Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Intraflagellar transport: A critical player in photoreceptor development and the pathogenesis of retinal degenerative diseases.

In vertebrate vision, photons are detected by highly specialized sensory cilia called outer segments. Photoreceptor outer segments form by remodeling the membrane of a primary cilium into a stack of flattened disks. Intraflagellar transport (IFT) is critical to the formation of most types of eukaryotic cilia including the outer segments. This review covers the state of knowledge of the role of IFT in the formation and maintenance of outer segments and the human diseases that result from mutations in genes encoding the IFT complex and associated motors.

The Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) oncogene is upregulated in pre-cystic epithelial cells revealing a new target for polycystic kidney disease.

Polycystic kidney disease (PKD) is an important cause of end stage renal disease, but treatment options are limited. While later stages of the disease have been extensively studied, mechanisms driving the initial conversion of renal tubules into cysts are not understood. To identify factors that promote the initiation of cysts we deleted polycystin-2 ( Pkd2 ) in mice and surveyed transcriptional changes before and immediately after cysts developed. We identified 74 genes which we term cyst initiation candidates (CICs). To identify conserved changes with relevance to human disease we compared these murine CICs to single cell transcriptomic data derived from patients with PKD and from healthy controls. Tumor-associated calcium signal transducer 2 ( Tacstd2 ) stood out as an epithelial-expressed gene whose levels were elevated prior to cystic transformation and further increased with disease progression. Human tissue biopsies and organoids show that TACSTD2 protein is low in normal kidney cells but is elevated in cyst lining cells. While TACSTD2 has not been studied in PKD, it has been studied in cancer where it is highly expressed in solid tumors while showing minimal expression in normal tissue. This property is being exploited by antibody drug conjugates that target TACSTD2 for the delivery of cytotoxic drugs. Our finding that Tacstd2 is highly expressed in cysts, but not normal tissue, suggests that it should be explored as a candidate for drug development in PKD. More immediately, our work suggests that PKD patients undergoing TACSTD2 treatment for cancer should be monitored for kidney effects. One Sentence Summary: The oncogene, tumor-associated calcium signal transducer 2 (Tacstd2) mRNA increased in abundance shortly after Pkd2 loss and may be a driver of cyst initiation in polycystic kidney disease.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Autonomous and non-cell autonomous etiology of ciliopathy associated structural birth defects.

Ciliopathies are associated with wide spectrum of structural birth defects (SBD), indicating important roles for cilia in development. Here we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140 , an intraflagellar transport protein regulating ciliogenesis. Ift140 deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula, randomized heart looping, congenital heart defects (CHD), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAG-Cre deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD was not observed with four Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathy.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

Arih2 regulates Hedgehog signaling through smoothened ubiquitylation and ER-associated degradation.

During Hedgehog signaling, the ciliary levels of Ptch1 and Smo are regulated by the pathway. At the basal state, Ptch1 localizes to cilia and prevents the ciliary accumulation and activation of Smo. Upon binding a Hedgehog ligand, Ptch1 exits cilia, relieving inhibition of Smo. Smo then concentrates in cilia, becomes activated and activates downstream signaling. Loss of the ubiquitin E3 ligase Arih2 elevates basal Hedgehog signaling, elevates the cellular level of Smo and increases basal levels of ciliary Smo. Mice express two isoforms of Arih2 with Arih2α found primarily in the nucleus and Arih2ß found on the cytoplasmic face of the endoplasmic reticulum (ER). Re-expression of ER-localized Arih2ß but not nuclear-localized Arih2α rescues the Arih2 mutant phenotypes. When Arih2 is defective, protein aggregates accumulate in the ER and the unfolded protein response is activated. Arih2ß appears to regulate the ER-associated degradation (ERAD) of Smo preventing excess and potentially misfolded Smo from reaching the cilium and interfering with pathway regulation.

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation.

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.

c-JUN n-Terminal Kinase (JNK) Signaling in Autosomal Dominant Polycystic Kidney Disease.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people and is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The mechanisms by which polycystin mutations induce cyst formation are not well understood, however pro-proliferative signaling must be involved for tubule epithelial cell number to increase over time. We recently found that the stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) pathway is activated in cystic disease and genetically removing JNK reduces cyst growth driven by a loss of Pkd2. This review covers the current state of knowledge of signaling in ADPKD with an emphasis on the JNK pathway.

Biallelic pathogenic variants in roundabout guidance receptor 1 associate with syndromic congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

Consensus nomenclature for dyneins and associated assembly factors.

Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.

c-Jun N-terminal kinase (JNK) signaling contributes to cystic burden in polycystic kidney disease.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD.

Loss of the ciliary protein Chibby1 in mice leads to exocrine pancreatic degeneration and pancreatitis.

Primary cilia protrude from the apical surface of many cell types and act as a sensory organelle that regulates diverse biological processes ranging from chemo- and mechanosensation to signaling. Ciliary dysfunction is associated with a wide array of genetic disorders, known as ciliopathies. Polycystic lesions are commonly found in the kidney, liver, and pancreas of ciliopathy patients and mouse models. However, the pathogenesis of the pancreatic phenotype remains poorly understood. Chibby1 (Cby1), a small conserved coiled-coil protein, localizes to the ciliary base and plays a crucial role in ciliogenesis. Here, we report that Cby1-knockout (KO) mice develop severe exocrine pancreatic atrophy with dilated ducts during early postnatal development. A significant reduction in the number and length of cilia was observed in Cby1-KO pancreta. In the adult Cby1-KO pancreas, inflammatory cell infiltration and fibrosis were noticeable. Intriguingly, Cby1-KO acinar cells showed an accumulation of zymogen granules (ZGs) with altered polarity. Moreover, isolated acini from Cby1-KO pancreas exhibited defective ZG secretion in vitro. Collectively, our results suggest that, upon loss of Cby1, concomitant with ciliary defects, acinar cells accumulate ZGs due to defective exocytosis, leading to cell death and progressive exocrine pancreatic degeneration after birth.

E3 ubiquitin ligase Wwp1 regulates ciliary dynamics of the Hedgehog receptor Smoothened.

The Hedgehog pathway, critical to vertebrate development, is organized in primary cilia. Activation of signaling causes the Hedgehog receptor Ptch1 to exit cilia, allowing a second receptor, Smo, to accumulate in cilia and activate the downstream steps of the pathway. Mechanisms regulating the dynamics of these receptors are unknown, but the ubiquitination of Smo regulates its interaction with the intraflagellar transport system to control ciliary levels. A focused screen of ubiquitin-related genes identified nine required for maintaining low ciliary Smo at the basal state. These included cytoplasmic E3s (Arih2, Mgrn1, and Maea), a ciliary localized E3 (Wwp1), a ciliary localized E2 (Ube2l3), a deubiquitinase (Bap1), and three adaptors (Kctd5, Skp1a, and Skp2). The ciliary E3, Wwp1, binds Ptch1 and localizes to cilia at the basal state. Activation of signaling removes both Ptch1 and Wwp1 from cilia, thus providing an elegant mechanism for Ptch1 to regulate ciliary Smo levels.

Loss of Primary Cilia Protein IFT20 Dysregulates Lymphatic Vessel Patterning in Development and Inflammation.

Microenvironmental signals produced during development or inflammation stimulate lymphatic endothelial cells to undergo lymphangiogenesis, in which they sprout, proliferate, and migrate to expand the vascular network. Many cell types detect changes in extracellular conditions via primary cilia, microtubule-based cellular protrusions that house specialized membrane receptors and signaling complexes. Primary cilia are critical for receipt of extracellular cues from both ligand-receptor pathways and physical forces such as fluid shear stress. Here, we report the presence of primary cilia on immortalized mouse and primary adult human dermal lymphatic endothelial cells in vitro and on both luminal and abluminal domains of mouse corneal, skin, and mesenteric lymphatic vessels in vivo. The purpose of this study was to determine the effects of disrupting primary cilia on lymphatic vessel patterning during development and inflammation. Intraflagellar transport protein 20 (IFT20) is part of the transport machinery required for ciliary assembly and function. To disrupt primary ciliary signaling, we generated global and lymphatic endothelium-specific IFT20 knockout mouse models and used immunofluorescence microscopy to quantify changes in lymphatic vessel patterning at E16.5 and in adult suture-mediated corneal lymphangiogenesis. Loss of IFT20 during development resulted in edema, increased and more variable lymphatic vessel caliber and branching, as well as red blood cell-filled lymphatics. We used a corneal suture model to determine ciliation status of lymphatic vessels during acute, recurrent, and tumor-associated inflammatory reactions and wound healing. Primary cilia were present on corneal lymphatics during all of the mechanistically distinct lymphatic patterning events of the model and assembled on lymphatic endothelial cells residing at the limbus, stalk, and vessel tip. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute corneal lymphangiogenesis resulting in increased lymphatic vessel density and branching. These data are the first functional studies of primary cilia on lymphatic endothelial cells and reveal a new dimension in regulation of lymphatic vascular biology.

Rab34 is necessary for early stages of intracellular ciliogenesis.

Primary cilia are sensory organelles present on most vertebrate cells and are critical for development and health. Ciliary dysfunction is associated with a large class of human pathologies collectively known as ciliopathies. These include cystic kidneys, blindness, obesity, skeletal malformations, and other organ anomalies. Using a proximity biotinylation with Ift27 as bait, we identified the small guanosine triphosphatase (GTPase) Rab34 as a ciliary protein. Rab34 localizes to the centrosomes near the mother centriole, the axoneme of developed cilia, and highly dynamic tubule structures in the centrosomal region. Rab34 is required for cilia formation in fibroblasts, where we find that Rab34 loss blocks ciliogenesis at an early step of ciliary vesicle formation. In inner medullary collecting duct (IMCD3) epithelial cells, the requirement is more complex, with Rab34 needed in cells grown at low density but becoming less important as cell density increases. Ciliogenesis can proceed by an internal pathway where cilia form in the cytoplasm before being displayed on the ciliary surface or cilia can assemble by an external pathway where the centriole docks on the plasma membrane before ciliary assembly. Fibroblasts are thought to use the internal pathway, although IMCD3 cells are thought to use the external pathway. However, we find that IMCD3 cells can use the internal assembly pathway and significant numbers of internally assembling cilia are observed in low-density cells. Together, our work indicates that Rab34 is required for internal assembly of cilia, but not for cilia built on the cell surface.

Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling.

In the absence of Hedgehog ligand, patched-1 (Ptch1) localizes to cilia and prevents ciliary accumulation and activation of smoothened (Smo). Upon ligand binding, Ptch1 is removed from cilia, and Smo is derepressed and accumulates in cilia where it activates signaling. The mechanisms regulating these dynamic movements are not well understood, but defects in intraflagellar transport components, including Ift27 and the BBSome, cause Smo to accumulate in cilia without pathway activation. We find that in the absence of ligand-induced pathway activation, Smo is ubiquitinated and removed from cilia, and this process is dependent on Ift27 and BBSome components. Activation of Hedgehog signaling decreases Smo ubiquitination and ciliary removal, resulting in its accumulation. Blocking ubiquitination of Smo by an E1 ligase inhibitor or by mutating two lysine residues in intracellular loop three causes Smo to aberrantly accumulate in cilia without pathway activation. These data provide a mechanism to control Smo's ciliary level during Hedgehog signaling by regulating the ubiquitination state of the receptor.

X Caps the Phosphate for Phospho-Rab GTPase Recognition in Ciliogenesis and Parkinson's Disease.

Some Rab GTPases, after activation by GDP to GTP exchange, are phosphorylated by the LRRK2 kinase implicated in Parkinson's disease. In the current issue of Structure, Waschbüsch et al. (2020) investigate the structural basis for recognition of active phospho-Rab GTPases by the RH2 domain of the effector protein RILPL2.

Abnormal fertility, acrosome formation, IFT20 expression and localization in conditional Gmap210 knockout mice.

GMAP210 (TRIP11) is a cis-Golgi network-associated protein and a Golgi membrane receptor for IFT20, an intraflagellar transport component essential for male fertility and spermiogenesis in mice. To investigate the role of GMAP210 in male fertility and spermatogenesis, floxed Gmap210 mice were bred with Stra8-iCre mice so that the Gmap210 gene is disrupted in spermatocytes and spermatids in this study. The Gmap210flox/flox: Stra8-iCre mutant mice showed no gross abnormalities and survived to adulthood. In adult males, testis and body weights showed no difference between controls and mutant mice. Low-magnification histological examination of the testes revealed normal seminiferous tubule structure, but sperm counts and fertility were significantly reduced in mutant mice compared with controls. Higher resolution examination of the mutant seminiferous epithelium showed that nearly all sperm had more oblong, abnormally shaped heads, while the sperm tails appeared to have normal morphology. Electron microscopy also revealed abnormally shaped sperm heads but normal axoneme core structure; some sperm showed membrane defects in the midpiece. In mutant mice, expression levels of IFT20 and other selective acrosomal proteins were significantly reduced, and their localization was also affected. Peanut-lectin, an acrosome maker, was almost absent in the spermatids and epididymal sperm. Mitochondrion staining was highly concentrated in the heads of sperm, suggesting that the midpieces were coiling around or aggregating near the heads. Defects in acrosome biogenesis were further confirmed by electron microscopy. Collectively, our findings suggest that GMAP210 is essential for acrosome biogenesis, normal mitochondrial sheath formation, and male fertility, and it determines expression levels and acrosomal localization of IFT20 and other acrosomal proteins.

WormCat: An Online Tool for Annotation and Visualization of Caenorhabditis elegans Genome-Scale Data.

The emergence of large gene expression datasets has revealed the need for improved tools to identify enriched gene categories and visualize enrichment patterns. While gene ontogeny (GO) provides a valuable tool for gene set enrichment analysis, it has several limitations. First, it is difficult to graph multiple GO analyses for comparison. Second, genes from some model systems are not well represented. For example, ∼30% of Caenorhabditis elegans genes are missing from the analysis in commonly used databases. To allow categorization and visualization of enriched C. elegans gene sets in different types of genome-scale data, we developed WormCat, a web-based tool that uses a near-complete annotation of the C. elegans genome to identify coexpressed gene sets and scaled heat map for enrichment visualization. We tested the performance of WormCat using a variety of published transcriptomic datasets, and show that it reproduces major categories identified by GO. Importantly, we also found previously unidentified categories that are informative for interpreting phenotypes or predicting biological function. For example, we analyzed published RNA-seq data from C. elegans treated with combinations of lifespan-extending drugs, where one combination paradoxically shortened lifespan. Using WormCat, we identified sterol metabolism as a category that was not enriched in the single or double combinations, but emerged in a triple combination along with the lifespan shortening. Thus, WormCat identified a gene set with potential. phenotypic relevance not found with previous GO analysis. In conclusion, WormCat provides a powerful tool for the analysis and visualization of gene set enrichment in different types of C. elegans datasets.