Sommer, Ralf J. (2011) International Worm Meeting "Molecular phylogeny and divergence time estimates for beetle-associated Pristionchus nematodes."

Sommer, Ralf J.

[International Worm Meeting, 2011]

Pristionchus pacificus has been established as a model system in evolutionary developmental biology (evo-devo) and for comparison to C. elegans. Recent studies expanded the evo-devo work to ecology and population genetics. P. pacificus and related species have a well-defined association with scarab beetles: Sampling of more than 20,000 scarab beetles from around the world resulted in the isolation of more than 8,000 Pristionchus isogenic female lines. Mating experiments and molecular sequence analysis identified 26 species to date. We provide a molecular phylogenetic framework based on nearly 11,000 characters. Studies on La Reunion, an Island with a unique P. pacificus biodiversity hotspot, provide a case study to link population genetics with ecology and evo-devo. We obtained more than 400 wild isolates of P. pacificus and carried out microsatellite analysis of 21 markers. These studies indicate four major biogeographic clades of P. pacificus. La Reunion represents the only geographic area with P. pacificus strains being present in all four clades. This finding is best explained by P. pacificus invading the island independently with different beetle vectors, such as Oryctes, Amneidus and Maladera (Herrmann et al., 2010). We assessed the molecular phylogeny of P. pacificus and used mutation accumulation line approaches to provide divergence time estimates. Mutation rates were assessed first, by mitochondrial DNA analysis and second, from representative microsatellite markers. Mitochondrial DNA analysis suggests a minimal divergence time for P. pacificus of 105 to 106 generations (Molnar et al., 2011). Similarly, microsatellite markers are used to robustly provide minimal divergence time estimates for closely related strains from La Reunion. This includes a first attempt for a serious analysis of the population size of P. pacificus.

Ralf Sommer (2006) Development & Evolution Meeting "Evolutionary Developmental Biology In Nematodes: Comparing Pristionchus pacificus To C. elegans"

Ralf Sommer

[Development & Evolution Meeting, 2006]

Cell fate specification and cell-cell signaling have been well studied during the development of C. elegans and provide a paradigm in evolutionary developmental biology. We have developed Pristionchus pacificus as a satellite organism in evolutionary developmental biology, in which forward and reverse genetic studies can be performed. Additionally, a genomic platform with an integrated physical and genetic map and an 8 X coverage of the P. pacificus genome sequence is available that allows detailed comparisons to C. elegans. The comparison between P. pacificus and C. elegans, two nematodes that shared a last common ancestor 200-300 million years ago, indicates major differences in the patterning mechanisms underlying developmental processes, i.e. vulva formation, gonad development and the regulation of dauer formation. Our analysis of vulva development indicates three major sets of differences. First, the two nematodes contain different molecular modules. For example, P. pacificus contains an ancestral HAIRY/GROUCHO module that is important in the regulation of vulva patterning and that no longer exists in C. elegans. Second, orthologous developmental control genes and signaling pathways have different functions. In C. elegans, EGF/RAS and Wnt signaling are crucial for vulva induction. In contrast in P. pacificus, mutations in Ppa-lin-17/Frizzled and Ppa-groucho/unc-37 result in a multivulva phenotype indicating a role in a negative signaling process. Third, at the cellular level, homologous cells perform species-specific cell-cell interactions reflecting the molecular differences in signaling activity. To complement our macro-evolutionary comparison, we initiated ecological studies. In contrast to C. elegans that lives as a soil-dwelling species, Pristionchus nematodes occur in strong association with beetles, in particular cockchafers, dung beetles and Colorado potato beetles. In this ecological context, we study the genetic control of dauer formation and olfaction to learn how during the evolution of this novel ecological association patterning mechanisms have changed. Interestingly, Pristionchus species but not C. elegans is attracted by various insect sex pheromones. Currently, we are using a genetic approach to determine the molecular mechanism of the nematode - insect interaction.

Susoy, Vladislav et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "EVOLUTIONARY DIVERSIFICATION THROUGH THE GAIN AND LOSS OF DEVELOPMENTAL PLASTICITY"

Sommer, Ralf J., Susoy, Vladislav, Ragsdale, Erik J.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Developmental plasticity is common among plants and animals, yet its role in mediating evolutionary processes is debated. Several species of Rhabditina, including Pristionchus pacificus, can execute two alternative mouth phenotypes, one of which is associated with predatory feeding. Studies in P. pacificus have revealed that environmental factors act through a conserved genetic pathway that enables a rapid developmental response to the environment (Bento et al., 2010). In addition, a developmental switch that controls the expression of the alternative mouth phenotypes was recently identified (Ragsdale et al., 2013). In contrast, little is known about the macroevolutionary potential of the mouth plasticity. We studied the relationship between plasticity and morphological change by a macroevolutionary analysis of nematode mouthparts when accompanied by a dimorphism. To test whether plasticity facilitates or hinders morphological change, we analyzed variation in form and complexity in 90 nematode species with or without a mouth dimorphism. Our analyses revealed a two-step process of morphological diversification associated with the gain and loss of plasticity. First, acquisition of a dimorphism was accompanied by an increase in complexity, including structural innovations such as moveable teeth. Second, the fixation of a single mouth-phenotype in several nematode lineages was associated with a decrease in mouth complexity but a sharp increase in evolutionary rates when measured as change of shape and size. Thus, plasticity facilitates phenotypic diversification by fostering evolutionary novelties, whereas subsequent loss of the dimorphism enables acceleration of evolution by releasing novel morphologies from developmental constraints. 1. G. Bento, A. Ogawa, R. J. Sommer. Nature 466, 494-497 (2010). 2. E. J. Ragsdale, M. R. Muller, C. Rodelsperger, R. J. Sommer, Cell 155, 922-933 (2013).

Wang, Xiaoyue et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A complex Network of Wnt Signaling with an unusual Wiring regulates the Induction of Vulva Formation in the Nematode P. pacificus."

Sommer, Ralf, Wang, Xiaoyue

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In C. elegans , vulva formation has been intensively studied and can serve as a paradigm for evo-devo studies. We have developed Pristionchus pacificus as a model organism in evo-devo . While in C. elegans EGF induces vulva development, forward and reverse genetic experiments revealed that Wnt signaling is crucial for vulva induction in P. pacificus . A Ppa-bar-1/-catenin mutant is completely vulvaless. Multiple Wnt ligands and receptors play a redundant role in vulva induction including Ppa-egl-20 /Wnt. Two striking features of P. pacificus Wnt signaling are a negative role of Ppa-lin-17/Frizzled , mutations in which result in a multivulva phenotype, and that a mutation in Ppa-egl-20 can suppress the Ppa-lin-17 phenotype. Such genetic interactions suggest a role of this Frizzled receptor in ligand sequestration. Interestingly, Ppa-egl-20 is only expressed in the rectal region, similar to C. elegans , representing the first inductive ligand for vulva induction that is expressed outside the gonad. To further elucidate the molecular mechanisms of P. pacificus vulva induction, we combined forward genetics and transformation technology. First, a transgenic approach using heat-shock induced Ppa-egl-20 revealed that this Wnt ligand is sufficient for vulva induction. Second, mutations in additional multivulva genes have been cloned. We will report genetic and biochemical experiments, which indicate that changes in ligand-receptor interactions were crucial for the evolution of Wnt signaling in nematodes. Together, this study shows that Wnt signaling during P. pacificus vulva induction i) represents an unusual complexity, ii) consists of a novel wiring involving ligand sequestration and iii) requires novel protein domains in o therwise conserved genes/proteins of the Wnt signaling pathway.

Ogawa, Akira et al. (2009) International Worm Meeting "Genetic analysis of dauer formation in Pristionchus pacificus reveals conserved and diverse aspects of signaling mechanisms."

Ogawa, Akira, Sommer, Ralf

[International Worm Meeting, 2009]

Despite the wealth of knowledge of C. elegans dauer formation, regulation of dauer formation in other nematode species is relatively poorly understood. To address how environmental regulation of dauer formation evolved, we are studying dauer formation in the satellite model nematode Pristionchus pacificus. P. pacificus shares with C. elegans most of the advantageous features of experimental organisms and has genetic tools such as forward and reverse genetics, a complete genome, and transgenic techniques. In the wild P. pacificus associates with scarab beetles as dauer larvae and occupies a distinct ecological niche from C. elegans. Since dauer formation play critical roles in the adaptive strategy of P. pacificus, one can expect many evolutionary novelties in P. pacificus dauer formation. Also genetic analysis of dauer formation in P. pacificus may lead to identification of conserved genetic components of dauer formation that are missed in C. elegans studies. This is because multiple genes may be involved in the same step of the regulatory cascade (redundancy) or there may be genes that are involved in both dauer formation and earlier development (pleiotropy). Such genes are hard to identify in conventional genetic and RNAi screens and studies in P. pacificus that may have different patterns of redundancy and pleiotropy might help identify such genes. So far we have shown that i) P. pacificus uses a distinct pheromone(s) from C. elegans to sense the population density ii) An endocrine module involving dafachronic acids (DAs) and DAF-12 controls dauer formation in P. pacificus. The latter finding was further extended to infective larva formation of the parasitic nematode Strongyloides papillosus. Exogenous administration of D7-DA completely suppresses the formation of infective larva in S. papillosus suggesting infective and dauer larvae share a common evolutionary origin. To elucidate the genes that control P. pacificus dauer formation we screened daf-c and daf-d mutants, and obtained more than 10 strains of each class. In a small-scale screen for daf-c mutants, we obtained several fully penetrant daf-c strains that are relatively rare in C. elegans, as well as partially penetrant strains. All the daf-c strains tested could be at least partially rescued by exogenous administration of D7-dafachronic acid, suggesting they have a mutation in a gene upstream of DA/DAF-12. Interestingly the fully penetrant daf-c strains we obtained did not respond to up to 250 nM of D4-dafachronic acid. Positional mapping of the gene responsible for one of the daf-c strains is ongoing.

Theska, Tobias et al. (2021) International Worm Meeting "NHR-1 and NHR-40 in C. elegans - an outgroup approach to the origin of a novel trait"

Theska, Tobias, Sommer, Ralf

[International Worm Meeting, 2021]

A longstanding prediction of evolutionary theory is that phenotypic plasticity, the ability of one genotype to produce different phenotypes based on environmental input during development, plays a significant role in the evolution of morphological novelty. Nematodes of the Diplogastridae family are characterized by the presence of such a novelty: they possess cuticular teeth at the base of their mouths. Additionally, these structures are also polyphenic; different environmental cues can induce development of either a predatory form with two large teeth or a bacterivorous morph with only one dorsal tooth. Previous work demonstrated that two nuclear receptors, Ppa-NHR-1 and Ppa-NHR-40, regulate the morphogenesis of these teeth in Pristionchus pacificus. Interestingly, nematodes outside of the Diplogastridae, like Caenorhabditis elegans, lack this morphological novelty and the associated phenotypic plasticity entirely. Instead, these worms are monomorphic with simple, triangular flaps at the base of their mouths. However, Ppa-nhr-1 and Ppa-nhr-40 are the only genes of the P. pacificus mouth-form regulatory network that retain 1:1 orthologs in C. elegans. Therefore, we investigate whether these transcription factors also have a conserved function in regulating the development of the C. elegans mouth. Using the CRISPR/Cas9 system, we generated small indels in the coding sequences of Cel-nhr-1 and Cel-nhr-40 and isolated frameshift mutants to ensure a loss-of-function. Additionally, we aim to investigate the localization of both nuclear receptors by tagging them with fluorescent protein markers (e.g., mNeonGreen). In order to identify potential mutant phenotypes, we quantify differences in mouth shape between wildtype and CRISPR-edited strains by applying landmark-based geometric morphometrics in combination with k-medoid and model-based clustering.This data will be complemented with future RNA-seq experiments, which will identify the regulatory targets of Cel-NHR-1 and Cel-NHR-40. Taken together, this data will allow a comparison of the cellular localization, the regulatory targets, and the functional relevance of NHR-1 and NHR-40 for stoma development between C. elegans and P. pacificus. Ultimately, this study will reveal whether these nuclear receptors share a conserved function in stoma development that predates the evolution of the morphological novelty, or if a neo-functionalization accompanied its origin.

Kienle, Simone et al. (2011) International Worm Meeting "Natural variation of gonad independent vulva induction among Pristionchus pacificus isolates."

Sommer, Ralf J., Kienle, Simone

[International Worm Meeting, 2011]

Pristionchus pacificus has been established as a model system in evolutionary developmental biology and evolutionary ecology. Previous studies have focused on macroevolutionary differences between the reference strain of P. pacificus (PS312) and Caenorhabditis elegans (N2). These studies have shown that, while the vulva in PS312 and N2 is formed from homologous precursor cells, different signaling pathways are required for vulva induction. In contrast to EGF/Ras signaling in N2, PS312 relies on Wnt signaling with an atypical wiring of this signaling system. To add a microevolutionary perspective to the evo-devo studies in P. pacificus, we isolated more than 150 different strains from around the world and examined natural variation, with particular emphasis on vulva induction. These strains showed high diversity, both at the molecular and the phenotypic level. We chose 19 genetically and geographically highly diverse strains and analysed vulva induction by gonad ablation experiments. In wild type PS312 animals, differentiation of the vulva precursor cells (VPCs) is completely abolished after Z(1,4) ablation, suggesting that vulva differentiation in this strain depends on the precursor cells of the somatic gonad. However, in most of the analysed P. pacificus strains, VPCs were still able to differentiate after ablation of the gonad. To identify the loci involved, we crossed the two isolates showing the most extreme phenotypes, PS312 and an isolate from Bolivia (RS5275). We obtained recombinant inbred lines (RILs) by selfing individual F2 and near isogenic lines (NILs) by continuously backcrossing the RILs to RS5275 animals. Gonad ablation experiments and genotyping of more than 200 RILs revealed a 430kb region on Chromosome I, corresponding to C. elegans Chromosome V, associated with the VPC differentiation trait. This interval contains 20 genes of which one is a known developmental control gene. Sequencing this gene for both strains revealed no substantial differences in the coding but high amount of variation in the non-coding regions. We used DNA-mediated transformation experiments to study the concentration dependence of this natural variation and to show how natural variation interacts with redundant cell fate specification mechanisms. Together, the microevolutionary perspective of P. pacificus vulva development is an effort to integrate evo-devo studies in evolutionary theory.

Wang, Xiaoyue et al. (2009) International Worm Meeting "DNA-mediated transformation in Pristionchus pacificus reveals novel functions of Wnt signaling in vulva induction."

Wang, Xiaoyue, Sommer, Ralf J.

[International Worm Meeting, 2009]

Cell fate specification and cell-cell signaling have been well studied during the development of C. elegans and provide a paradigm for evolutionary developmental biology. To identify the molecular basis of evolutionary alterations in development, we have developed Pristionchus pacificus as a satellite organism, in which i) forward and reverse genetic studies can be performed, ii) a 10 X coverage of the genome is available and iii) DNA-mediated transformation allows experimental manipulation (Schlager et al., 2009). One developmental process that has been studied intensively is vulva formation and functional differences were identified in the molecular mechanisms of vulva induction between C. elegans and P. pacificus. While in C. elegans EGF is the key initiation signal of vulva development, forward and reverse genetic experiments revealed that Wnt signaling is crucial for vulva induction in P. pacificus. A Ppa-bar-1/b-catenin mutant is completely vulvaless. Reverse-genetic analysis indicated that multiple Wnt ligands and Fz-like receptors play a redundant role in vulva induction, including Ppa-egl-20/Wnt. Two striking features are a negative role of Ppa-lin-17/Frizzled, mutations in which result in a multivulva phenotype, and the function of Ppa-egl-20, that when mutated can suppress the Ppa-lin-17 phenotype. Such genetic interactions suggest a role of this Frizzled receptor in ligand sequestration. Interestingly, Ppa-egl-20 is only expressed in the rectal region, similar to C. elegans, representing the first inductive ligand for vulva induction that is expressed outside the gonad. To further elucidate the molecular mechanisms of P. pacificus vulva induction, we are combining forward genetics and transformation technology. First, another multivulva mutant, formerly known as limu-1, has been cloned and displays a novel aspect of Wnt signaling function. Second, a transgenic approach using heat-shock induced Ppa-bar-1 and Ppa-egl-20 reveals the temporal aspects of Wnt signaling. These studies show that at least three distinct Wnt signaling mechanisms are at work, which control i) vulva induction, ii) the polarity of the secondary cell fates and iii) the fate of the epidermal cell P8.p. In particular Ppa-lin-17 has distinct functions in all three decisions, most of which are unknown from Cel-lin-17. This study provides the first molecular insight into vulva induction by a signaling center outside of the gonad and thereby extends our understanding of signaling mechanisms in nematodes. References: Schlager, B. et al. (2009) Molecular cloning of a dominant Roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus. Genesis in Press.

Lightfoot, James W. et al. (2013) International Worm Meeting "The Role of Pharyngeal Glands in Nematode Feeding and Diet."

Lightfoot, James W., Sommer, Ralf J.

[International Worm Meeting, 2013]

Nematode pharyngeal glands have historically been associated with feeding as well as cuticle moulting, although little is understood of the molecular biology underlying these processes. By utilising two evolutionary divergent nematode species with differing diets and feeding traits we are elucidating the roles of the pharyngeal gland cells and their function in feeding, diet and other cellular processes. The model organisms C. elegans and P. pacificus provide an apt system with which to analyse the pharyngeal glands, as while both nematode species contain several pharyngeal gland cells, C. elegans is predominantly a bacterial feeder, whereas P. pacificus is capable of predating other nematode species in addition to its bacterial feeding habits. Accordingly, gland cells display an evolutionary divergence in both quantity and morphology between species. The pharynx of C. elegans contains five pharyngeal gland cells, a large dorsal gland (g1D) exiting at the mouth opening, two ventral glands (g1V) emptying into the median bulb and two ventral glands (g2) exiting into the terminal bulb. The pharynx of P. pacificus contains three gland cells, lacking the g2 glands, and additionally the g1D is morphologically distinct as the gland duct exits through the predatory dorsal tooth used to eviscerate other nematodes before feeding. Furthermore, substantial divergence in the function of pharyngeal glands between these two nematode species has been predicted. The dorsal gland in the bacterial feeding nematode C. elegans is stimulated through inputs from the isthmus while the dorsal gland in the bacterial and predatory feeder, P. pacificus receives inputs from the corpus. This dramatic shift in regulation may indicate an alteration in gland cell regulation towards additional predatory functions. Cell ablation experiments removing g1D cells however reveal much functional conservation as animals become starved due to a bacterial plug blocking the pharynx in both species. Therefore, we have begun generating the transcriptome of the pharyngeal gland cells using mRNA tagging followed by RNA-seq with which we will pursue the genetic candidates behind gland cell function and the expanded diet evident in P. pacificus..

Anderton SM et al. (1996) East Coast Worm Meeting "Comparisons of serotonin containing neurons in freeliving nematodes: correlation (or not) of ventral cord neurons with vulval precursor cell patterns."

Anderton SM, Loer CM

[East Coast Worm Meeting, 1996]

We are interested in how patterns of serotonergic neurons are specified in the nematode. In C. elegans, six hypodermal cells (P38.p) are competent to produce vulva in the central body of the hermaphrodite. In this same body region in the ventral nerve cord (VNC) of the male, P38.aap normally divide to produce two neurons, one of which contains serotonin (CP neurons, P38.aapp). We wondered whether related species of worms that have a different number of vulval precursor cells than C. elegans might also alter the pattern of serotonergic neurons in the male. We first examined Pristionchus pactficus which in the hermaphrodite has only 4 Pn.p ectoblasts, P58.p; anterior and posterior Pn.p cells undergo programmed cell death (R. Sommer, personal communication). We found four serotonin-immunoreactive (JR) CP-like neurons in male Pristionchus VNC, correctly positioned to be P58.aapp. In the hermaphrodite VNC, we saw four cells, two anterior and two posterior to the vulva, and appearing to innervate the vulval region. These are likely to be 7C cells, only four of which (P58.aap) survive in Pristionchus (R. Sommer, pers. comm.). VC cells in C. elegans occasionally stain with serotonin antisera. With this simple picture in mind of a correlation between Pn.p cell fates in the hermaphrodite and Pn.aapp fate in the male, we examined several other species of worms with varying numbers of Pn.p cells (Sommer & Sternberg, 1995, Dev. Biol. 167: 61; Sommer & Sternberg, 1994, Science 265: 114). The simple-minded hypothesis did not hold up beyond Pristionchus. Panagrolaimus, also with P58.p cells in the hermaphrodite, had 8 CP-like somas in the male VNC (4 faint in the anterior, 4 bright in the posterior). Essentially the same pattern was seen in the male Rhabditella, in which the hermaphrodite has 5 Pn.p cells (P48.p). Both Pelodera and Teratorhabditis (each with P48.p in the hermaphrodite), looked like C. elegans, with 6 CP-like cells in the male VNC. Each of these species sometimes showed other faintly staining somas, paired with the CP-like neurons. Similar cells are sometimes seen in C. elegans and are believed to be CA neurons (Pn.aapa). We will present a complete pattern of serotoninIR cells in males and hermaphrodites for each of these species. We wish to thank Ralf Sommer for providing the non-C. elegans strains used and sharing unpublished information about them. Another undergraduate, Nicole Hinson, did the first staining with Pristionchus during a summer HHMI fellowship.