The nonsense mediated decay (NMD) pathway degrades mRNAs bearing premature translation termination codons. In mammals, SMG-8 has been implicated in the NMD pathway, in part by its association with SMG-1 kinase. Here we use four independent assays to show that C. elegans smg-8 is not required to degrade nonsense-containing mRNAs. We examine the genetic requirement for smg-8 to destabilize the endogenous, natural NMD targets produced by alternative splicing of rpl-7a and rpl-12. We test smg-8 for degradation of the endogenous, NMD target generated by unc-54(r293), which lacks a normal polyadenylation site. We probe the effect of smg-8 on the exogenous NMD target produced by myo-3::GFP, which carries a long 3′ untranslated region that destabilizes mRNAs. None of these known NMD targets is influenced by smg-8 mutations. In addition, smg-8 animals lack classical Smg mutant phenotypes such as a reduced brood size or abnormal vulva. We conclude that smg-8 is unlikely to encode a component critical for NMD.

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

Author Summary

The correct wiring of the nervous system depends on the ability of axons to properly interpret extracellular cues that guide them to their targets. The Eph receptor tyrosine kinases (RTKs) have roles in guiding axons, but their signaling pathways are not completely understood. In this study, we used the nematode Caenorhabditis elegans to study how the VAB-1 Eph RTK regulates the growth cone structure for axon guidance. Genetic and molecular data show that VAB-1 regulates the conserved molecules NCK-1, WSP-1/N-WASP, and UNC-34/Ena. Our study provides a model of how the VAB-1 Eph RTK modulates the growth cone structure to inhibit axonal outgrowth. We show that activated VAB-1 can inhibit an NCK-1/UNC-34 interaction by binding to the NCK-1 SH2 domain. We also show that NCK-1 and WSP-1 can physically interact and that VAB-1/NCK-1 and WSP-1 form a complex in vitro. We suggest that the VAB-1 Eph RTK can contribute to the termination of axon outgrowth by two methods: 1) The VAB-1/NCK-1/WSP-1 complex activates ARP-2/3 to change the actin growth cone dynamics to that of a branched structure thus reducing the number of filopodia, and 2) VAB-1 inhibits axon extension by inhibiting UNC-34/Ena's function in actin polymerization.

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.

Author Summary

The generation of a neuron from a fertilized egg requires a multi-step cascade of molecules acting from within and outside that cell to direct it towards a neuronal fate, rather than, say, a muscle cell. These cascades are not fully understood. In this study we systematically eliminate the function of almost all genes in the C. elegans genome, one by one, to determine what it takes to build a neuron. We identified 245 genes that affect the development of a specific sensory neuron pair, e.g. the neurons were not generated or the neurons were generated but the terminal fate was not correctly specified. We characterize in more detail the transcription factor hlh-14, which we find is required to generate multiple neurons, and the COMPASS histone methyltransferase complex, which we find to have a surprisingly specific role in the specification of a molecular and functional left-right asymmetry in this sensory neuron pair. Our study represents the first genome-wide analysis of a single neuronal cell fate decision. Further characterization of the genes identified here will enhance our understanding, and thus our capacity for treatment and prevention, of human neurological disorders.

Central regulators of cell fate, or selector genes, establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different sets of target genes at various times and in diverse cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. We have used the Nuclear Spot Assay and GFP reporters to examine PHA-4 interactions with target promoters in living embryos and with single cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen of candidate nuclear factors identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx, but emr-1 did not modulate PHA-4 binding in the intestine. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, may facilitate promoter access and productive transcription. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells, preventing target gene expression in that organ. PHA-4 binding within the pharynx is limited by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.

Author Summary

Central regulators of cell fate establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different target genes in different cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. Here we examine PHA-4 interactions with target promoters in living embryos and with single-cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, facilitates promoter access. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells and is limited in the pharynx by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.

Planaria continue to blossom as a model system for understanding all aspects of regeneration. They provide an opportunity to understand how the replacement of missing tissues from preexisting adult tissue is orchestrated at the molecular level. When amputated along any plane, planaria are capable of regenerating all missing tissue and rescaling all structures to the new size of the animal. Recently, rapid progress has been made in understanding the developmental pathways that control planarian regeneration. In particular Wnt/beta-catenin signaling is central in promoting posterior fates and inhibiting anterior identity. Currently the mechanisms that actively promote anterior identity remain unknown. Here, Smed-prep, encoding a TALE class homeodomain, is described as the first gene necessary for correct anterior fate and patterning during planarian regeneration. Smed-prep is expressed at high levels in the anterior portion of whole animals, and Smed-prep(RNAi) leads to loss of the whole brain during anterior regeneration, but not during lateral regeneration or homeostasis in intact worms. Expression of markers of different anterior fated cells are greatly reduced or lost in Smed-prep(RNAi) animals. We find that the ectopic anterior structures induced by abrogation of Wnt signaling also require Smed-prep to form. We use double knockdown experiments with the S. mediterranea ortholog of nou-darake (that when knocked down induces ectopic brain formation) to show that Smed-prep defines an anterior fated compartment within which stem cells are permitted to assume brain fate, but is not required directly for this differentiation process. Smed-prep is the first gene clearly implicated as being necessary for promoting anterior fate and the first homeobox gene implicated in establishing positional identity during regeneration. Together our results suggest that Smed-prep is required in stem cell progeny as they form the anterior regenerative blastema and is required for specifying anterior cell fates and correct patterning.

Author Summary

Understanding the genetic basis of tissue regeneration (remaking) from adult structures is an important long-term goal for biomedical science. The widespread nature of regenerative phenomena in different animals allows us to study evolution's answers to coordinating this process. We use the relatively simple and experimentally tractable planarian model to study this process. After almost any amputation these animals unerringly replace all missing tissues. This ability has two key components. Firstly, planaria have a population of stem cells capable of rapidly dividing and becoming all the cell types that are missing, such as muscle, gut, and brain cells, after amputation. Secondly, the genetic information in these stem cells and the remaining tissue is able to coordinate the regeneration process so that new structures are the correct size and in the correct place. This allows the production of a fully functional individual at the end of the regeneration process. We are specifically interested in how structures end up in the correct place in new tissue they form. Here we discover and describe the role of a gene, called Smed-prep, particularly central to this process. Smed-prep is required to coordinate the regeneration of the planarian brain, arguably the most exciting part of planarian regeneration.

SUMMARY

BACKGROUND

FoxA factors are critical regulators of embryonic development and post-embryonic life, but little is know about the upstream pathways that modulate their activity [1]. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos, and to control growth and longevity after birth [2–5]. We previously identified the AAA+ ATPase homologue ruvb-1 as a potent suppressor of pha-4 mutations [6].

RESULTS

Here we show that ruvb-1 is a component of the TOR pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity [7, 8]. We find that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan [9–11], but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations.

CONCLUSION

The data suggest that pha-4 and the CeTOR pathway antagonize one another to regulate post-embryonic development and adult longevity. We suggest a model in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity or disease.

Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4α type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system.

Author Summary

The function of many important biological structures requires the construction of very complex cellular shapes. For example, mammalian kidneys or related renal systems in other animals rely on the formation of elongated tubes that maximize surface area to facilitate the exchange of ions between the body and excreted fluid. Defects in kidney development or function may lead to kidney failure or polycystic kidney disease. Mechanisms involved in orchestrating the formation and function of the elaborate tube structures in renal systems are still poorly characterized. Here, we show a novel transcription factor involved in the growth and elongation of an excretory tube in C. elegans. This factor helps manage tube development by regulating genes involved in ion transport and membrane fusion, likely helping to balance the growth of the inner and outer portions of the excretory tube as this structure elongates. This transcription factor shares significant homology with a mammalian protein that participates in hormone signaling and is present in the kidney tubules, suggesting that elongation and growth of tube structures may rely on a new kind of hormonal communication that occurs between distant parts of the cell; this signaling mechanism may be important for appropriate kidney development in humans.

Dietary restriction (DR) extends lifespan in various species and also slows the onset of age-related diseases. Previous studies from flies and yeast have demonstrated that the target of rapamycin (TOR) pathway is essential for longevity phenotypes resulting from DR. TOR is a conserved protein kinase that regulates growth and metabolism in response to nutrients and growth factors. While some of the downstream targets of TOR have been implicated in regulating lifespan, it is still unclear whether additional targets of this pathway also modulate lifespan. It has been shown that the hypoxia inducible factor-1 (HIF-1) is one of the targets of the TOR pathway in mammalian cells. HIF-1 is a transcription factor complex that plays key roles in oxygen homeostasis, tumor formation, glucose metabolism, cell survival, and inflammatory response. Here, we describe a novel role for HIF-1 in modulating lifespan extension by DR in Caenorhabditis elegans. We find that HIF-1 deficiency results in extended lifespan, which overlaps with that by inhibition of the RSKS-1/S6 kinase, a key component of the TOR pathway. Using a modified DR method based on variation of bacterial food concentrations on solid agar plates, we find that HIF-1 modulates longevity in a nutrient-dependent manner. The hif-1 loss-of-function mutant extends lifespan under rich nutrient conditions but fails to show lifespan extension under DR. Conversely, a mutation in egl-9, which increases HIF-1 activity, diminishes the lifespan extension under DR. This deficiency is rescued by tissue-specific expression of egl-9 in specific neurons and muscles. Increased lifespan by hif-1 or DR is dependent on the endoplasmic reticulum (ER) stress regulator inositol-requiring protein-1 (IRE-1) and is associated with lower levels of ER stress. Therefore, our results demonstrate a tissue-specific role for HIF-1 in the lifespan extension by DR involving the IRE-1 ER stress pathway.

Author Summary

Dietary restriction (DR) is one of the most robust environmental manipulations that extend lifespan in various species. DR has also been shown to slow the onset of a number of age-related diseases. Studies in model organisms like C. elegans can be used to uncover biological mechanisms that determine the beneficial effects of DR. Previous studies suggest that the nutrient-sensing target of rapamycin (TOR) pathway is required for DR-mediated lifespan extension. However, the downstream mechanisms by which TOR modulates lifespan remain unclear. In mammalian cells, TOR and the downstream S6 kinase (S6K) activate expression of the hypoxia-inducible factor-1 (HIF-1), which is frequently up-regulated in various tumors. Using C. elegans as a model system, we characterized novel functions of HIF-1 in aging. We find that inhibition of HIF-1 extends lifespan under rich nutrient conditions, whereas enhanced levels of HIF-1 only allow partial lifespan extension by DR. We also demonstrated that increased lifespan by hif-1 or DR depends on the endoplasmic reticulum (ER) stress regulator inositol-requiring protein-1 (IRE-1) and is associated with lower levels of ER stress, which is caused by overloading of misfolded/unfolded proteins to ER. Thus, our results support the idea that HIF-1–mediated changes in protein homeostasis play a key role in the lifespan extension by DR.

Summary

Background

The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP.

Results

We report that the C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf. Here, we show that TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase and that phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation.

Conclusions

The C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase.

Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPRmt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPRmt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPRmt. Activation of the UPRmt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPRmt, suggesting that surveillance-activated defenses specifically inhibit the UPRmt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.

Author Summary

Cellular respiration takes place in the mitochondria. Reactive oxygen species (ROS) are a damaging byproduct of cellular respiration. In adverse conditions, when the load of ROS becomes critical for mitochondrial function, a stress-response pathway, the unfolded protein response of the mitochondria (UPRmt), is triggered. This can be monitored by the activation of the hsp-6 gene. We used the model organism Caenorhabditis elegans to screen for genes required for the activation of hsp-6 and found 54 novel candidates. Surprisingly, most of the genes we identified serve basic cellular functions and are not plausible candidates for regulatory functions. However, this group of genes was recently shown to trigger a cellular surveillance–mediated stress response, sensing pathogen invasion and toxin attack, and forcing the animals to escape from environmental hazards. This mechanism partially requires signaling through the kinase KGB-1. In worms in which KGB-1 was inactivated, UPRmt was not longer interrupted by downregulation of the cellular surveillance activating genes. We suggest this surveillance system as a regulator of the UPRmt that prevents its activation when pathogen attack is sensed.

In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage–induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem.

Author Summary

As women age, the quality of their oocytes declines, causing the eggs they make to have a higher chance of producing a miscarriage or a child with birth defects. We used the roundworm C. elegans to study this problem. We show that oocyte quality declines in these small animals during aging, much as in mammals. Furthermore, our results show that the programmed deaths of many developing oocytes help maintain the quality of the oocytes that survive, resulting in better eggs. These cell deaths appear to regulate the way resources are allocated in the aging germ line. Since many oocytes die in humans as well as in nematodes, our studies point to the possibility of improving oocyte quality by manipulating cell death in the germ line.

An integrated gene network for Caenorhabditis elegans encompasses most protein-coding genes.

An integrated gene network for Caenorhabditis elegans using data from multiple genome-wide screens encompasses most protein-coding genes and can accurately predict their phenotypes.

A key question in development is how pluripotent progenitors are progressively restricted to acquire specific cell fates. Here we investigate how embryonic blastomeres in C. elegans develop into foregut (pharynx) cells in response to the selector gene PHA-4/FoxA. When pha-4 is removed from pharyngeal precursors, they exhibit two alternative responses. Before late-gastrulation (8E stage), these cells lose their pharyngeal identity and acquire an alternative fate such as ectoderm (Specification stage). After the Specification stage, mutant cells develop into aberrant pharyngeal cells (Morphogenesis/Differentiation stage). Two lines of evidence suggest that the Specification stage depends on transcriptional repression of ectodermal genes by pha-4. First, pha-4 exhibits strong synthetic phenotypes with the B class synMuv gene tam-1 (Tandam Array expression Modifier 1) and with a mediator of transcriptional repression, the NuRD complex (NUcleosome Remodeling and histone Deacetylase). Second, pha-4 associates with the promoter of the ectodermal regulator lin-26 and is required to repress lin-26 expression. We propose that restriction of early blastomeres to the pharyngeal fate depends on both repression of ectodermal genes and activation of pharyngeal genes by PHA-4.

SUMMARY

During organogenesis, pluripotent precursor cells acquire a defined identity such as muscle or nerve. The transition from naïve precursor towards the differentiated state is characterized by sequential waves of gene expression that are determined by regulatory transcription factors. A key question is how transcriptional circuitry dictates the succession of events that accompanies developmental competence, cell fate specification and differentiation. To address this question, we have examined how anterior muscles are established within the C. elegans foregut (pharynx). We find that the T-box transcription factor tbx-2 is essential to form anterior pharyngeal muscles from the ABa blastomere. In the absence of tbx-2 function, ABa-derived cells initiate development normally: they receive glp-1/Notch signaling cues, activate the T-box gene TBX-38 and express the organ selector gene PHA-4/FoxA. However, these cells subsequently arrest development, extinguish PHA-4 and fail to activate PHA-4 target genes. tbx-2 mutant cells do not undergo apoptosis and there is no evidence for adoption of an alternative fate. TBX-2 is expressed in ABa descendants and depends on activation by pha-4 and repression by glp-1/Notch signaling. Our analysis suggests that a positive feedback loop between tbx-2 and pha-4 is required for ABa-derived precursors to commit to pharyngeal muscle fate.

Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, we have found that the cytoplasmic Dynein motor is an essential regulator of apico–basal polarity. Our data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. We have mapped the sdt mRNA localization signal to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are only found during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico–basal organization. Moreover, we introduce a unique mechanism in which alternative splicing of a coding exon is used to control mRNA localization during development.

Author Summary

Cells within epithelial sheets are highly polarized with distinct apical and basolateral membrane domains. This cellular organization is critical to both epithelial form and function, and a failure to maintain epithelial polarity is often linked to tumor progression. The protein network that establishes and maintains the two membrane domains relies on the precise subcellular localization of its molecular components, but little is known about how these proteins are targeted to their sites of action. We have shown that the localization of the apical determinant protein Stardust depends on the microtubule motor Dynein. While investigating the relationship between Dynein and Stardust, we also made two unexpected observations about stardust mRNA regulation. First, the mechanism by which Dynein localizes Stardust may depend, in part, on the apical targeting of the stardust mRNA. Second, some stardust mRNA is apically localized during early stages of epithelial development, but the selective removal of the apical localization signal leads to the sole production of uniformly localized transcripts in mature epithelial cells. Together, these results introduce roles for Dynein in apico–basal polarity regulation and raise important questions about the role of mRNA localization in the targeting of polarity determinant proteins and epithelial maturation.

Accumulated research has demonstrated the beneficial effects of dietary restriction on extending lifespan and increasing cellular stress resistance. However, reducing nutrient intake has also been shown to direct animal behaviors toward food acquisition. Under food-limiting conditions, behavioral changes suggest that neuronal and muscle activities in circuits that are not involved in nutrient acquisition are down-regulated. These dietary-regulated mechanisms, if understood better, might provide an approach to compensate for defects in molecules that regulate cell excitability. We previously reported that a neuromuscular circuit used in Caenorhabditis elegans male mating behavior is attenuated under food-limiting conditions. During periods between matings, sex-specific muscles that control movements of the male's copulatory spicules are kept inactive by UNC-103 ether-a-go-go–related gene (ERG)–like K+ channels. Deletion of unc-103 causes ∼30%–40% of virgin males to display sex-muscle seizures; however, when food is deprived from males, the incidence of spontaneous muscle contractions drops to 9%–11%. In this work, we used genetics and pharmacology to address the mechanisms that act parallel with UNC-103 to suppress muscle seizures in males that lack ERG-like K+ channel function. We identify calcium/calmodulin-dependent protein kinase II as a regulator that uses different mechanisms in food and nonfood conditions to compensate for reduced ERG-like K+ channel activity. We found that in food-deprived conditions, calcium/calmodulin-dependent protein kinase II acts cell-autonomously with ether-a-go-go K+ channels to inhibit spontaneous muscle contractions. Our work suggests that upregulating mechanisms used by food deprivation can suppress muscle seizures.

Author Summary

We investigated the mechanisms used during dietary stress conditions that regulate the behavioral output of excitable cells. In the roundworm Caenorhabditis elegans, males must display the proper behavioral response to potential food sources or mating partners. This regulation is disrupted by loss of ERG-like K+ channel function. K+ channel defects cause a percentage of males to contract their genitalia muscles permanently, even in the absence of mating cues. For the percentage of K+ channel–defective males that do not display spontaneous sex-muscle seizure, we show that abnormal muscle contraction is attenuated by the calcium/calmodulin-dependent protein kinase II (CaMKII). Under food-deprived conditions, we find that CaMKII acts with the ether-a-go-go K+ channel to further suppress spontaneous sex-muscle seizures. We speculate that in food-abundant conditions, ether-a-go-go–related gene–like K+ channels act in parallel with a CaMKII-regulated pathway(s) to attenuate sexual behavior until mating signals are presented. If one of these mechanisms is disrupted, the other can compensate. In the absence of food, CaMKII and ether-a-go-go K+ channels further hyperpolarize the genital neuromuscular circuitry, changing the behavioral state so that the male will forage for food rather than mate.

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.

Author Summary

Microtubules and microtubule-dependent motor proteins segregate chromosomes during mitosis and also promote cellular organization in nondividing cells. An essential motor protein complex called cytoplasmic dynein powers many aspects of microtubule-dependent transport, but it is currently unclear how dynein is regulated such that it can execute different processes. We have performed a genome-wide screen to isolate genes that are involved in dynein-dependent processes. We determined that 20 of the 49 genes we identified specifically influenced the viability of dynein mutant strains but not the viability of other C. elegans mutants. Many of the proteins that specifically influence dynein localized to subcellular sites where the dynein heavy chain has been reported by others to be found. Additionally, we identified four dynein components that appear to negatively regulate the force-generating dynein heavy chain. The identification and initial characterization of this group of genes represents a route to identify genes that are not themselves essential but do participate in essential processes.

Regulation of spatio-temporal gene expression in diverse cell and tissue types is a critical aspect of development. Progression through Caenorhabditis elegans vulval development leads to the generation of seven distinct vulval cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), each with its own unique gene expression profile. The mechanisms that establish the precise spatial patterning of these mature cell types are largely unknown. Dissection of the gene regulatory networks involved in vulval patterning and differentiation would help us understand how cells generate a spatially defined pattern of cell fates during organogenesis. We disrupted the activity of 508 transcription factors via RNAi and assayed the expression of ceh-2, a marker for vulB fate during the L4 stage. From this screen, we identified the tailless ortholog nhr-67 as a novel regulator of gene expression in multiple vulval cell types. We find that one way in which nhr-67 maintains cell identity is by restricting inappropriate cell fusion events in specific vulval cells, namely vulE and vulF. nhr-67 exhibits a dynamic expression pattern in the vulval cells and interacts with three other transcriptional regulators cog-1 (Nkx6.1/6.2), lin-11 (LIM), and egl-38 (Pax2/5/8) to generate the composite expression patterns of their downstream targets. We provide evidence that egl-38 regulates gene expression in vulB1, vulC, vulD, vulE, as well as vulF cells. We demonstrate that the pairwise interactions between these regulatory genes are complex and vary among the seven cell types. We also discovered a striking regulatory circuit that affects a subset of the vulval lineages: cog-1 and nhr-67 inhibit both one another and themselves. We postulate that the differential levels and combinatorial patterns of lin-11, cog-1, and nhr-67 expression are a part of a regulatory code for the mature vulval cell types.

Author Summary

During development, in which the single-celled egg generates a whole organism, cells become different from each other and form patterns of types of cells. It is these spatially defined fate patterns that underlie the formation of complex organs. Regulatory molecules called transcription factors influence the fate patterns that cells adopt. Understanding the role of these transcription factors and their interactions with other genes could tell us how cells establish a certain pattern of cell fates. This study focuses on studying how the seven cell types of the Caenorhabditis elegans vulva arise. This organ is one of the most intensively studied, and while the signaling network that initiates vulval development and sets the gross pattern of cell differentiation is well understood, the network of transcription factors that specifies the final cell fates is not understood. Here, we identify nhr-67, a new transcription factor that regulates patterning of cell fates in this organ. Transcription factors do not necessarily act alone, and we explore how NHR-67 works with three other regulatory factors (each with human homologs) to specify the different properties of the vulval cells. We also demonstrate that the interconnections of these transcription factors differ between these seven diverse cell types, which may partially account for how these cells acquire a certain pattern of cell fates.

The histone variant H2A.Z is evolutionarily conserved and plays an essential role in mice, Drosophila, and Tetrahymena. The essential function of H2A.Z is unknown, with some studies suggesting a role in transcriptional repression and others in activation. Here we show that Caenorhabditis elegans HTZ-1/H2A.Z and the remodeling complex MYS-1/ESA1–SSL-1/SWR1 synergize with the FoxA transcription factor PHA-4 to coordinate temporal gene expression during foregut development. We observe dramatic genetic interactions between pha-4 and htz-1, mys-1, and ssl-1. A survey of transcription factors reveals that this interaction is specific, and thus pha-4 is acutely sensitive to reductions in these three proteins. Using a nuclear spot assay to visualize HTZ-1 in living embryos as organogenesis proceeds, we show that HTZ-1 is recruited to foregut promoters at the time of transcriptional onset, and this recruitment requires PHA-4. Loss of htz-1 by RNAi is lethal and leads to delayed expression of a subset of foregut genes. Thus, the effects of PHA-4 on temporal regulation can be explained in part by recruitment of HTZ-1 to target promoters. We suggest PHA-4 and HTZ-1 coordinate temporal gene expression by modulating the chromatin environment.

Synopsis

During development, a single fertilized egg gives rise to the different cell types within an embryo. These different cell types are characterized by the different genes that they express. A critical question in biology is how embryonic cells activate genes at the appropriate time and place to generate the different cell types. In this paper, the authors explore temporal regulation of gene expression during formation of an organ, namely the Caenorhabditis elegans foregut. They have discovered that foregut genes require a variant of the canonical H2A histone for the onset of transcription. This variant, called H2A.Z, or htz-1 in C. elegans, promotes transcription by modifying how DNA is packaged within cells. Their data suggest that a key regulator of foregut development, the transcription factor PHA-4, recruits HTZ-1 to pharyngeal promoters, and this association contributes to their timely activation.

The sensory systems of multicellular organisms are designed to provide information about the environment and thus elicit appropriate changes in physiology and behavior. In the nematode Caenorhabditis elegans, sensory neurons affect the decision to arrest during development in a diapause state, the dauer larva, and modulate the lifespan of the animals in adulthood. However, the mechanisms underlying these effects are incompletely understood. Using whole-genome microarray analysis, we identified transcripts whose levels are altered by mutations in the intraflagellar transport protein daf-10, which result in impaired development and function of many sensory neurons in C. elegans. In agreement with existing genetic data, the expression of genes regulated by the transcription factor DAF-16/FOXO was affected by daf-10 mutations. In addition, we found altered expression of transcriptional targets of the DAF-12/nuclear hormone receptor in the daf-10 mutants and showed that this pathway influences specifically the dauer formation phenotype of these animals. Unexpectedly, pathogen-responsive genes were repressed in daf-10 mutant animals, and these sensory mutants exhibited altered susceptibility to and behavioral avoidance of bacterial pathogens. Moreover, we found that a solute transporter gene mct-1/2, which was induced by daf-10 mutations, was necessary and sufficient for longevity. Thus, sensory input seems to influence an extensive transcriptional network that modulates basic biological processes in C. elegans. This situation is reminiscent of the complex regulation of physiology by the mammalian hypothalamus, which also receives innervations from sensory systems, most notably the visual and olfactory systems.

Author Summary

The senses provide animals with information about their environment, which affects not only their behavior but also their internal state and physiological outputs. How this information is processed is still unclear. In this study, we used mutant C. elegans roundworms that had defective sensory neurons to investigate how changes in sensation alter the expression of genes and regulate physiology, specifically the worms' choice to hibernate during growth and their longevity as fully-grown adults. We showed that defects in sensory neurons change the pattern of gene expression and regulate these outputs through known hormonal pathways, including insulin/IGF-1 and steroid pathways. We also identified a new regulator of longevity, MCT-1, that is predicted to transport small metabolites and hormones in the body. Unexpectedly, we found that sensory impairment altered yet another physiological output, the response to infectious agents. It prevented the worms from avoiding infectious bacteria and reduced the expression of potentially protective factors, but also increased the worms' resistance to infection, suggesting a complex network of responses to environmental stimuli. Understanding how sensory information is relayed in this relatively simple organism may inform our understanding of sensory processing in higher organisms like mammals.

The Caenorhabditis elegans one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another C. elegans DUB gene, usp-46, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in Schizosaccharomyces pombe are also required in C. elegans raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.

Author Summary

In eukaryotes, modification of proteins by the covalent ligation of a protein called ubiquitin is an important regulatory mechanism. In this study we found that deubiquitylation enzymes, which are known to cleave ubiquitin off of target proteins, are required for asymmetry in one-cell embryos of the nematode C. elegans. In one-cell embryos the establishment of asymmetry depends on a signal from the centrosome, a microtubule-organizing center. This signal breaks homogeneity in the contractile cytoskeleton located at the cortex of the embryo. We have identified three deubiquitylation enzymes that are necessary for the centrosome to properly localize adjacent to the cortex to perform its symmetry-breaking role. Furthermore, a homologous group of enzymes in fission yeast also regulates cell polarity. Our results suggest that a novel mechanism of centrosome localization regulated by ubiquitylation exists in C. elegans; this mechanism is masked by genetic redundancy and may be an evolutionarily conserved mechanism for cell asymmetry.

We have investigated the cis-regulatory network that mediates temporal gene expression during organogenesis. Previous studies demonstrated that the organ selector gene pha-4/FoxA is critical to establish the onset of transcription of Caenorhabditis elegans foregut (pharynx) genes. Here, we discover additional cis-regulatory elements that function in combination with PHA-4. We use a computational approach to identify candidate cis-regulatory sites for genes activated either early or late during pharyngeal development. Analysis of natural or synthetic promoters reveals that six of these sites function in vivo. The newly discovered temporal elements, together with predicted PHA-4 sites, account for the onset of expression of roughly half of the pharyngeal genes examined. Moreover, combinations of temporal elements and PHA-4 sites can be used in genome-wide searches to predict pharyngeal genes, with more than 85% accuracy for their onset of expression. These findings suggest a regulatory code for temporal gene expression during foregut development and provide a means to predict gene expression patterns based solely on genomic sequence.

Computational analysis combined with validation by reporter gene studies is uncovering the code for temporal gene regulation in the C. elegans foregut - a model for organogenesis

Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are “stored" in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is surprisingly little understood. A key player in Caenorhabditis elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1–dependent repression prevents precocious translation of OET–promoting mRNAs. Secondly, GLD-1– and CGH-1–dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET–promoting mRNAs, and consequently the oocyte-to-embryo transition, might be compromised.

Author Summary

One of the most striking developmental events is the oocyte-to-embryo transition that, in the absence of Pol II–dependent transcription, depends on regulated translation of maternal mRNAs. Prior to their activation, these maternal mRNAs need to be “stored" in the egg cytoplasm in a repressed but stable form. Surprisingly little is known about how the stored mRNAs are stabilized. The STAR family of RNA–binding proteins includes the C. elegans GLD-1, which controls many aspects of germ cell development. To obtain a comprehensive picture of GLD-1–dependent mRNA regulation, we performed a genome-wide survey of translational repression and mRNA stability of GLD-1 targets. This uncovered a potential role of GLD-1 in mRNA storage, as we found that GLD-1 both represses and stabilizes a subpopulation of its targets. The stabilization also involves a DDX6-like RNA helicase, CGH-1, which is a component of repressive germ granules and processing bodies. Remarkably, the GLD-1 and CGH-1 stabilized mRNAs encode regulators of the oocyte-to-embryo transition, providing an insight into how these functionally related mRNAs are specifically stabilized during germ cell formation. These findings have potential implications for oocyte quality and reproductive fitness, and for mRNA storage in other cell types such as neurons.

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7–independent positive regulation of hbl-1 through its 3′UTR to control L2 stage-specific cell fates; and second, a let-7–dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.

Author Summary

As tissues form, different cell types are generated from a common pool of undifferentiated cells. The mechanisms that control this developmental timing are largely unknown. In the nematode Caenorhabditis elegans, the heterochronic genes control a succession of cell fates in progressively differentiating tissues of the larva. Two of these genes, lin-28 and let-7, are evolutionarily conserved in animals where they have roles in pluripotency and differentiation. The LIN-28 protein is known to bind to and block the maturation of the small RNA encoded by let-7. This mechanism would seem to explain lin-28's role in development. Here we show that lin-28's primary activity in C. elegans—the proper timing of second larval stage cell fates—does not require let-7 or related genes. In explaining this discrepancy, we provide evidence that lin-28 has two distinct activities controlling successive cell fates. This situation is remarkably like that of lin-14, which acts one stage earlier. The overlap of their activities by one stage may reflect a fundamental feature of this cell fate succession mechanism. Furthermore, the two-step mechanism explains observations that mammalian Lin28 also has genetically separable activities. Therefore, lin-28's two successive activities may be essential to its evolutionarily conserved role in developmental timing.

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

Author Summary

In metazoans, soma and germline have specialized functions that require differential tissue-specific gene expression. In C. elegans, explicit chromatin marks deposited by the MES-4 histone methyltransferase and the MRG-1 chromodomain protein allow germline expression of particular suites of target genes. Conversely, the expression of germline-specific genes is repressed in somatic cells by other chromatin regulatory factors, including the retinoblastoma pathway genes. We characterized the distinct profiles of somatic misexpression of normally germline-specific genes in these mutants and mapped out three chromatin complexes that prevent misexpression. We demonstrate that one of the complexes closely counteracts the activity of MES-4 and MRG-1, whereas another complex interacts with additional regulators that are yet to be identified. We show that these intersecting chromatin complexes prevent the upregulation of a suite of germline-specific as well as ubiquitous small RNA pathway genes, which contributes to the enhanced RNAi response in retinoblastoma pathway mutant worms. We suggest that this function of the retinoblastoma pathway chromatin factors to prevent germline-associated gene expression programs in the soma and the upregulation of small RNA pathways may also underlie their role as tumor suppressors.