L4 Larval Stage Research Articles

Microchip device for measurement of body volume of C. elegans as bioindicator application

We propose a micro fluidic device to analyze Caenorhabditis elegans (C. elegans) as a bioindicator. In this device, we assessed the body volume of C. elegans using a microchip with a capacitance sensor. This device is composed of two key parts. One is a tapered micro channel. The tapered micro channel is used to immobilize C. elegans. The other is a pair of electrodes. The electrodes are used to measure the capacitance change. These two components form a sensing space; a capacitance change occurs when C. elegans is placed in the sensing space. In this study, we performed two kinds of experiments. First, to check the correlation between the body volume and the capacitance change, a total of 15 C. elegans were measured; from L2 larval stage to adult stage. Second, to confirm the possibility of the proposed microchip as a device that can analyze the condition of C. elegans as a bioindicator, a total of 35 C. elegans were measured. To measure the body volume as a reference, first, the body length and the diameter of C. elegans were measured by an optical microscope and then the volume was calculated from those two measured values. The capacitance change was measured by the proposed microchip. By comparison between the result of two methods, we confirmed the correlation between the calculated values. The possibility of the proposed microchip as the device to analyze the effect of cadmium on C. elegans as a bioindicator was established.

Apoptosis-mediated in vivo toxicity of hydroxylated fullerene nanoparticles in soil nematode Caenorhabditis elegans

Although a number of manufactured nanoparticles are applied for the medical and clinical purposes, the understanding of interaction between nanomaterials and biological systems are still insufficient. Using nematode Caenorhabditis elegans model organism, we here investigated the in vivo toxicity or safety of hydroxylated fullerene nanoparticles known to detoxify anti-cancer drug-induced oxidative damages in mammals. The survival ratio of C. elegans rapidly decreased by the uptake of nanoparticles from their L4 larval stage with resulting in shortened lifespan (20d). Both reproduction rate and body size of C. elegans were also reduced after exposure to 100μgmL−1 of fullerol. We found ectopic cell corpses caused by apoptotic cell death in the adult worms grown with fullerol nanoparticles. By the mutation of core pro-apoptotic regulator genes, ced-3 and ced-4, these nanoparticle-induced cell death were significantly suppressed, and the viability of animals consequently increased despite of nanoparticle uptake. The apoptosis-mediated toxicity of nanoparticles particularly led to the disorder of digestion system in the animals containing a large number of undigested foods in their intestine. These results demonstrated that the water-soluble fullerol nanoparticles widely used in medicinal applications have a potential for inducing apoptotic cell death in multicellular organisms despite of their antioxidative detoxifying property.

Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans

We describe a novel screen to isolate pharyngeal cell morphology mutants in Caenorhabditis elegans using myo-2::GFP to rapidly identify abnormally shaped pharynxes in EMS (Ethyl Methanesulfonate) mutagenized worms. We observed over 83 C. elegans lines with distinctive pharyngeal phenotypes in worms surviving to the L1 larval stage, with phenotypes ranging from short pharynx, unattached pharynx, missing cells, asymmetric morphology, and non-adherent pharynx cells. Thirteen of these mutations have been chromosomally mapped using Single Nucleotide Polymorphisms (SNPs) and deficiency strain complementation. Our studies have focused on genetically mapping and functionally testing two phenotypes, the short pharynx and the loss of muscle cohesion phenotypes. We have also identified new alleles of sma-1, and our screen suggests many genes directing pharynx assembly and structure may be either pharynx specific or less critical in other tissues.

STR-33, a Novel G Protein-coupled Receptor That Regulates Locomotion and Egg Laying in Caenorhabditis elegans

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

Use of mixed cultures of biocontrol agents to control sheep nematodes

Biological control is a promising non-chemical approach for the control of gastrointestinal nematodes of sheep. Use of combinations of biocontrol agents have been reported to be an effective method to increase the efficacy of biological control effects. In this study, combinations of either two Bacillus thuringiensis (Bt) or Clonostachys rosea (C. rosea) isolates and Bt+C. rosea isolates were evaluated in vitro in microtitre plates for their biocontrol activity on sheep nematodes. The Baermann technique was used to extract the surviving L3 larval stages of intestinal nematodes and counted under a dissecting microscope to determine the larval counts. Results indicate that there was a significant reduction of nematode counts due to combination of biocontrol agents (P<0.001). Combinations of Bt isolates reduced nematodes counts by 72.8%, 64% and 29.8%. The results revealed a control level of 57% when C. rosea isolates P3+P8 were combined. Combination of Bt and C. rosea isolates B10+P8 caused the greatest mortality of 76.7%. Most combinations were antagonistic, with only a few combinations showing an additive effect. None were synergistic. The isolate combinations were more effective than when isolates were used alone.

Efficacy of Emodepside plus Toltrazuril (Procox® Oral Suspension for Dogs) against Toxocara canis, Uncinaria stenocephala and Ancylostoma caninum in Dogs

The efficacy of emodepside plus toltrazuril (Procox® oral suspension for dogs) against different species of gastrointestinal nematodes (Toxocara canis, Ancylostoma caninum, Uncinaria stenocephala) was evaluated in nine randomised,blinded and placebo-controlled laboratory studies in naturally or experimentally infected dogs. The product was used at the proposed minimum dose of 0.45 mg emodepside and 9 mg toltrazuril per kg body weight. Efficacy was calculated based on worm counts after necropsy. Worm burdens in the control dogs ranged between 0 and 409 worms of the respective stage for T. canis and between 4 and 655 worms for hookworms. The studies demonstrated 100 % efficacy of emodepside/toltrazuril suspension against mature adult, ≥ 94.7 %efficacy against immature adult and 99.3 % efficacy against the L4 larval stage of T. canis. The efficacy against mature adult A. caninum was ≥ 99.5 % and the efficacy against mature adult U. stenocephala was 100 %. All differences between treatment and control groups were statistically significant and no gender effect was found. It can be concluded that the emodepside/toltrazuril suspension represents a safe and highly effective product in dogs with nematode (T. canis, hookworms) infection.

The POU transcription factor UNC‐86 controls the timing and ventral guidance of Caenorhabditis elegans axon growth

The in vivo mechanisms that coordinate the timing of axon growth and guidance are not well understood. In the Caenorhabditis elegans hermaphrodite specific neurons (HSNs), the lin-4 microRNA controls the stage of axon initiation independent of the UNC-40 and SAX-3 ventral guidance receptors. lin-4 loss-of-function mutants exhibit marked delays in axon outgrowth, while lin-4 overexpression leads to precocious growth in the L3 larval stage. Here, we show that loss of the POU transcription factor UNC-86 not only results in penetrant ventral axon growth defects in in the HSNs, but also causes processes to extend in the L1, three stages earlier than wild-type. This temporal shift is not dependent on UNC-40 or SAX-3, and does not require the presence of lin-4. We propose that unc-86(lf) HSN axons are misguided due to the temporal decoupling of axon initiation and ventral guidance responses.

Association of oxidative stress with the formation of reproductive toxicity from mercury exposure on hermaphrodite nematode Caenorhabditis elegans

Here we selected HgCl 2 to investigate the mechanism of Hg toxicity on reproduction in hermaphrodite nematodes. Accompanied with decrease of brood size, Hg exposure caused severe deficits in egg number in uterus, egg laying and reproductive structures, including gonad arms and vulva, and formation of protruding phenotype for vulva. Meanwhile, Hg exposure induced severe stress response and oxidative damage in gonad and vulva. Pre-treatment with vitamin E, a potent antioxidant, at the L2-larval stage prevented the oxidative damage and formation of reproductive deficits in Hg exposed nematodes; however, pre-treatment with paraquat, a regent generating superoxide anions, induced more severe reproductive deficits in Hg exposed nematodes. Moreover, Hg exposure increased expression of clk-2 and isp-1 genes, whose mutations decrease ROS production, and decreased expression of mev-1 and gas-1 genes, whose mutations increase ROS production. Thus, oxidative stress may be essential for the induction of reproductive deficits in Hg exposed hermaphrodite nematodes.

Ceramide glucosyltransferase of the nematode Caenorhabditis elegans is involved in oocyte formation and in early embryonic cell division

Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle.

Metallothioneins Are Required for Formation of Cross-Adaptation Response to Neurobehavioral Toxicity from Lead and Mercury Exposure in Nematodes

Metallothioneins (MTs) are small, cysteine-rich polypeptides, but the role of MTs in inducing the formation of adaptive response is still largely unknown. We investigated the roles of metallothionein genes (mtl-1 and mtl-2) in the formation of cross-adaptation response to neurobehavioral toxicity from metal exposure in Caenorhabditis elegans. Pre-treatment with mild heat-shock at L2-larva stage effectively prevented the formation of the neurobehavioral defects and the activation of severe stress response in metal exposed nematodes at concentrations of 50 and 100 µM, but pre-treatment with mild heat-shock did not prevent the formation of neurobehavioral defects in 200 µM of metal exposed nematodes. During the formation of cross-adaptation response, the induction of mtl-1 and mtl-2 promoter activity and subsequent GFP gene expression were sharply increased in 50 µM or 100 µM of metal exposed Pmtl-1::GFP and Pmtl-2::GFP transgenic adult animals after mild heat-shock treatment compared with those treated with mild heat-shock or metal exposure alone. Moreover, after pre-treatment with mild heat-shock, no noticeable increase of locomotion behaviors could be observed in metal exposed mtl-1 or mtl-2 mutant nematodes compared to those without mild heat-shock pre-treatment. The defects of adaptive response to neurobehavioral toxicity induced by metal exposure formed in mtl-1 and mtl-2 mutants could be completely rescued by the expression of mtl-1 and mtl-2 with the aid of their native promoters. Furthermore, over-expression of MTL-1 and MTL-2 at the L2-larval stage significantly suppressed the toxicity on locomotion behaviors from metal exposure at all examined concentrations. Therefore, the normal formation of cross-adaptation response to neurobehavioral toxicity induced by metal exposure may need the enough accumulation of MTs protein in animal tissues.

Aluminum nanoparticle exposure in L1 larvae results in more severe lethality toxicity than in L4 larvae or young adults by strengthening the formation of stress response and intestinal lipofuscin accumulation in nematodes

Toxicity of Al 2O 3-NPs, as compared to that of Al 2O 3, to L1-larval, L4-larval or young adult nematodes was evaluated. When exposure was performed at L1-larval stage, the significant increases of lethality, stress response, and intestinal lipofuscin autofluorescence were observed in 6.3–203.9 mg/L of Al 2O 3-NPs exposed nematodes. In contrast, when exposure was performed at L4-larval or young adult stage, the significant increases of lethality and intestinal lipofuscin autofluorescence were observed in 12.7–203.9 mg/L of Al 2O 3-NPs exposed nematodes, and the significant inductions of stress response were detected in 25.5–203.9 mg/L of Al 2O 3-NPs exposed nematodes. Moreover, the lethality was significantly correlated with the stress response and the intestinal lipofuscin autofluorescence in Al 2O 3-NPs exposed nematodes. These data imply that Al 2O 3-NPs exposure in L1 larvae causes more severe lethality toxicity than in L4 larvae or young adults by strengthening the formation of stress response and intestinal lipofuscin accumulation in nematodes.

A Developmental Timing Switch Promotes Axon Outgrowth Independent of Known Guidance Receptors

To form functional neuronal connections, axon outgrowth and guidance must be tightly regulated across space as well as time. While a number of genes and pathways have been shown to control spatial features of axon development, very little is known about the in vivo mechanisms that direct the timing of axon initiation and elongation. The Caenorhabditis elegans hermaphrodite specific motor neurons (HSNs) extend a single axon ventrally and then anteriorly during the L4 larval stage. Here we show the lin-4 microRNA promotes HSN axon initiation after cell cycle withdrawal. Axons fail to form in lin-4 mutants, while they grow prematurely in lin-4–overexpressing animals. lin-4 is required to down-regulate two inhibitors of HSN differentiation—the transcriptional regulator LIN-14 and the “stemness” factor LIN-28—and it likely does so through a cell-autonomous mechanism. This developmental switch depends neither on the UNC-40/DCC and SAX-3/Robo receptors nor on the direction of axon growth, demonstrating that it acts independently of ventral guidance signals to control the timing of HSN axon elongation.

Role of matrix metalloproteinase ZMP-2 in pathogen resistance and development in Caenorhabditis elegans

The genome of Caenorhabditis elegans includes six homologs of matrix metalloproteinases (MMPs). The C. elegans MMP gene zmp-1 has recently been shown to be involved in anchor cell invasion during post-embryonic vulval development. Here, we identified H19M22.3 ( zmp-2) as a pleiotropic MMP gene regulating disease resistance, molting, larval development, and fecundity. Zmp-2(RNAi) nematodes showed significant lifespan reduction during infection with pathogenic Photorhabdus luminescence. Moreover, we observed molting defects indicating a direct or regulative role in extracellular matrix degradation during ecdysis, delayed larval to adult development, and reduced offspring production in hermaphrodite adults. GFP-expressing nematodes revealed predominant expression of zmp-2 in multiple cells during embryogenesis; in hypodermal, muscle, and somatic gonad cells during larval development; and in developing and mature spermathecae in the L4 larval stage and adults. These results give evidence for pleiotropic roles of zmp-2 and provide novel insights into evolutionarily conserved and derived MMP functions in C. elegans.

Mutant FAR protein construction and their potential as vaccine candidates

Fatty acid and retinol binding proteins are a family of proteins found in free living and parasitic nematodes. It is our hypothesis that parasitic nematodes secrete FAR into host tissues, resulting in localized immunosuppression, and hence chronic parsitic infection. Heligmosomoides polygyrus is a mouse model for hookworm that is commonly used in the laboratory for the investigation of the cellular responses to chronic helminth infections. Our laboratory is investigating a protein retrieved from the L4 larval stage of this parasite, a protein believed to be involved in assisting the parasite to form and maintain chronic infections. We have termed this protein Hp‐FAR‐1, for H. polygyrus Fatty Acid and Retinol Binding Protein 1. Creation of mutant FAR proteins that do not bind retinol are of interest for the development of potential vaccine candidates. The purpose of this study was to compare Ce‐FAR‐4, a non‐functional FAR protein from C. elegans, with parasitic FAR proteins. The primary amino acid structures of both parasitic and Ce‐FAR‐4 were analyzed for regions of conservation. Specifically, we target regions that were conserved among the parasitic proteins, including Hp‐FAR‐1, but not in Ce‐FAR‐4; hypothesizing that mutating the conserved regions of Hp‐FAR‐1to the amino acids found in Ce‐FAR‐4 would inhibit retinol binding.

Does onchocerciasis transmission take place in hypoendemic areas? a study from the North Region of Cameroon

Community-directed treatment with ivermectin (CDTI) for onchocerciasis control is targeted to meso and hyperendemic areas in Africa. Below the threshold, communities are considered hypoendemic and, mass treatment is not recommended. As policy begins to shift from control to elimination, the role of hypoendemic areas in maintaining Onchocerca volvulus needs to be re-examined. The study determined whether independent transmission occurs in a hypoendemic area in the North region of Cameroon. Ten 'high risk' communities along the River Mayo Douka system in Ngong Health District, at least 20 km from the nearest CDTI program were studied. Six hundred and forty-nine adults (over 20 years of age) and 561 children (under 10 years) were examined for nodules and microfilaria. A subsample of 334 adults was examined for onchocercal ocular morbidity. Simulium flies from 4 collection points were captured over 3 months annually for 2 years and dissected for larval stages of O. volvulus. Nodule and microfilariae (mf) prevalence among adults was 12.20% and 2.91%, and 9.2% and 0.48% among children, respectively. Blindness because of onchocerciasis was insignificant, although low rates of chronic onchocercal ocular disease (<2%) were observed. Four (0.16 percent) of 255 flies collected in 2008 were infected with L3 larval stage, and 1 black fly of 39 collected in 2009 had two L2 larval stage morphologically consistent with O. volvulus. Ngong is a 'hypoendemic' focus with likely low grade indigenous transmission in isolation from meso/hyperendemic areas. Consequently, transmission from hypoendemic areas could contribute to rapid disease recrudescence in the post-treatment phase of adjacent former meso and hyperendemic areas.

An ortholog of the ecdysone receptor protein (EcR) from the parasitic nematode Haemonchus contortus

High concentrations (≥4.2 mM) of 20E inhibit the development of Haemonchus contortus eggs to the L3 larval stage. We report the cloning of cDNA encoding an EcR ortholog (HcEcR) from H. contortus mRNA expressed during L3. Phylogenetically, this and the putative EcR from Brugia malayi form a separate branch between arthropod EcRs and liver X receptors. Two isoforms of HcEcR differ in the inclusion/omission of a 3-residue segment in the A/B domain. Single nucleotide polymorphisms at 49 positions can be grouped into two major patterns in the A/BC segment and two in the DE/F segment. Some 35% of the highly conserved ecdysteroid-contacting residues in insect EcRs are also conserved in the HcEcR ligand binding domain, but it contains unusual residue choices at other ligand-contacting positions. Recombinant co-expression of HcEcR DE/F segments with a phthirapteran USP DE/F segment in insect cells resulted in stable proteins which did not heterodimerize or bind [ 3H]ponasterone A.

Fatty acid composition and gene expression profiles are altered in aryl hydrocarbon receptor-1 mutant Caenorhabditis elegans

The aryl hydrocarbon receptor (AHR) is a eukaryotic transcription factor that plays an essential role in neuronal, immune, vascular, hepatic and hematopoietic development. In mammals, AHR induces metabolism-associated genes in response to xenobiotics. AHR is evolutionarily conserved, and the C. elegans AHR ortholog likely shares many physiologic functions with the mammalian version. While the role of AHR in development is known, the molecular basis of AHR action is less well understood. To understand the physiologic role of AHR in C. elegans, a combination of fatty acid profiling, transcriptomics, and phenotyping approaches was used. Fatty acid profiles from L4 larval stage whole animals indicated that C17isoA, C18:1n9t, C20:3n6 and C20:4n6 were significantly increased in an ahr-1 mutant compared to wild-type. Consistent with these changes, we observed a significant 5.8 fold increase in fat-7, and 1.7–1.9 fold increases in elo-5, nhr-49, and mdt-15 gene expression during the L4 stage. The ahr-1(ju145) mutant displayed deficits in growth and development including a reduced number of eggs laid, a higher proportion of dead embryos, delay in time to reach L4 stage, and movement deficits including a fewer number of body bends and a longer defecation cycle. To understand global effects of AHR-1 on transcription, microarray analysis was performed on L1 stage animals. Expression changes (324 under- and 238 over-expressed) were found in genes associated with metabolism, growth, and development. These results indicate a role for C. elegans AHR in regulating fatty acid composition and in contributing to some aspects of development. Since the transcriptional control of AHR targets may be evolutionarily conserved, these results provide a deeper understanding of the molecular actions of AHR in a model invertebrate system that may be informative for higher organisms.

Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies.

Developmental studies in multicellular model organisms such as Caernohabditis elegans rely extensively on the ability to cultivate and image animals repeatedly at the cell or subcellular level. However, standard high-resolution imaging techniques require the use of anaesthetics for immobilization, and may have undesirable side effects on development. Thus such techniques are not ideal in allowing the same animals to grow and be imaged throughout development to observe specific developmental processes. In this paper, we present a microfluidic system designed to overcome these difficulties. The system allows for long-term culture of C. elegans starting at L1 larval stage and repeated high-resolution imaging at physiological temperatures without using anaesthetics. We use a commercially available biocompatible polymer, Pluronic F127 for immobilization; this polymer is capable of a reversible thermo-sensitive sol-gel transition within approximately 2 degrees C, which is well-controlled in the microfluidic chip. The gel phase is sufficient to immobilize the animals. While animals are not imaged, they are cultured in individual chambers in media containing nutrients required for development. We show here that this method facilitates time-lapse studies of single animals at high-resolution and lends itself to live imaging experiments on developmental processes and dynamic events.

Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans

Kinesin-3 motor UNC-104/KIF1A is essential for transporting synaptic precursors to synapses. Although the mechanism of cargo binding is well understood, little is known how motor activity is regulated. We mapped functional interaction domains between SYD-2 and UNC-104 by using yeast 2-hybrid and pull-down assays and by using FRET/fluorescence lifetime imaging microscopy to image the binding of SYD-2 to UNC-104 in living Caenorhabditis elegans. We found that UNC-104 forms SYD-2-dependent axonal clusters (appearing during the transition from L2 to L3 larval stages), which behave in FRAP experiments as dynamic aggregates. High-resolution microscopy reveals that these clusters contain UNC-104 and synaptic precursors (synaptobrevin-1). Analysis of motor motility indicates bi-directional movement of UNC-104, whereas in syd-2 mutants, loss of SYD-2 binding reduces net anterograde movement and velocity (similar after deleting UNC-104's liprin-binding domain), switching to retrograde transport characteristics when no role of SYD-2 on dynein and conventional kinesin UNC-116 motility was found. These data present a kinesin scaffolding protein that controls both motor clustering along axons and motor motility, resulting in reduced cargo transport efficiency upon loss of interaction.

The C. elegans tailless/Tlx homolog nhr-67 regulates a stage-specific program of linker cell migration in male gonadogenesis

Cell migration is a common event during organogenesis, yet little is known about how migration is temporally coordinated with organ development. We are investigating stage-specific programs of cell migration using the linker cell (LC), a migratory cell crucial for male gonadogenesis of C. elegans. During the L3 and L4 larval stages of wild-type males, the LC undergoes changes in its position along the migratory route, in transcriptional regulation of the unc-5 netrin receptor and zmp-1 zinc matrix metalloprotease, and in cell morphology. We have identified the tailless homolog nhr-67 as a cell-autonomous, stage-specific regulator of timing in LC migration programs. In nhr-67-deficient animals, each of the L3 and L4 stage changes is either severely delayed or never occurs, yet LC development before the early L3 stage or after the mid-L4 stage occurs with normal timing. We propose that there is a basal migration program utilized throughout LC migration that is modified by stage-specific regulators such as nhr-67.