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Molting Defects Caused by RNAi N2 larvae were fed bacteria expressing dsRNA corresponding to the indicated genes, or control bacteria not expressing dsRNA of a worm gene (A). Panels A–D show the anterior, whereas (E) shows the mid-body of a larva. Black arrowheads mark unshed cuticle. White arrowhead (C) indicates the buccal capsule. Nomarski optics. DOI: 10.1371/journal.pbio.0030312.g001
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Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-inter...
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... identify a full set of endocrine and enzymatic regulators of molting in C. elegans, we screened a combined library of 18,578 bacterial clones that each express a double-stranded RNA designed to silence one of the 19,427 predicted worm genes via RNAi [26][27][28]. About 25 L1-stage larvae were fed each clone and later examined for molting defects, indicated by the adherence of cuticle from the pre-molt larval stage to the body of the worm (the Mlt phenotype; Figure 1). Gene inactivations observed to prevent molting in the primary library screen were tested again by feeding the bacterial clones to approximately 50 wild-type (N2) and 50 rrf-3(pk1426) mutant larvae, a genetic background where RNAi is more effective [29]. ...
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... genes fbn-1, noah-1, and noah-2 were assigned names based on homology to genes of mammals or insects (Table S1). Figure 1 shows examples of molting-defective larvae produced by RNAi. Most often, larvae were observed incarcerated in sheaths of old cuticle extending from the anterior end of the worm, as shown for a mlt-11(RNAi) larva ( Figure 1B). ...
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... genes fbn-1, noah-1, and noah-2 were assigned names based on homology to genes of mammals or insects (Table S1). Figure 1 shows examples of molting-defective larvae produced by RNAi. Most often, larvae were observed incarcerated in sheaths of old cuticle extending from the anterior end of the worm, as shown for a mlt-11(RNAi) larva ( Figure 1B). The nature of molting defects caused by particular gene inactivations suggested that the correspond- ing proteins function in a specific anatomical place or stage of ecdysis. ...
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... similar type of molting defect has been observed in animals lacking the DNA binding protein PEB-1 [35]. Many mlt- 9(RNAi) larvae fail to shed cuticle lining the buccal cavity, causing the lips to evert ( Figure 1C). Unshed cuticle often forms coronal constrictions on nas-37(RNAi) larvae ( Figure 1E), possibly when animals flip on their long axis during molting. ...
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... mlt- 9(RNAi) larvae fail to shed cuticle lining the buccal cavity, causing the lips to evert ( Figure 1C). Unshed cuticle often forms coronal constrictions on nas-37(RNAi) larvae ( Figure 1E), possibly when animals flip on their long axis during molting. Inactivation of the collagenase gene nas-37 also prevents the breakdown of old cuticle at the anterior tip of the worm, thereby blocking escape from the old exoskeleton (unpublished data) [23,24]. ...
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... from all eight of the gfp fusion genes was observed in epithelial cells that synthesize cuticle (Figures 2 and S1). The qua-1, nas-37, mlt-9, mlt-11, acn-1, mlt-8, and mlt-10 fusion genes were each expressed in the hypodermis, including the major body syncytium, hyp7, and hypodermal cells in the head and tail (Figure 2A-C and 2E-F) (unpub- lished data). ...
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... protease NAS-37 and anti-protease MLT-11 may therefore be required, respec- tively, to induce or repress fusion of the seam cells. The gfp fusion gene for the exoribonuclease xrn-2 was expressed in specialized myoepithelial cells that secrete the pharyngeal cuticle ( Figures 2D and S1H), consistent with the defect of xrn-2(RNAi) larvae in shedding cuticle from the pharynx. Particular fusion genes were also expressed in specialized interfacial cells that secrete cuticle, including the rectal epithelia (Figure 2A), the excretory duct and pore cells ( Figure S1A-B), the vulval epithelium ( Figure S1D), and the rectal gland ( Figure S1E), as well as support cells for sensory neurons ( Figure S1A and S1F). ...
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... gfp fusion gene for the exoribonuclease xrn-2 was expressed in specialized myoepithelial cells that secrete the pharyngeal cuticle ( Figures 2D and S1H), consistent with the defect of xrn-2(RNAi) larvae in shedding cuticle from the pharynx. Particular fusion genes were also expressed in specialized interfacial cells that secrete cuticle, including the rectal epithelia (Figure 2A), the excretory duct and pore cells ( Figure S1A-B), the vulval epithelium ( Figure S1D), and the rectal gland ( Figure S1E), as well as support cells for sensory neurons ( Figure S1A and S1F). ...
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... gfp fusion gene for the exoribonuclease xrn-2 was expressed in specialized myoepithelial cells that secrete the pharyngeal cuticle ( Figures 2D and S1H), consistent with the defect of xrn-2(RNAi) larvae in shedding cuticle from the pharynx. Particular fusion genes were also expressed in specialized interfacial cells that secrete cuticle, including the rectal epithelia (Figure 2A), the excretory duct and pore cells ( Figure S1A-B), the vulval epithelium ( Figure S1D), and the rectal gland ( Figure S1E), as well as support cells for sensory neurons ( Figure S1A and S1F). ...
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... gfp fusion gene for the exoribonuclease xrn-2 was expressed in specialized myoepithelial cells that secrete the pharyngeal cuticle ( Figures 2D and S1H), consistent with the defect of xrn-2(RNAi) larvae in shedding cuticle from the pharynx. Particular fusion genes were also expressed in specialized interfacial cells that secrete cuticle, including the rectal epithelia (Figure 2A), the excretory duct and pore cells ( Figure S1A-B), the vulval epithelium ( Figure S1D), and the rectal gland ( Figure S1E), as well as support cells for sensory neurons ( Figure S1A and S1F). ...
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... gfp fusion gene for the exoribonuclease xrn-2 was expressed in specialized myoepithelial cells that secrete the pharyngeal cuticle ( Figures 2D and S1H), consistent with the defect of xrn-2(RNAi) larvae in shedding cuticle from the pharynx. Particular fusion genes were also expressed in specialized interfacial cells that secrete cuticle, including the rectal epithelia (Figure 2A), the excretory duct and pore cells ( Figure S1A-B), the vulval epithelium ( Figure S1D), and the rectal gland ( Figure S1E), as well as support cells for sensory neurons ( Figure S1A and S1F). ...
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... particular fusion genes were expressed in neurons and gland cells that might produce or respond to endocrine signals regulating molting. For example, xrn-2p::gfp was expressed in several anterior neurons, including sensory neurons, as well as the PVT neuron that projects along the ventral cord, and the M5 pharyngeal neuron (Figures 2G, S1G, and S1H). Expression of xrn-2 in the M5 neuron might be relevant to molting because M5 innervates gland cells whose secretions are thought to expedite release of the pharyngeal cuticle [19,35]. ...
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... mlt-8 reporter was expressed, in larvae, in a single posterior neuron that remains to be identified. Interestingly, particular fusion genes, such as acn-1p::gfp-pest, were also expressed in the excretory gland cell of larvae ( Figure S1A-C). The gland cell is active during ecdysis [63] and is thought to contribute material for the new surface coat. ...
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... of the organism, modulate secretion of prothoracicotropic hormone in various arthropods, suggesting extensive sensory input to the neuroendocrine secretions that govern molting [3]. However, little is known about the circuits that initiate, terminate, or set the pace of the molting cycle in any Ecdysozoan. Although an endocrine trigger for nematode molting has yet to be identified, several lines of evidence implicate steroid hormones in Caenorhabditis elegans molting. Molting of C. elegans requires cholesterol, the biosynthetic precursor of all steroid hormones, as well as the low-density lipoprotein (LDL) receptor-like protein LRP-1, which is thought to endocytose sterols from the growth medium [11]. A sterol- modifying enzyme synthesized in the intestine, LET-767, is also essential for molting, consistent with the production or modification of a hormone derived from steroids [12]. The best evidence of a hormonal cue for molting of C. elegans is the requirement for two nuclear hormone receptors (NHRs), NHR-23 and NHR-25, orthologous, respectively, to the ecdysone-responsive gene products DHR3 and Ftz-F1 of Drosophila melanogaster [13–16]. Ecdysone itself, however, is unlikely to serve as a molting hormone in nematodes because ecdysteroids have not been detected in any free-living nematode [17], and because orthologs of the ecdysone receptor components EcR and USP (Ultraspiracle) have not been identified in the complete genome of C. elegans [18]. Molting of C. elegans involves the synthesis and secretion of a new exoskeleton underneath the old one, separation of the old exoskeleton from the epidermis (apolysis), and escape from the old exoskeleton (ecdysis) [19]. At the end of each stage, larvae become inactive for a brief period of time known as lethargus that coincides with separation of the old exoskeleton from the epidermis. Next, particular behaviors promote ecdysis; larvae flip on their long axis to loosen the body cuticle, expel the anterior half of the pharyngeal cuticle, and ultimately escape the old exoskeleton via a forward thrust [19]. The exoskeleton of nematodes, called the cuticle, is a collagenous extracellular matrix secreted by underlying epithelial cells, known as the hypodermis and seam cells, and also by specialized interfacial cells that line openings of the body, including the buccal cavity, pharynx, vulva, rectum, and sensilia [20]. Lipids and glycolipids comprise the outer- most layer of the cuticle, whereas glycoproteins, thought to be secreted by gland cells, form the surface coat [21]. Elasticity of the cuticle permits growth during each larval stage, but particular structures, such as the buccal cavity, grow salta- tionally at molts [22]. The distinction between collagen in the nematode exoskeleton and chitin in the insect exoskeleton suggests that the enzymatic cascades that mediate release of the exoskeleton in nematodes may be distinct from those that release the exoskeleton in arthropods. Although two collagenases essential for molting have been identified in C. elegans [23,24], the full ensemble of signaling proteins and extracellular matrix enzymes required to remodel the exoskeleton has yet to be illuminated. Human diseases caused by parasitic nematodes affect tropical regions of Africa, Asia, and South America. The World Health Organization estimates that 120 million people endure lymphatic filariasis (elephantiasis), due to infection by the filarial nematodes Wuchereria bancrofti or Brugia malayi, and that 18 million people endure onchocerciasis (African river blindness), due to infection by Onchocerca volvulus [25]. Ascaris, hookworms, and whipworms are also important pathogens, infecting approximately 1 billion people. Parasitic nematodes further damage livestock and lay waste to $80 billion of crop plants annually. One promising approach to the discovery of new targets for anti-nematode drugs, vaccines, and pesticides is the identification of nematode-specific genes essential for the viability of larvae. In the screen described here, a large number of nematode-specific genes essential for molting were identified, and some encode attractive drug targets. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference (RNAi) screen, providing a broad view of functions essential for molting in a model Ecdysozoan. We further develop models for the genetic regulation of molting based on the location, timing, and order of expression of particular molting genes. To identify a full set of endocrine and enzymatic regulators of molting in C. elegans, we screened a combined library of 18,578 bacterial clones that each express a double-stranded RNA designed to silence one of the 19,427 predicted worm genes via RNAi [26–28]. About 25 L1-stage larvae were fed each clone and later examined for molting defects, indicated by the adherence of cuticle from the pre-molt larval stage to the body of the worm (the Mlt phenotype; Figure 1). Gene inactivations observed to prevent molting in the primary library screen were tested again by feeding the bacterial clones to approximately 50 wild-type (N2) and 50 rrf-3(pk1426) mutant larvae, a genetic background where RNAi is more effective [29]. Inactivation of 159 genes (Tables 1 and S1–S4) interfered with molting. Tables 1 and S1 show genes whose inactivation produced molting defects in 10% to 100% of wild-type (N2) or rrf-3(pk1426) mutant larvae. Eighty-seven other genes were assigned a lower priority based on gene annotation (Tables S2 and S3) or the low penetrance of molting defects observed after RNAi (Table S4). The blind identification of nine genes previously described to cause an arrest at a molt, including lrp-1, nhr-23, nhr-25, nas-37, nas-36, skp-1, rme-8, acn-1, and bli-4 [11,13–16,23,24,30–33], verified the efficacy of this RNAi- based strategy for isolating bona fide molting genes. An additional 28 of the 159 gene inactivations were independently described as causing an arrest at a molt in broad screens that identified many loss-of-function phenotypes using RNAi [26,27,34]. Further, the observation of molting defects in larvae with mutations in qua-1 (unpublished data), lrp-1 [11], and nas-37 [23] verified that RNAi faithfully recapitulates loss- of-function phenotypes in the molting pathway. The names mlt-8, mlt-9, and mlt-11 were assigned, respectively, to W08F4.6, F09B12.1 , and W01F3.3 after expression data verified a primary function in molting. The genes fbn-1, noah-1, and noah-2 were assigned names based on homology to genes of mammals or insects (Table S1). Figure 1 shows examples of molting-defective larvae produced by RNAi. Most often, larvae were observed incarcerated in sheaths of old cuticle extending from the anterior end of the worm, as shown for a mlt-11(RNAi) larva (Figure 1B). The nature of molting defects caused by particular gene inactivations suggested that the corresponding proteins function in a specific anatomical place or stage of ecdysis. For example, L4-stage larvae fail to shed cuticle from the pharynx after RNAi of xrn-2 (unpublished data). A similar type of molting defect has been observed in animals lacking the DNA binding protein PEB-1 [35]. Many mlt- 9(RNAi) larvae fail to shed cuticle lining the buccal cavity, causing the lips to evert (Figure 1C). Unshed cuticle often forms coronal constrictions on nas-37(RNAi) larvae (Figure 1E), possibly when animals flip on their long axis during molting. Inactivation of the collagenase gene nas-37 also prevents the breakdown of old cuticle at the anterior tip of the worm, thereby blocking escape from the old exoskeleton (unpublished data) [23,24]. Although Mlt larvae typically arrest development, some gradually escape from the old cuticle, only to fail again at the next molt, a phenomenon observed often after RNAi of qua-1 (unpublished data). The majority of genes we identified are likely to act at all four molts, because their inactivation prevents molting from several larval stages. Moreover, although feeding L1-stage larvae dsRNA for particular genes, such as mlt-8 or acn-1, prevents development beyond the L3 stage (Table S5), feeding the same dsRNAs to older larvae also disrupts the final molt (unpublished data). The majority of gene inactivations also disrupt molting from the dauer stage, an alternative L3 stage that is adapted for survival in unfavorable conditions and resembles the infective form of parasitic nematodes (Table S6). The majority of genes we identified are conserved in parasitic nematodes responsible for human, animal, and plant diseases (Table S7). Many of the genes, including mlt-8 and mlt-9, are conserved only in nematodes; similar proteins are readily identified among the predicted products of cDNAs or genomic sequences from parasitic and free-living nematodes (Table S7), but not in the translated genomes of D. melanogaster or Homo sapiens (Table S1). In contrast, the genes noah-1 and noah-2, which specify putative extracellular matrix components, are conserved in insects and nematodes, but not in humans, and thus show the phylogenetic conservation signature expected for molting genes common to Ecdysozoans. In this section, we discuss how the annotations of particular genes uncovered by RNAi implicate the corresponding proteins in basic aspects of the molting cycle. Based on experimental evidence of a steroidal pathway, as well as the evolutionary relationship between arthropods and nematodes [1], we expect that endocrine cues periodically initiate molting in C. elegans, stimulating the synthesis of a new cuticle and release of the old one. We expected to isolate many genes essential for apolysis or ecdysis because we screened for larvae arrested at the final stage of the molt. However, we also anticipated the identification of genes required for the production of, or response to, hormonal cues for molting, because the loss of either nhr-23 in C. elegans or EcR in D. ...
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... (apolysis), and escape from the old exoskeleton (ecdysis) [19]. At the end of each stage, larvae become inactive for a brief period of time known as lethargus that coincides with separation of the old exoskeleton from the epidermis. Next, particular behaviors promote ecdysis; larvae flip on their long axis to loosen the body cuticle, expel the anterior half of the pharyngeal cuticle, and ultimately escape the old exoskeleton via a forward thrust [19]. The exoskeleton of nematodes, called the cuticle, is a collagenous extracellular matrix secreted by underlying epithelial cells, known as the hypodermis and seam cells, and also by specialized interfacial cells that line openings of the body, including the buccal cavity, pharynx, vulva, rectum, and sensilia [20]. Lipids and glycolipids comprise the outer- most layer of the cuticle, whereas glycoproteins, thought to be secreted by gland cells, form the surface coat [21]. Elasticity of the cuticle permits growth during each larval stage, but particular structures, such as the buccal cavity, grow salta- tionally at molts [22]. The distinction between collagen in the nematode exoskeleton and chitin in the insect exoskeleton suggests that the enzymatic cascades that mediate release of the exoskeleton in nematodes may be distinct from those that release the exoskeleton in arthropods. Although two collagenases essential for molting have been identified in C. elegans [23,24], the full ensemble of signaling proteins and extracellular matrix enzymes required to remodel the exoskeleton has yet to be illuminated. Human diseases caused by parasitic nematodes affect tropical regions of Africa, Asia, and South America. The World Health Organization estimates that 120 million people endure lymphatic filariasis (elephantiasis), due to infection by the filarial nematodes Wuchereria bancrofti or Brugia malayi, and that 18 million people endure onchocerciasis (African river blindness), due to infection by Onchocerca volvulus [25]. Ascaris, hookworms, and whipworms are also important pathogens, infecting approximately 1 billion people. Parasitic nematodes further damage livestock and lay waste to $80 billion of crop plants annually. One promising approach to the discovery of new targets for anti-nematode drugs, vaccines, and pesticides is the identification of nematode-specific genes essential for the viability of larvae. In the screen described here, a large number of nematode-specific genes essential for molting were identified, and some encode attractive drug targets. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference (RNAi) screen, providing a broad view of functions essential for molting in a model Ecdysozoan. We further develop models for the genetic regulation of molting based on the location, timing, and order of expression of particular molting genes. To identify a full set of endocrine and enzymatic regulators of molting in C. elegans, we screened a combined library of 18,578 bacterial clones that each express a double-stranded RNA designed to silence one of the 19,427 predicted worm genes via RNAi [26–28]. About 25 L1-stage larvae were fed each clone and later examined for molting defects, indicated by the adherence of cuticle from the pre-molt larval stage to the body of the worm (the Mlt phenotype; Figure 1). Gene inactivations observed to prevent molting in the primary library screen were tested again by feeding the bacterial clones to approximately 50 wild-type (N2) and 50 rrf-3(pk1426) mutant larvae, a genetic background where RNAi is more effective [29]. Inactivation of 159 genes (Tables 1 and S1–S4) interfered with molting. Tables 1 and S1 show genes whose inactivation produced molting defects in 10% to 100% of wild-type (N2) or rrf-3(pk1426) mutant larvae. Eighty-seven other genes were assigned a lower priority based on gene annotation (Tables S2 and S3) or the low penetrance of molting defects observed after RNAi (Table S4). The blind identification of nine genes previously described to cause an arrest at a molt, including lrp-1, nhr-23, nhr-25, nas-37, nas-36, skp-1, rme-8, acn-1, and bli-4 [11,13–16,23,24,30–33], verified the efficacy of this RNAi- based strategy for isolating bona fide molting genes. An additional 28 of the 159 gene inactivations were independently described as causing an arrest at a molt in broad screens that identified many loss-of-function phenotypes using RNAi [26,27,34]. Further, the observation of molting defects in larvae with mutations in qua-1 (unpublished data), lrp-1 [11], and nas-37 [23] verified that RNAi faithfully recapitulates loss- of-function phenotypes in the molting pathway. The names mlt-8, mlt-9, and mlt-11 were assigned, respectively, to W08F4.6, F09B12.1 , and W01F3.3 after expression data verified a primary function in molting. The genes fbn-1, noah-1, and noah-2 were assigned names based on homology to genes of mammals or insects (Table S1). Figure 1 shows examples of molting-defective larvae produced by RNAi. Most often, larvae were observed incarcerated in sheaths of old cuticle extending from the anterior end of the worm, as shown for a mlt-11(RNAi) larva (Figure 1B). The nature of molting defects caused by particular gene inactivations suggested that the corresponding proteins function in a specific anatomical place or stage of ecdysis. For example, L4-stage larvae fail to shed cuticle from the pharynx after RNAi of xrn-2 (unpublished data). A similar type of molting defect has been observed in animals lacking the DNA binding protein PEB-1 [35]. Many mlt- 9(RNAi) larvae fail to shed cuticle lining the buccal cavity, causing the lips to evert (Figure 1C). Unshed cuticle often forms coronal constrictions on nas-37(RNAi) larvae (Figure 1E), possibly when animals flip on their long axis during molting. Inactivation of the collagenase gene nas-37 also prevents the breakdown of old cuticle at the anterior tip of the worm, thereby blocking escape from the old exoskeleton (unpublished data) [23,24]. Although Mlt larvae typically arrest development, some gradually escape from the old cuticle, only to fail again at the next molt, a phenomenon observed often after RNAi of qua-1 (unpublished data). The majority of genes we identified are likely to act at all four molts, because their inactivation prevents molting from several larval stages. Moreover, although feeding L1-stage larvae dsRNA for particular genes, such as mlt-8 or acn-1, prevents development beyond the L3 stage (Table S5), feeding the same dsRNAs to older larvae also disrupts the final molt (unpublished data). The majority of gene inactivations also disrupt molting from the dauer stage, an alternative L3 stage that is adapted for survival in unfavorable conditions and resembles the infective form of parasitic nematodes (Table S6). The majority of genes we identified are conserved in parasitic nematodes responsible for human, animal, and plant diseases (Table S7). Many of the genes, including mlt-8 and mlt-9, are conserved only in nematodes; similar proteins are readily identified among the predicted products of cDNAs or genomic sequences from parasitic and free-living nematodes (Table S7), but not in the translated genomes of D. melanogaster or Homo sapiens (Table S1). In contrast, the genes noah-1 and noah-2, which specify putative extracellular matrix components, are conserved in insects and nematodes, but not in humans, and thus show the phylogenetic conservation signature expected for molting genes common to Ecdysozoans. In this section, we discuss how the annotations of particular genes uncovered by RNAi implicate the corresponding proteins in basic aspects of the molting cycle. Based on experimental evidence of a steroidal pathway, as well as the evolutionary relationship between arthropods and nematodes [1], we expect that endocrine cues periodically initiate molting in C. elegans, stimulating the synthesis of a new cuticle and release of the old one. We expected to isolate many genes essential for apolysis or ecdysis because we screened for larvae arrested at the final stage of the molt. However, we also anticipated the identification of genes required for the production of, or response to, hormonal cues for molting, because the loss of either nhr-23 in C. elegans or EcR in D. melanogaster can result in a terminal failure to ecdyse [14,36]. Also, a breakdown in the coordination of signaling events associated with molting might trigger an aberrant ecdysis and thereby cause arrest at that stage. The identification of several transcription factors suggests that molting of C. elegans requires extensive changes in gene expression, similar to how transcriptional cascades promote molting and metamorphosis of insects [2]. Particular transcription factors likely alter gene expression in epithelial cells, possibly in response to endocrine cues. Annotated DNA binding proteins and transcription factors required for molting include three zinc-finger proteins, specified by F10C1.5, F25H8.6, and lir-1 [37], that resemble, respectively, Drosophila Doublesex, BED subfamily members, and the C. elegans transcription factor LIN-26, as well as two NHRs, NHR-23 and NHR-25, that were previously implicated in molting [13,15]. NHR-23 and NHR-25 are the best candidates for transducing hormonal signals, because the NHRs are expressed in epithelial cells and conserved in insects [13,15,16,30]. In theory, NHRs required for molting might regulate the expression of zinc-finger transcription factors identified in this screen, just as particular NHRs activate zinc- finger proteins in the transcriptional cascades coupled to insect metamorphosis [2]. The xrn-2 gene encodes a 5 9 -3 9 exoribonuclease that is conserved from yeast to humans [38] and is essential for molting in C. elegans . The homologous enzyme, Rat1p, is required for degradation of nuclear pre-mRNAs as well as the 5 9 processing of ribosomal and ...
Citations
... In the model nematode Caenorhabditis elegans, some Ecd-response genes (Hr3 and Ftzf1) are involved in molting regulation (Fig. 1B, nhr-23 and nhr-25, respectively) 15-18 , suggesting their evolutionarily conserved roles. Interestingly, however, other genes, such as E75, Hr4, Hr78, and Hr38, are unrelated to molting regulation in C. elegans (Fig. 1B) 18 , and molting in C. elegans is independent from Ecd hormone as they lost Ecr (Ecd receptor) genes [19][20][21] . In contrast to C. elegans, Ecr was identified in some parasitic nematodes such as Brugia malayi and Dirofilaria immitis 22,23 . ...
Molting is a defining feature of the most species-rich animal taxa, the Ecdysozoa, including arthropods, tardigrades, nematodes, and others. In pancrustaceans, such as insects and decapods, molting is regulated by the ecdysteroid (Ecd) hormone and its downstream cascade. However, whether the regulation of molting predates the emergence of the arthropods and represents an ancestral machinery of ecdysozoans remains unclear. Therefore, we investigated the role of Ecd in the molting process of the tardigrade Hypsibius exemplaris. We show that the endogenous Ecd level periodically increases during the molting cycle of H. exemplaris. The pulse treatment with exogenous Ecd induced molting while an antagonist of the Ecd receptor suppressed the molting. Our spatial and temporal gene expression analysis revealed the putative regulatory organs and Ecd downstream cascades. We demonstrate that tardigrade molting is regulated by Ecd hormone, supporting the ancestry of Ecd-dependent molting in panarthropods. Further, we were able to identify the putative neural center of molting regulation in tardigrades, which may represent an ancestral state of panarthropods homologous to the protocerebrum of pancrustaceans. Together, our results suggest that Ecd-dependent molting evolved 100 million years earlier than previously suggested.
... To generate synchronized populations of L1 larvae, gravid adults were subjected to treatment with bleach, after which eggs were washed at least five times and allowed to hatch in M9 buffer overnight at room temperature with gentle rotation (PORTA-DE-LA- RIVA et al. 2012). Hatched larvae were transferred to NGM/OP50 plates, and pharyngeal pumping or Pmlt-10::GFP-PEST (FRAND et al. 2005) expression was recorded every hour at 20°C using an Olympus MVX10 MacroView microscope equipped with a 2´ objective. Molting was defined as when ³50% animals (n = 10) were pumping or when >50% expressed Pmlt-10::GFP-PEST. ...
... Using two different strong loss-of-function alleles of catp-1 (ok2585 and kr17), we observed a slight lengthening of both L1 and L2 by ~1.1-to 1.2-fold in catp-1 mutants but no strong or specific effect at the L2 stage ( Figure 3A, C). As a further assessment, we similarly timed molting cycles using the Pmlt-10::GFP-PEST marker, which peaks in expression for several hours during each molting cycle (FRAND et al. 2005). Based on this marker, catp-1 mutants displayed an ~1.2-fold increase in the length of L1 but exhibited little or no increase in the length of L2 ( Figure 3B, C). ...
The conserved C. elegans protein kinases NEKL-2 and NEKL-3 regulate multiple steps of membrane trafficking and are required for larval molting. Through a forward genetic screen we identified a loss-of-function mutation in catp-1 as a suppressor of molting defects in synthetically lethal nekl-2; nekl-3 double mutants. catp-1 is predicted to encode a membrane-associated P4-type ATPase involved in Na+K+ exchange. Moreover, a mutation predicted to abolish CATP-1 ion-pump activity also suppressed nekl-2; nekl-3 mutants. Endogenously tagged CATP-1 was primarily expressed in epidermal (hypodermal) cells within punctate structures located at or near the apical plasma membrane. Through whole genome sequencing, we identified two additional nekl-2; nekl-3 suppressor strains containing coding-altering mutations in catp-1 but found that neither mutation, when introduced into nekl-2; nekl-3 mutants using CRISPR methods, was sufficient to elicit robust suppression of molting defects. Our data also suggested that the two catp-1 isoforms, catp-1a and catp-1b, may in some contexts be functionally redundant. On the basis of previously published studies, we tested the hypothesis that loss of catp-1 may suppress nekl-associated defects by inducing partial entry into the dauer pathway. Contrary to expectations, however, we failed to obtain evidence that loss of catp-1 suppresses nekl-2; nekl-3 defects through a dauer-associated mechanism or that loss of catp-1 leads to entry into the pre-dauer L2d stage. As such, loss of catp-1 may suppress nekl-associated molting and membrane trafficking defects by altering electrochemical gradients within membrane-bound compartments.
... lin-42 gene exhibits a clear developmental rhythm at the mRNA level 10 , and lin-42 mutants exhibit an arhythmic molting cycle 9,12 . Furthermore, the C. elegans Ror homolog, nhr-23, controls rhythmic transcriptions of molting-related genes [13][14][15][16] and is hence indispensable for developmental progression 15,17 . Thus, in C. elegans, it has been believed that the ancestral circadian clock had evolved into the developmental clock 8 . ...
... We discovered that the expression of Ror/nhr-23-targeted genes exhibited adult circadian rhythms in a specific phase, while they exhibited developmental rhythms during larval development. We then showed that Ror/nhr-23 is essential for the adult circadian rhythms, as well as for the larval developmental rhythms 13,17,24 . These results suggest that bilaterian ancestral circadian clock genes might have evolved to count multiple periods in C. elegans. ...
... Hereafter, we call these genes "cluster ii genes". Since these cluster ii genes were likely to be regulated by the same transcription factor, we investigated the detail of these genes and found that some of them (dpy-2, noah-1, mlt-8, mlt-10, mlt-11) were reported to be regulated by nhr-23 [13][14][15][16]24 , the homolog of the clock gene Ror. To examine whether other cluster ii genes are also downstream of ROR/NHR-23, we explored the genome sequence and a previous comprehensive ChIP-seq dataset 27 . ...
Animals have internal clocks that generate biological rhythms. In mammals, clock genes such as Period form the circadian clock to generate approximately 24-h biological rhythms. In C. elegans, the clock gene homologs constitute the “developmental clock”, which has an 8-h period during larval development to determine the timing of molting. Thus, the ancestral circadian clock has been believed to evolve into the oscillator with a shorter period in C. elegans. However, circadian rhythms have also been observed in adult C. elegans, albeit relatively weak. This prompts the question: if the clock gene homologs drive the developmental rhythm with 8-h period, which genes generate the circadian rhythms in C. elegans? In this study, we discovered that nhr-23, a homolog of the mammalian circadian clock gene Ror, is essential for circadian transcriptional rhythms in adult C. elegans. Interestingly, nhr-23 was also known to be essential for the molting clock. The bilaterian ancestral circadian clock genes might have evolved to function over multiple periods depending on developmental contexts rather than a single 8-h period in C. elegans.
... acn-1 mRNA levels were measured by RNA-seq in a synchronized population of spe-9(lf) animals cultured at 25°C to prevent progeny production. acn-1 mRNA levels were high in larvae, consistent with previous reports (Fig. 6B) (Brooks et al., 2003;Frand et al., 2005;Oskouian et al., 2005;Metheetrairut et al., 2017). Interestingly, acn-1 mRNA levels declined in young adults but then remained relatively constant throughout Development • Accepted manuscript adulthood (Fig. 6B). ...
The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in C. elegans, Drosophila, and rodents, but its mechanism is not well defined. Here we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril-hypersensitive mutants. We identified a missense mutation that causes a partial loss-of-function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNAi promoted dauer larvae formation, suggesting acn-1 is a daf gene. Captopril-mediated lifespan extension was abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 influence lifespan by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.
... These proteins have not been characterized extensively. However, its orthologue mlt-11 in C. elegans is known to be associated with molting 72 . Knockdown of mlt-11 by RNAi induced Larval (L1) arrest and defects during larval molting [74][75][76] . ...
This study employed subtractive proteomics and immunoinformatics to analyze the Wuchereria bancrofti proteome and identify potential therapeutic targets, with a focus on designing a vaccine against the parasite species. A comprehensive bioinformatics analysis of the parasite's proteome identified 51 probable therapeutic targets, among which "Kunitz/bovine pancreatic trypsin inhibitor domain-containing protein" was identified as the most promising vaccine candidate. The candidate protein was used to design a multi-epitope vaccine, incorporating B-cell and T-cell epitopes identified through various tools. The vaccine construct underwent extensive analysis of its antigenic, physical, and chemical features, including the determination of secondary and tertiary structures. Docking and molecular dynamics simulations were performed with HLA alleles, Toll-like receptor 4 (TLR4), and TLR3 to assess its potential to elicit the human immune response. Immune simulation analysis confirmed the predicted vaccine’s strong binding affinity with immunoglobulins, indicating its potential efficacy in generating an immune response. However, experimental validation and testing of this multi-epitope vaccine construct would be needed to assess its potential against W. bancrofti and even for a broader range of lymphatic filarial infections given the similarities between W. bancrofti and Brugia.
... The C. elegans genome is thought to contain 2 fibrillin homologs, fbn-1 and mua-3. These show a maximum identity of 45% (mua-3 isoform a and fbn-1 isoform h) and 32% (mua-3 isoform b) (Bercher et al. 2001;Frand et al. 2005) with human fibrillin-1. These genes are believed to affect C. elegans tissue equivalent to connective tissue in higher eukaryotes (Bercher et al. 2001;Frand et al. 2005). ...
... These show a maximum identity of 45% (mua-3 isoform a and fbn-1 isoform h) and 32% (mua-3 isoform b) (Bercher et al. 2001;Frand et al. 2005) with human fibrillin-1. These genes are believed to affect C. elegans tissue equivalent to connective tissue in higher eukaryotes (Bercher et al. 2001;Frand et al. 2005). A proposed C. elegans model for MFS has been developed (Fotopoulos et al. 2015) in which a temperature sensitive deletion allele of mua-3 was shown to interact with a TGFB2 homolog as well as a collagen. ...
The fibrillinopathies represent a group of diseases in which the 10–12 nm extracellular microfibrils are disrupted by genetic variants in one of the genes encoding fibrillin molecules, large glycoproteins of the extracellular matrix. The best-known fibrillinopathy is Marfan syndrome, an autosomal dominant condition affecting the cardiovascular, ocular, skeletal, and other systems, with a prevalence of around 1 in 3,000 across all ethnic groups. It is caused by variants of the FBN1 gene, encoding fibrillin-1, which interacts with elastin to provide strength and elasticity to connective tissues. A number of mouse models have been created in an attempt to replicate the human phenotype, although all have limitations. There are also natural bovine models and engineered models in pig and rabbit. Variants in FBN2 encoding fibrillin-2 cause congenital contractural arachnodactyly and mouse models for this condition have also been produced. In most animals, including birds, reptiles, and amphibians, there is a third fibrillin, fibrillin-3 (FBN3 gene) for which the creation of models has been difficult as the gene is degenerate and nonfunctional in mice and rats. Other eukaryotes such as the nematode C. elegans and zebrafish D. rerio have a gene with some homology to fibrillins and models have been used to discover more about the function of this family of proteins. This review looks at the phenotype, inheritance, and relevance of the various animal models for the different fibrillinopathies.
... We investigated the site of crystal formation using a third approach. Previous work showed that RNAi targeting mlt-9 results in animals that remain attached to their shed anterior exocuticle and pharyngeal cuticle [16,27]. We investigated whether attached shed pharyngeal cuticle would accumulate fluorescent crystals when incubated in wact-469 for 3 hours and found that it did (Fig 2S and 2T). ...
... We previously described our mlt-9(RNAi)-related methodology [16], which is modified from the work of Frand et al [27]. Briefly, a bacterial culture expressing dsRNA of mlt-9 (referred to here as mlt-9(RNAi)) [75] was started from a single colony in 30 mL LB broth containing 100 μg/mL ampicillin for 18 hrs at 37˚C at 200 rpm. ...
Author summary
An infinite number of small molecules have the potential to threaten life. Not surprisingly then, animals have evolved multiple mechanisms to defend against such threats. One defense mechanism employed by many animals is the creation of an outer protective layer called the cuticle. Lipids within the cuticle act as a barrier to retard small molecule passage into the underlying tissues. Water-loving small molecules cannot traverse a lipid barrier and fat-loving molecules can get trapped within the barrier. How this lipid barrier is established is incompletely understood. Here, we describe our discovery of a conserved protein called PGP-14 that is expressed in the tissue that makes the cuticle that protects the mouth and pharynx of the nematode worm C. elegans. We show that PGP-14 peaks in expression at the end of new cuticle synthesis and is necessary for lipid deposition within it. Without PGP-14, many small molecules adversely accumulate within the animal and consequently kill it. Hence, PGP-14 is a key component employed by the animal to help protect it from small molecule threats.
... These dynamic matrix changes are controlled in part by oscillatory gene expression programs, with pre-cuticle genes peaking relatively early in each molt cycle and different cuticle collagen genes peaking at early, intermediate, or late timepoints, consistent with an extended period of cuticle synthesis and assembly [35][36][37]. The molt cycle also is controlled by various post-transcriptional mechanisms such as regulated trafficking and proteolysis [38][39][40][41][42][43]. A BMP1-related astacin proteinase, DPY-31, has been implicated in C-terminal processing of SQT-3 cuticle collagen [44][45][46], while one or more furin/PCSKs are thought to be responsible for N-terminal processing of many cuticle collagens at the CFCS. ...
Author summary
Extracellular matrices coat cell surfaces to affect many aspects of animal biology. Collagens are among the most common constituents of such matrices and often form fibrillar- or mesh-like structures. It is important that these structures form only at the right time and place in the extracellular environment, and not prematurely during intracellular trafficking. Type I collagen matrix assembly typically occurs after proteolytic cleavage to remove both the N- and C-terminal ends of collagen molecules, and defects in this cleavage can cause a variety of human matrix disorders. Nevertheless, many questions remain about how N-terminal cleavage impacts collagen matrix assembly, particularly within other families of collagens that may be regulated differently than Type I collagen. The nematode Caenorhabditis elegans has an exoskeleton or cuticle that is composed of many collagens, and sequential secretion and membrane-proximal assembly of different collagens has been supposed to explain the eventual layered structure of the cuticle. Here we visualized tagged collagens during cuticle assembly and investigated the roles of N-terminal cleavage by characterizing mutants in the predicted collagen protease BLI-4 (a member of the furin/PCSK family) and also mutants in the predicted N-terminal cleavage sites within the tagged cuticle collagens. We observed that collagen secretion normally precedes cuticle matrix assembly by several hours, requiring a new way of thinking about cuticle layer formation. Furthermore, in both bli-4 mutants and N-terminal cleavage site mutants, collagens were not secreted efficiently and instead formed intracellular puncta. These data reveal an unexpected role for N-terminal cleavage in cuticle collagen secretion, which could be relevant to the cell biology of human matrix disorders.
... Besides having a role in force transmission and cytoskeleton adaptation, ECM proteins in the vicinity of ceHDs also influence ECM remodeling. For instance, the zona pellucida domaincontaining ECM proteins NOAH-1 and NOAH-2 are important for maintaining mechanoreceptor potentials and cuticular ECM remodeling (Frand et al., 2005;Vuong-Brender et al., 2017). During C. elegans development, cuticular ECM remodeling occurs during molting. ...
... During C. elegans development, cuticular ECM remodeling occurs during molting. Intriguingly, loss of unc-52, pat-3 or unc-95 ( paxillin in mammals) results in ECM-associated molting defects (Frand et al., 2005;Zaidel-Bar et al., 2010). By contrast, mutations in the muscle myosin unc-54, which is important for muscle contraction, or mutations in unc-13, important for neurotransmitter release, did not induce cuticular collagen expression (Broday et al., 2007). ...
Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.
... Furthermore, seam cells displayed fusion defects, indicating acn-1 plays a role in 81 establishing cell fates. Consistent with this analysis of molting, Ruvkun and colleagues identified acn-1 in 82 a genome wide RNAi screen for molting defects (Frand et al., 2005). Analysis of ACN-1 transcriptional 83 and translation reporters revealed that ACN-1 is expressed in hypodermal seam and excretory gland 84 cells in embryos and larvae, as well as in the developing vulva and male tail (Brooks et al., 2003;Frand et 85 al., 2005). ...
... acn-1 mRNA levels were 304 measured by RNA-seq in a synchronized population of spe-9(lf) animals raised at 25°C to prevent 305 progeny production. acn-1 mRNA levels were high in larvae, consistent with previous reports (Fig. 6B) 306 (Brooks et al., 2003;Frand et al., 2005;Oskouian et al., 2005;Metheetrairut et al., 2017). Interestingly, 307 acn-1 mRNA levels declined in young adults but then remained relatively constant throughout 308 adulthood (Fig. 6B). ...
The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in C. elegans , Drosophila , and rodents, but its mechanism is not well defined. Here we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril hypersensitive mutants. We identified a missense mutation that causes a partial loss-of-function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNAi promoted dauer larvae formation, suggesting acn-1 is a daf gene. Captopril-mediated lifespan extension xwas abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 control aging by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.
Summary Statement
Captopril and acn-1 control aging. By demonstrating they regulate dauer formation and interact with daf genes, including a new DAF-2(A261V) mutant corresponding to a human disease variant, we clarified the mechanism.
