MLT-10 defines a family of DUF644 and proline-rich repeat proteins involved in the molting cycle of Caenorhabditis elegans.
ABSTRACT: The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X(2)-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10.
Project description:The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.
Project description: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-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development.
Project description:Molting is an essential process in the nematode Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL-MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL-MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL-MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL-MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL-MLT and CDC-42-SID-3 activities in the epidermis.
Project description:Melatonin (MLT) plays an important role in regulating the physiological cycle of seasonal breeding animals. Melatonin receptor I (MT1) is effectively expressed in the cambium layer of deer antler. However, the function and metabolic mechanism of MLT/MT1 signaling in the mesenchymal cells of sika deer remain to be further elucidated. In this work, we detected the effects of MLT/MT1 signaling on mesenchymal cells proliferation and the interaction between MLT/MT1 and IGF1/IGF1-R signaling. The results show that (1) deer antler mesenchymal cells actually express MT1; (2) exogenous melatonin significantly promotes mesenchymal cells proliferation, while MT1 knock-down significantly impairs the positive effects of melatonin; and (3) melatonin significantly enhanced IGF1/IGF1-R signaling, as both the expression of IGF1 and IGF-1R increased, while MT1 knock-down significantly decreased IGF1-R expression and IGF1 synthesis. In summary, these data verified that MLT/MT1 signaling plays a crucial role in antler mesenchymal proliferation, which may be mediated by IGF1/IGF1-R.
Project description:Extracellular leucine-rich repeat (eLRR) proteins are a highly diverse superfamily of membrane-associated or secreted proteins. In the membrane-associated eLRR proteins, the leucine-rich repeat motifs interact with the extracellular matrix and other ligands. Characterizing their functions in animal model systems is key to deciphering their activities in various developmental processes.In this study, we identify pan-1 as a critical regulator of C. elegans larval development. pan-1 encodes both transmembrane and cytoplasmic isoforms that vary in the presence and number of leucine-rich repeats. RNAi experiments reveal that pan-1 is required for developmental processes that occur during the mid to late larval stages. Specifically, pan-1 loss of function causes a late larval arrest with a failure to complete development of the gonad, vulva, and hypodermis. pan-1 is also required for early larval ecdysis and execution of the molting cycle at the adult molt. We also provide evidence that pan-1 functionally interacts with the heterochronic gene lin-29 during the molting process.We show that PAN-1 is a critical regulator of larval development. Our data suggests that PAN-1 promotes developmental progression of multiple tissues during the transition from a larva to a reproductive adult. We further demonstrate that the activity of PAN-1 is complex with diverse roles in the regulation of animal development.
Project description:Spermatogenesis in all animal species occurs within a syncytium. Only at the very end of spermatogenesis are individual sperm cells resolved from this syncytium in a process known as individualization. Individualization in Drosophila begins as a membrane-cytoskeletal complex known as the individualization complex (IC) assembles around the sperm heads and proceeds down the flagella, removing cytoplasm from between the sperm tails and shrink-wrapping each spermatid into its own plasma membrane as it travels. The mulet (mlt) mutation results in severely disrupted ICs, indicating that the mlt gene product is required for individualization. Inverse PCR followed by cycle sequencing maps all known P-insertion alleles of mlt to two overlapping genes, CG12214 (the Drosophila tubulin-binding cofactor E-like homolog) and KCNQ (a large voltage-gated potassium channel). However, since the alleles of mlt map to the 5'-UTR of CG12214 and since CG12214 is contained within an intron of KCNQ, it was hypothesized that mlt and CG12214 are allelic. Indeed, CG12214 mutant testes exhibited severely disrupted ICs and were indistinguishable from mlt mutant testes, thus further suggesting allelism. To test this hypothesis, alleles of mlt were crossed to CG12214 in order to generate trans-heterozygous males. Testes from all trans-heterozygous combinations revealed severely disrupted ICs and were also indistinguishable from mlt mutant testes, indicating that mlt and CG12214 fail to complement one another and are thus allelic. In addition, complementation testing against null alleles of KCNQ verified that the observed individualization defect is not caused by a disruption of KCNQ. Finally, since a population of spermatid-associated microtubules known to disappear prior to movement of the IC abnormally persists during individualization in CG12214 mutant testes, this work implicates TBCE-like in the removal of these microtubules prior to IC movement. Taken together, these results identify mlt as CG12214 and suggest that the removal of microtubules by TBCE-like is a necessary pre-requisite for proper coordinated movement of the IC.
Project description:Precise staging of Caenorhabditis elegans is essential for developmental studies in different environmental conditions. In favorable conditions, larvae develop continuously through four larval stages separated by molting periods. Distinguishing molting from intermolt larvae has been achieved using transgenes with molting reporters, therefore requiring strain constructions, or careful observation of individuals for pharyngeal pumping or behavioral quiescence. In unfavorable conditions, larvae can enter the stress-resistant and developmentally arrested dauer larva stage. Identifying dauer larvae has been based on their ability to withstand detergent selection, precluding identification of recovering animals or of mutants with defects in dauer morphogenesis. Here, we describe a simple method to distinguish molting larvae or dauer larvae from intermolt larvae that bypasses the limitations of current methods. Fluorescent latex beads are mixed with the bacterial food source and ingested by intermolt larvae and adults. Molting and dauer larvae do not feed, and therefore lack beads in their digestive tract. The presence of beads can be determined using a dissecting microscope at magnifications as low as 100 ×, or by using a wormsorter for high-throughput experiments. We find that continuously developing bead-lacking larvae display hallmarks of molting, including expression of the mlt-10::gfp molting marker and a lack of pharyngeal pumping. Furthermore, wild-type and mutant dauer larvae produced by any of three common methods are accurately identified by a lack of beads. Importantly, this method is effective in SDS-sensitive mutant backgrounds and can identify recovering dauer larvae, a stage for which there is no other method of positive selection.
Project description:Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.
Project description:During their development, Caenorhabditis elegans larvae go through four developmental stages. At the end of each larval stage, nematodes molt. They synthesize a new cuticle and shed the old cuticle. During the molt, larvae display a sleep-like behavior that is called lethargus. We wanted to determine how gene expression changes during the C. elegans molting cycle. We performed transcriptional profiling of C. elegans by selecting larvae displaying either sleep-like behavior during the molt or wake behavior during the intermolt to identify genes that oscillate with the molting-cycle. We found that expression changed during the molt and we identified 520 genes that oscillated with the molting cycle. 138 of these genes were not previously reported to oscillate. The majority of genes that had oscillating expression levels appear to be involved in molting, indicating that the majority of transcriptional changes serve to resynthesize the cuticle. Identification of genes that control sleep-like behavior during lethargus is difficult but may be possible by looking at genes that are expressed in neurons. 22 of the oscillating genes were expressed in neurons. One of these genes, the dopamine transporter gene dat-1, was previously shown in mammals and in C. elegans to control sleep. Taken together, we provide a dataset of genes that oscillate with the molting and sleep-wake cycle, which will be useful to investigate molting and possibly also sleep-like behavior during lethargus.
Project description:Molting is of great importance for the survival and development of nematodes. Nematode astacins (NAS), a large family of zinc metalloproteases, have been proposed as novel anthelmintic targets due to their multiple roles in biological processes of parasitic nematodes. In this study, we report a well conserved <i>nas-33</i> gene in nematodes of clade V and elucidate how this gene is involved in the molting process of the free-living nematode <i>Caenorhabditis elegans</i> and the parasitic nematode <i>Haemonchus contortus</i>. A predominant transcription of <i>nas-33</i> is detected in the larval stages of these worms, particularly in the molting process. Knockdown of this gene results in marked molecular changes of genes involved in cuticle synthesis and ecdysis, compromised shedding of the old cuticle, and reduced worm viability in <i>H. contortus</i>. The crucial role of <i>nas-33</i> in molting is closely associated with a G protein beta subunit (GPB-1). Suppression of both <i>nas-33</i> and <i>gpb-1</i> blocks shedding of the old cuticle, compromises the connection between the cuticle and hypodermis, and leads to an increased number of sick and dead worms, indicating essentiality of this module in nematode development and survival. These findings reveal the functional role of <i>nas-33</i> in nematode molting process and identify astacins as novel anthelmintic targets for parasitic nematodes of socioeconomic significance.