificus as a satellite organism in evolutionary developmental biology (Sommer et al. 1996, Sommer and Sternberg 1996, Hong and Sommer 2006). P. pacificus shows many substantial differences from C. elegans in the development of the vulva and at the same time fulfils many requirements for a model organism: P. pacificus is a hermaphroditic species that can feed on E. coli and has a 3-4 day generation time (20 0). Large-scale mutagenesis screens have been performed in P. pacificus and many mutants defective in sex determination, vulva and gonad formation have been isolated (Eizinger and Sommer 1997, Pires-daSilva and Sommer 2004, Rudel et al 2005). More recently, a genomic initiative including the generation of a genetic linkage map and a physical map has complemented the developmental and genetic studies (Srinivasan et al. 2002, 2003). A whole genome-sequencing project is currently ongoing and should result in a draft of the complete genome sequence in 2007 (www.pristionchus.org).

macro-evolution: vulva formation differs strongly between p. pacificus and c. elegans

Detailed experimental, genetic and molecular studies identified at least four major differences between vulva development in P. pacificus and C. elegans. First, non-vulval cells in the anterior and posterior body region fuse with the surrounding hypodermis in C. elegans, but die of programmed cell death in P. pacificus (Figure 9.2) (Eizinger and Sommer 1997). Second, vulva induction relies on a continuous interaction

Figure 9.2 Schematic summary of vulva formation in P. pacificus. A, Cell fate specification of the 12 ventral epidermal cells. P(1-4,9-11).p die of programmed cell death during late embryogenesis. P(5-7).p form the vulva with a 2°-1°-2° pattern. P8.p (shaded oval) has a special fate designated as 4°. B, Cell lineage pattern of the vulval precursor cells. P(5,7).p generate seven progeny each, whereas P6.p generates six progeny. P8.p does not divide and finally fuses with hyp7. C, Model for cell-cell interactions during vulva development in P. pacificus. P8.p provides lateral inhibition to P(5,7).p, mediated by the mesoblast M (chequered circle). Lateral inhibition influences the 1° vs. 2° cell fate decision of P(5,7).p. P8.p also provides a negative signal (black bars), which influences the vulva vs. non-vulva cell fate decision. For clarity, negative signalling is shown here as an interaction between P8.p and P(5-7).p. It is possible that indirect interactions involving

Figure 9.2 Schematic summary of vulva formation in P. pacificus. A, Cell fate specification of the 12 ventral epidermal cells. P(1-4,9-11).p die of programmed cell death during late embryogenesis. P(5-7).p form the vulva with a 2°-1°-2° pattern. P8.p (shaded oval) has a special fate designated as 4°. B, Cell lineage pattern of the vulval precursor cells. P(5,7).p generate seven progeny each, whereas P6.p generates six progeny. P8.p does not divide and finally fuses with hyp7. C, Model for cell-cell interactions during vulva development in P. pacificus. P8.p provides lateral inhibition to P(5,7).p, mediated by the mesoblast M (chequered circle). Lateral inhibition influences the 1° vs. 2° cell fate decision of P(5,7).p. P8.p also provides a negative signal (black bars), which influences the vulva vs. non-vulva cell fate decision. For clarity, negative signalling is shown here as an interaction between P8.p and P(5-7).p. It is possible that indirect interactions involving

between several cells of the somatic gonad and the vulval precursor cells (VPCs) rather than an interaction between the VPCs and the AC as in C. elegans (Sigrist and Sommer 1999). Third, P8.p represents a novel cell type in P. pacificus and is involved in multiple cell-cell interactions during vulva formation, not known from C. elegans or other nematodes (Jungblut and Sommer 2000). For instance, P8.p inhibits P5.p and P7.p to adopt the 1° cell fate, a process called 'lateral inhibition'. Additional experiments indicated that the mesoblast M is also involved in lateral inhibition and that P8.p and M interact to inhibit P(5,7).p from taking a 1° fate (Jungblut and Sommer 2000). No interaction between P8.p and the M cell has been observed in C. elegans. Finally, a negative signalling system provided by the VPCs themselves prohibits precocious vulva differentiation and counteracts vulva induction by the somatic gonad (Jungblut and Sommer 2000, Zheng et al. 2005).

P. pacificus vulva defective mutants have been isolated in large-scale mutagenesis screens and the phenotypes of mutations have

Figure 9.2 (cont.) other cells could exist. Inductive signalling from the somatic gonad is a continuous process (black arrows). Lateral signalling occurs between P6.p and P8.p (not indicated) and perhaps also between P6.p and P(5,7).p (dotted arrows). D, Summary of cell ablation experiments. After ablation of P(6,7).p, P5.p adopts a 2° fate in the presence of P8.p. After ablation of P(6-8).p, P5.p predominantly has a 1° fate, indicating that the presence of P8.p influences the cell fate decision of P5.p. E, Comparison of the function of the homeotic gene lin-39 between C. elegans and P. pacificus. In C. elegans lin-39 mutant animals, the central body region shows a homeotic transformation, and P(3-8).p fuse with the surrounding hypodermis like their anterior and posterior lineage homologues. P. pacificus lin-39 mutant animals also show a homeotic transformation and P(5-8).p die of programmed cell death. If the first function of Cel-lin-39 is rescued by providing lin-39 under the control of a heat-shock promoter, P(3-8).p have a 3° fate because Cel-lin-39 is required during vulva induction. The first function of Ppa-lin-39 can be overcome by generating a Ppa-lin-39 Ppa-ced-3 double mutant. Ppa-CED-3 is a general regulator of programmed cell death and mutations in Ppa-ced-3 result in animals unable to undergo apoptosis. Such double mutants form a normal vulva indicating that in contrast to Cel-lin-39, Ppa-lin-39 is not required during vulva induction. The size of the vulva equivalence group in P. pacificus is regulated by two genes, Ppa-hairy and Ppa-groucho. Mutations in these genes result in the survival of P(3,4).p, but not P(1,2,9-11).p. Biochemical studies have shown that Ppa-HAIRY and Ppa-GROUCHO form a heterodimer that regulates the activity of the Hox gene Ppa-lin-39. See text for details. X, programmed cell death; F, cell fusion; D, ectopic vulva differentiation.

helped in elucidating the molecular mechanisms of evolutionary change. So far, major differences involve the function of the Hox genes Ppa-lin-39 and Ppa-mab-5, the basic helix-loop-helix (bHLH) gene Ppa-hairy and the Wnt signalling genes Ppa-lin-17/Frizzled and Ppa-groucho (Eizinger and Sommer 1997, Jungblut and Sommer 1998, Sommer et al. 1998, Zheng et al. 2005, Schlager et al. 2006). In the following, we highlight three of these functional differences.

Vulva formation is initiated in P. pacificus and C. elegans by the formation of the vulva equivalence group (VEG). Genetic studies in the 1990s have shown that both organisms use the Hox gene lin-39, which is most similar to the Drosophila genes Scr and Dfd, for the establishment of the VEG (Clark et al. 1993, Wang et al. 1993, Eizinger and Sommer

1997). Mutations in Cel-lin-39 and Ppa-lin-39 result in the VPCs adopting the fate of their anterior and posterior lineage homologues. That is, in C. elegans these cells fuse with the surrounding hypodermis in the L1 stage and in P. pacificus they undergo programmed cell death (Figure 9.2E). However, later studies indicated substantial differences with regard to an additional function of LIN-39 in vulva formation in C. elegans. It has been shown that Cel-lin-39 is required during the L3 stage in response to EGF/RAS signalling, and mutants in which the first function of Cel-lin-39 has been rescued are induction vulvaless (Maloof and Kenyon

1998). Thus, Cel-LIN-39 is actively involved in vulva induction. In contrast, Ppa-lin-39; Ppa-ced-3 double mutants, in which cell death no longer occurs, form a normal vulva indicating that Ppa-LIN-39 is dispensable for vulva induction (Figure 9.2E) (Sommer et al. 1998). This observation represented the first fundamental difference in a function of a homologous developmental control gene in nematode development between different species (Eizinger et al. 1999).

More recent studies have revealed major differences in the signalling systems acting during vulva formation of the two species. The first mutation with a multivulva phenotype in P. pacificus was originally isolated as ped-7. Positional cloning identified ped-7 to encode Ppa-lin-17, one of the Frizzled-type Wnt receptors in nematodes (Zheng et al. 2005). Ppa-lin-17/Frizzled shows conserved functions during vulva formation (i.e. the regulation of cell lineage symmetry of the posterior cell P7.p) as well as divergent functions: in P. pacificus, Ppa-lin-17/Fz is part of the negative signalling system that counteracts vulva formation. In contrast, no such negative signalling function is known from C. elegans.

The most substantial differences so far have been identified by the molecular cloning of two genes that regulate the size of the VEG in P.

pacificus. Mutations in two genes result in the survival of P(3,4).p and expand the size of the VEG in the anterior region. These mutations have an atavistic phenotype, resembling nearly exactly the pattern known from C. elegans (Figure 9.2E). Molecular cloning revealed that one of these two genes encodes for a bHLH-like protein of the HAIRY-type (Schlager et al 2006). Further studies revealed that Ppa-HAIRY forms a heterodimer with the product of the second gene, Ppa-groucho, which when mutated also causes the survival of P(3,4).p. Ppa-HAIRY and Ppa-GROUCHO form a heterodimer and Ppa-HAIRY binds to HES-binding sites in the Ppa-lin-39 promoter. Ppa-lin-39 is up-regulated in Ppa-hairy mutants, further indicating that Ppa-HAIRY and Ppa-GROUCHO restrict the size of the VEG in P. pacificus by repressing the Hox gene Ppa-lin-39. Interestingly, the Ppa-hairy gene does not have a 1:1 orthologue in C. elegans. Thus, a Ppa-HAIRY and Ppa-GROUCHO module that is absent in C. elegans regulates the size of the VEG in P. paci-ficus (Schlager et al. 2006). Phylogenetic considerations strongly suggest that the pattern observed in P. pacificus and all other diplogasterid nema-todes represents a derived character and that the HAIRY/GROUCHO module was involved in the evolutionary restriction of the VEG.

ecology

Ultimately, the development of organisms has to be studied in the context of the natural environment, and several recent studies have pointed towards the importance of ecology in developmental biology (Gilbert 2001, Dusheck 2002, Herrmann et al. 2006a). In general, little is known about the ecology of model organisms in developmental biology. This holds true also for the nematode laboratory organisms, P. pacificus and C. elegans. For example, the environmental niche of the model organism C. elegans is largely unknown and it is only recently that studies have indicated that C. elegans occurs predominantly in compost heaps (Barrière and Félix 2005, Kiontke and Sudhaus 2005). We have recently shown that nematodes of the genus Pristionchus live in close association with scarab beetles and the Colorado potato beetle in Western Europe and the United States (Herrmann et al. 2006a,b). Intensive samplings in Europe in 2004 and 2005 generated 371 isolates that fell into six species, most of which are morphologically indistinguishable from one another. The two hermaphroditic species P. entomo-phagus and P. maupasi accounted for 226 of these 371 (60%) isolates and occurred on dung beetles and cockchafers, respectively.

A relationship between nematodes and their hosts, such as seen in the case of Pristionchus and scarab beetles, has long been known as 'necromeny' (Sudhaus 1976). In general, existing types of nematode-insect associations can be divided into three types (Sudhaus 1976). In phoresy, nematodes attach themselves to passing insects/invertebrates for transportation, an interaction that is usually not species-specific (Kiontke and Sudhaus 2005). For example, C. elegans can occasionally exhibit phoretic behaviour through unspecific associations with invertebrates. In necromeny, nematodes not only use insects for transport, but also subsequently as food. This association is much more specific than a phoretic relationship. Finally, entomopathogenic nematodes are obligate parasites preying on insects. It has been suggested that necromeny is an intermediate step preceding true parasitism. In this context it will be interesting to study the association of Pristionchus with beetles in more detail to learn more about the pre-adaptations towards parasitism (Herrmann et al. 2006a).

The Pristionchus-beetle association represents a unique opportunity to couple research in evolutionary developmental biology with ecology. Some scarab beetles and the Colorado potato beetles can be cultured in the laboratory, and thus the nematode-beetle interaction can be studied under laboratory conditions. Such studies will be of importance for understanding many aspects of the biology of the nematode, e.g. the genetic regulation of dauer formation and olfaction. Ultimately, knowledge of the natural environment of Pristionchus and the development of nematode cultures under similar conditions will also be of importance for the analysis of the evolution of developmental processes, because any adaptation (like the developmental adaptations discussed above) results from environmental conditions to which organisms are exposed.

micro-evolution

In the long run, the comparison of developmental processes between P. pacificus and C. elegans will benefit from knowledge of the natural environment of these nematodes. However, micro-evolutionary studies are necessary to bridge macro-evolution of development (as represented by the comparison between P. pacificus and C. elegans) and ecology. To this end we have initiated micro-evolutionary studies to better understand, first, the population structure of P. pacificus and second, how developmental characters change among wild isolates.

Sampling efforts by various nematologists throughout the world and detailed field studies as described above have revealed many strains of P. pacificus. However, in contrast to many other Pristionchus species for which we could identify specific beetle hosts, no strong beetle association has yet been observed for P. pacificus. Instead, sampling efforts during the past 10 years have identified more than 15 strains of P. pacificus from several countries in Africa, Asia, Europe and North America (Srinivasan et al. 2001, Zauner et al 2007). However, P. pacificus collections were rare in all samples from Europe, North America and South Africa. At the same time, the available strains indicate that P. pacificus is a cosmopolitan species and interestingly, P. pacificus is currently the only species in the genus Pristionchus with such a worldwide distribution (Figure 9.3).

The availability of multiple strains of P. pacificus from different countries and continents allows for the initiation of population genetic investigations. Micro-evolutionary studies can help address many questions of importance for a comprehensive understanding of the biology of this organism. Are the strains of P. pacificus in permanent genetic contact with one another? Do males occur in nature, and is out-crossing observed in the natural environment? We have started to use mitochon-drial sequence data for various P. pacificus strains to address these and related questions. Mitochondrial sequence analysis of more than 3300 bp has shown that the molecular diversity between P. pacificus strains is much higher than between C. elegans strains (Zauner et al. 2007). At the same time, single stranded conformational polymorphism analysis and amplified fragment length polymorphism (AFLP) studies of P. pacificus strains reveal genome-wide linkage disequilibrium indicating

Figure 9.3 Pristionchus pacificus is a cosmopolitan species.

low out-crossing rates. The highly diverse molecular signatures of P. pacificus strains hint at a long-lasting colonisation of new habitats, likely to be in association with a beetle host.

Do the different P. pacificus strains also show differences in their development, i.e. the formation of the vulva? Some of the P. pacificus strains have been compared with one another and several differences in developmental properties have been observed. The strain of P. pacificus that serves as the basis for genetic and molecular studies is PS312 from Pasadena (California, USA). Mitochondrial sequence data revealed that this strain is untypical for American isolates of the species. While all other North American strains fall into a distinct mitochondrial clade, PS312 from California is more closely related to strains from Poland (RS106) and Hawaii (JU138). Surprisingly, these molecular signatures do not correlate with vulva developmental characters. The strongest difference in vulva development was observed in the strain from Poland RS106 (Srinivasan et al. 2001). Specifically, the developmental competence of individual VPCs differs between the strain from Poland and most of the other investigated strains. For example, if P(6,7).p are ablated, P5.p will adopt a 2° fate as long as P8.p is present and prevents P5.p from taking a 1° fate (Figure 9.2D). This result is observed in the majority of P(6,7).p-ablated animals for the laboratory strain PS312 and most other strains of P. pacificus. However, after similar ablations in RS106 from Poland, P5.p can have a 1° fate in the majority of the treated worms (Srinivasan et al. 2001). Considering that AFLP analysis and mitochondrial haplotype analysis have revealed that PS312 and RS106 are nearly identical at the molecular level, these developmental differences suggest that small molecular alterations (mutations), which have yet to be identified, can account for developmental novelties. Building on these promising observations, we hope to create a micro-evolutionary analysis of vulva development between P. pacificus strains and closely related Pristionchus species.

conclusions

We have summarised here three different lines of investigation that try to use the nematode P. pacificus and related species of the same genus for studies in different areas of evolutionary biology. Genetic and developmental studies of vulva development in P. pacificus, when compared with the knowledge available from C. elegans, show how developmental processes change at a macro-evolutionary level and how genetic alterations can create developmental novelty. Studies on the ecology of

Pristionchus nematodes provide the first indication of what the natural environment of these organisms looks like. In the long run, such investigations can help to provide a basis for an understanding of those adaptations that in response to the environment have shaped morphological and developmental patterns that differ between species. And finally, micro-evolutionary comparisons of different P. pacificus strains might reveal the actual molecular nature of developmental 'mutations' that ultimately result in novel patterns and structures. We envisage coupling genetic analysis to environmental studies by testing, for example, vulval patterning mutants for their effect in the beetle environment rather than under agar-plate (laboratory) conditions. Also, we plan to test different isolates from the wild for their genetic and developmental variations. We hope that by combining macroevolutionary, micro-evolutionary and ecological studies, we can contribute to a much-needed synthesis in evolutionary biology.

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