Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

Although the arrangement of internal organs in most metazoans is profoundly left–right (L/R) asymmetric with a predominant handedness, rare individuals show full (mirror-symmetric) or partial (heterotaxy) reversals. While the nematode Caenorhabditis elegans is known for its highly determinate development, including stereotyped L/R organ handedness, we found that L/R asymmetry of the major organs, the gut and gonad, varies among natural isolates of the species in both males and hermaphrodites. In hermaphrodites, heterotaxy can involve one or both bilaterally asymmetric gonad arms. Male heterotaxy is probably not attributable to relaxed selection in this hermaphroditic species, as it is also seen in gonochoristic Caenorhabditis species. Heterotaxy increases in many isolates at elevated temperature, with one showing a pregastrulation temperature-sensitive period, suggesting a very early embryonic or germline effect on this much later developmental outcome. A genome-wide association study of 100 isolates showed that male heterotaxy is associated with three genomic regions. Analysis of recombinant inbred lines suggests that a small number of loci are responsible for the observed variation. These findings reveal that heterotaxy is a widely varying quantitative trait in an animal with an otherwise highly stereotyped anatomy, demonstrating unexpected plasticity in an L/R arrangement of the major organs even in a simple animal.This article is part of the themed issue ‘Provocative questions in left–right asymmetry’.

Anatomical left-right (L/R) handedness asymmetry in C. elegans is established in the four-cell embryo as a result of anteroposterior skewing of transverse mitotic spindles with a defined handedness. This event creates a chiral embryo and ultimately an adult body plan with fixed L/R positioning of internal organs and components of the nervous system. While this “dextral” configuration is invariant in hermaphrodites, it can be reversed by physical manipulation of the early embryo or by mutations that interfere with mitotic spindle orientation, which leads to viable, mirror-reversed (sinistral) animals. During normal development of the C. elegans male, the gonad develops on the right of the midline, with the gut bilaterally apposed on the left. However, we found that in males of the laboratory N2 strain and Hawaiian (“Hw”) wild isolate, the gut/gonad asymmetry is frequently reversed in a temperature-dependent manner, independent of normal embryonic chirality. We also observed sporadic errors in gonad migration occurring naturally during early larval stages of these and other wild strains; however, the incidence of such errors does not correlate with the frequency of L/R gut/gonad reversals in these strains. Analysis of N2/Hw hybrids and recombinant inbred advanced intercross lines (RIAILs) indicate that the L/R organ reversals are likely to result from recessively acting variations in multiple genes. Thus, unlike the highly reproducible L/R asymmetries of most structures in hermaphrodites, the L/R asymmetry of the male C. elegans body plan is less rigidly determined and subject to natural variation that is influenced by a multiplicity of genes.

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BACKGROUND The current paradigm in inflammatory bowel disease (IBD) diagnostics is based on peripheral biomarkers such as C-reactive protein and fecal calprotectin that achieve low sensitivity and specificity for intestinal inflammation. Extracellular vesicles (EVs) are lipid-enveloped particles involved in inter tissue and cell-cell interactions. They also have unique properties reflecting the metabolic and phenotypic nature of the producer cells. Recent data revealed that surface proteins on intestinal epithelial cells-derived EVs can be detected in the peripheral blood. We posit that lipid profiling of circulating EVs (PBEs) can be used to discriminate active IBD from healthy subjects and further classify different stages of IBD (PCT Patent Pending: 13177N/2194P5). METHODS Patients diagnosed with Ulcerative Colitis (UC, n=50) or normal controls (n=50) were recruited at the UK IBD clinic or colonoscopy suite. During their colonoscopy or regular outpatient labs, 2 tubes of blood (20-30 mL) were drawn into K2-EDTA tubes. The blood samples were immediately stored on ice and then centrifuged at 1,500 g for 15 minutes at 4 °C within 30 minutes. The clarified plasma was immediately aliquoted and frozen in liquid N2. PBEs were isolated from plasma using size-exclusion microcolumns. Isolated exosomal preparations were lysed in cold acetonitrile, followed by extraction using a modified Folch method. The lipid fraction was carefully aspirated and dried in a Vacufuge before reconstitution in a chloroform/methanol mixture containing butylated hydroxytoluene. The extracted lipids from active UC and healthy control patient plasma were analyzed using direct infusion ultrahigh resolution Orbitrap mass spectrometry. We used statistical tools LASSO and Random Forest to select informative lipid features and build classification models. The performance of the classifiers was quantified by the receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) based on 10-fold cross-validation. RESULTS Three PBE lipids identified by LASSO were also identified using the Random Forest method along with 7 additional lipids. Discriminating lipid classifiers between active UC and normal patients identified by both LASSO and Random Forest included phosphatidylcholines, plasmalogens, and sphingolipids. An AUC of 0.86 discriminated active UC from normal patients using the Random Forest method and 0.80 using the LASSO method. CONCLUSION These results are the first-ever depiction of harnessing the diagnostic utility of PBEs through lipid profiling. Differences in PBE lipid composition accurately discriminated active UC patients from normal patients paving the way for a diagnostic liquid biopsy for patients with IBD.