Castiglioni VG et al. (2024) Infect Genet Evol "Experimental evolution of an RNA virus in Caenorhabditis elegans."

Olmo-Uceda MJ, Martin S, Castiglioni VG, Felix MA, Elena SF, Gonzalez R

[Infect Genet Evol, 2024]

The discovery of Orsay virus (OrV), the first virus infecting wild populations of Caenorhabditis elegans, has boosted studies of viral immunity pathways in this nematode. Considering the many advantages that C. elegans offers for fundamental research in host-pathogen interactions, this pathosystem has high potential to become a model system for experimental virus evolution studies. However, the evolutionary constraints - i.e, the balance between genetic variation, selection, drift and historical contingency- operating in this pathosystem have barely been explored. Here we describe for the first time an evolution experiment of two different OrV strains in C. elegans. Comparison of the two ancestral strains showed differences in infectivity and sequence, and highlighted the importance of consistently normalize viral inocula for meaningful comparisons among strains. After 10 serial passages of evolution, we report slight changes in infectivity and non-synonymous mutations fixed in the evolved viral populations. In addition, we observed numerous minor variants emerging in the viral population. These minor variants were not randomly distributed along the genome but concentrated in polymorphic genomic regions. Overall, our work established the grounds for future experimental virus evolution studies using Caenorhabditis nematodes.

Castiglioni VG et al. (2022) J Biol Chem "Identification and characterization of Crumbs polarity complex proteins in C. elegans."

Schmidt R, Kroll JR, Boxem M, van Beuzekom H, Castiglioni VG, Stucchi R, Ramalho JJ, Altelaar M

[J Biol Chem, 2022]

Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1, and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues, and therefore not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.

Castiglioni, V.G. et al. (2019) International Worm Meeting "The role of cortical polarity protein PAR-6 in larval epithelia."

Castiglioni, V.G., Pires, H.R., Rosas, R., Boxem, M.

[International Worm Meeting, 2019]

The PAR complex, which includes PAR-6, PKC-3 and PAR-3, is essential for polarity in a broad variety of cell types. The PAR proteins were first identified in the C. elegans one cell embryo, where they are essential for the first asymmetric division, and were later found to be involved in polarization of epithelia and embryonic junction formation. Studies in Drosophila and mammals have also shown that the PAR complex controls epidermal homeostasis and stem cell fate. Although the role of PAR-6 in embryonic development has been extensively studied, little is known about its function in larval tissues. Our aim is to understand the role of PAR-6 in postembryonic epithelia. As RNAi experiments fail to show strong defects upon par-6 depletion, we combined the use of CRISPR-Cas9 and the Auxin Inducible Degradation (AID) system, which allows us to deplete PAR-6 in a time and tissue specific manner. We observe that PAR-6 is apically enriched in larval epithelia, and ubiquitous degradation shows that PAR-6 is essential for postembryonic development. To understand whether PAR-6 has different roles depending on the function of the tissue, we studied the effects of PAR-6 depletion in two different tissues: the intestine and the seam cells. PAR-6 expression in the intestine, where there is no cell division during larval stages, is constant throughout development. However, PAR-6 expression in the seam cells is dynamic and varies during the cell cycle. Our results show that PAR-6 is essential for the correct localization of PKC-3 and DLG-1 in epithelia. We observe that PAR-6 expression in the intestine is not necessary for the postembryonic development and function of the intestine. However, PAR-6 is essential for proper development of the seam and the hypodermis, as well as for molting. PAR-6 is required for the correct timing and pattern of seam cell divisions, especially in the symmetric L2 division. We are currently working to identify the mechanism, in seam cell division, differentiation or homeostasis, where PAR-6 is required. Our work shows tissue specific requirements for PAR-6 in postembryonic C. elegans epithelia, and a novel function for PAR-6 in the control of seam cell homeostasis.

Castiglioni VG et al. (2024) Sci Adv "Story of an infection: Viral dynamics and host responses in the Caenorhabditis elegans-Orsay virus pathosystem."

Muñ\;oz-Sá\;nchez JC, Villena-Gimé\;nez A, Elena SF, Legarda EG, Castiglioni VG, Olmo-Uceda MJ

[Sci Adv, 2024]

Orsay virus (OrV) is the only known natural virus affecting <i>Caenorhabditis elegans</i>, with minimal impact on the animal's fitness due to its robust innate immune response. This study aimed to understand the interactions between <i>C. elegans</i> and OrV by tracking the infection's progression during larval development. Four distinct stages of infection were identified on the basis of viral load, with a peak in capsid-encoding RNA2 coinciding with the first signs of viral egression. Transcriptomic analysis revealed temporal changes in gene expression and functions induced by the infection. A specific set of up-regulated genes remained active throughout the infection, and genes correlated and anticorrelated with virus accumulation were identified. Responses to OrV mirrored reactions to other biotic stressors, distinguishing between virus-specific responses and broader immune responses. Moreover, mutants of early response genes and defense-related processes showed altered viral load progression, uncovering additional players in the antiviral defense response.

Castiglioni VG et al. (2020) Elife "Epidermal PAR-6 and PKC-3 are essential for larval development of C. elegans and organize non-centrosomal microtubules."

Kerver J, Rosas Bertolini R, Pires HR, Castiglioni VG, Boxem M, Riga A

[Elife, 2020]

The cortical polarity regulators PAR-6, PKC-3, and PAR-3 are essential for the polarization of a broad variety of cell types in multicellular animals. In <i>C. elegans</i>, the roles of the PAR proteins in embryonic development have been extensively studied, yet little is known about their functions during larval development. Using inducible protein degradation, we show that PAR-6 and PKC-3, but not PAR-3, are essential for postembryonic development. PAR-6 and PKC-3 are required in the epidermal epithelium for animal growth, molting, and the proper pattern of seam-cell divisions. Finally, we uncovered a novel role for PAR-6 in organizing non-centrosomal microtubule arrays in the epidermis. PAR-6 was required for the localization of the microtubule organizer NOCA-1/Ninein, and defects in a <i>noca-1</i> mutant are highly similar to those caused by epidermal PAR-6 depletion. As NOCA-1 physically interacts with PAR-6, we propose that PAR-6 promotes non-centrosomal microtubule organization through localization of NOCA-1/Ninein.

Salomon MP et al. (2009) Genetics "Comparing mutational and standing genetic variability for fitness and size in Caenorhabditis briggsae and C. elegans."

Bour W, Phillips N, Baer CF, Salomon MP, Ostrow D, Sylvestre T, Upadhyay A, Keller TE, Blanton D, Levy L

[Genetics, 2009]

The genetic variation present in a species depends on the interplay between mutation, population size, and natural selection. At mutation-(purifying) selection balance (MSB) in a large population, the standing genetic variance for a trait (VG) is predicted to be proportional to the mutational variance for the trait (VM); VM is proportional to the mutation rate for the trait. The ratio VM/VG predicts the average strength of selection (S) against a new mutation. Here we compare VM and VG for lifetime reproductive success (approximately fitness) and body volume in two species of self-fertilizing rhabditid nematodes, Caenorhabditis briggsae and C. elegans, which the evidence suggests have different mutation rates. Averaged over traits, species, and populations within species, the relationship between VG and VM is quite stable, consistent with the hypothesis that differences among groups in standing variance can be explained by differences in mutational input. The average (homozygous) selection coefficient inferred from VM/VG is a few percent, smaller than typical direct estimates from mutation accumulation (MA) experiments. With one exception, the variance present in a worldwide sample of these species is similar to the variance present within a sample from a single locale. These results are consistent with specieswide MSB and uniform purifying selection, but genetic draft (hitchhiking) is a plausible alternative possibility.

Chen JS et al. (1997) J Mol Evol "Extensive sequence conservation among insect, nematode, and vertebrate vitellogenins reveals ancient common ancestry."

Chen JS, Raikhel AS, Sappington TW

[J Mol Evol, 1997]

The eggs of most oviparous animals are provisioned with a class of protein called vitellogenin (Vg) which is stored as the major component of yolk. Until recently, deduced amino acid sequences were available only from vertebrate and nematode Vgs, which proved to be homologous. The sequences of several insect Vgs are now known, but early attempts at pairwise alignments with vertebrate and nematode Vgs have been problematic, leading to conflicting conclusions about how closely insect Vgs are related to the others. In this paper we demonstrate that insect VE sequences can be confidently aligned with one another along their entire lengths and with multiple vertebrate and nematode Vg sequences along most of their spans. Although divergence is high, conservation among insect, vertebrate, and nematode Vg sequences is widespread with a preponderance of glycine, proline, and cysteine residues among strictly conserved amino acids, establishing conclusively that Vgs from the three phyla are homologous. Areas of least-certain alignment are primarily in and around insect and vertebrate polyserine domains which are not homologous. Phylogenetic reconstructions of Vgs based on sequence identities indicate that the insect lineage is the most diverged and that the mammalian serum protein, apolipoprotein B-100, arose from a Vg ancestor after the nematode/vertebrate divergence.

Farhadifar R et al. (2016) Genetics "Mutation Is a Sufficient and Robust Predictor of Genetic Variation for Mitotic Spindle Traits in Caenorhabditis elegans."

Andersen EC, Ponciano JM, Baer CF, Needleman DJ, Farhadifar R

[Genetics, 2016]

Different types of phenotypic traits consistently exhibit different levels of genetic variation in natural populations. There are two potential explanations: either mutation produces genetic variation at different rates, or natural selection removes or promotes genetic variation at different rates. Whether mutation or selection is of greater general importance is a longstanding unresolved question in evolutionary genetics. We report mutational variances (VM) for 19 traits related to the first mitotic cell division in C. elegans, and compare them to the standing genetic variances (VG) for the same suite of traits in a worldwide collection C. elegans Two robust conclusions emerge. First, the mutational process is highly repeatable: the correlation between VM in two independent sets of mutation accumulation lines is ~0.9. Second, VM for a trait is a good predictor of VG for that trait: the correlation between VM and VG is ~0.9. This result is predicted for a population at mutation-selection balance; it is not predicted if balancing selection plays a primary role in maintaining genetic variation.

Farhadifar, Reza et al. (2015) International Worm Meeting "Mutation is a sufficient predictor of genetic variation for mitotic spindle traits in C. elegans."

Ponciano, Jose Miguel, Farhadifar, Reza, Andersen, Erik, Needleman, Daniel, Baer, Charles

[International Worm Meeting, 2015]

Seemingly similar phenotypic traits can vary considerably in the extent of genetic variation in the trait. If two traits in the same set of organisms have different levels of genetic variation, there are two potential underlying causes: mutation and/or selection. Traits may differ in the mutational target they present, or in the rate at which those loci mutate. Traits may also differ in the average effect that mutations have on the trait. Alternatively, selection may differ between traits.We investigated these issues by comparing the input of genetic variation by mutation to the standing genetic variation found in a worldwide collection of C. elegans for a suite of 19 traits related to the first mitotic cell division. The results are striking: the mutational variance (VM) explains 90% of the variation in the standing genetic variance (VG). One of the traits - embryo length - has been previously shown to be under stabilizing selection. The strong positive relationship between VM and VG remains after accounting for variation in embryo length. Mutations were allowed to accumulate in two separate experiments in two different genetic backgrounds (N2 and PB306). The correlation between VM in the two experiments was ~0.9, which indicates that for a given set of traits, the mutational process produces highly repeatable outcomes.The ratio of VG/VM can be interpreted as the number of generations of mutation required to produce that amount of variation in the population. For 18 of the 19 traits (embryo length notwithstanding) the ratio of VG/VM is on the order of a few hundred generations. That result is consistent with molecular evidence suggesting that C. elegans has recently undergone a species-wide selective sweep that purged most of the standing genetic variation.We conclude that variation among traits in the amount of genetic variation in the population is a predictable function of the mutational input, and that the role of direct and/or linked natural selection is secondary. This result has implications for understanding the genetic architecture of complex traits in any organism, including humans.

Riga A et al. (2021) PLoS Genet "Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells."

Boxem M, Pires HR, Riga A, Schmidt R, Cravo J, Castiglioni VG, van den Heuvel S

[PLoS Genet, 2021]

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.