Basham SE et al. (1998) West Coast Worm Meeting "MUTATIONS IN ooc-5 AND ooc-3 DISRUPT P1 NUCLEAR ROTATION IN EMBRYOS"

Rose LS, Basham SE

[West Coast Worm Meeting, 1998]

Division of a parent cell containing a polar distribution of fate determining molecules can produce daughters with distinct cell fates. For this process to work efficiently, polarity and spindle orientation must be coordinated. C. elegans embryos are highly polarized and display two distinct patterns of spindle orientation. Divisions in the AB lineage occur in a typical orthogonal pattern of cleavage while some divisions in the P lineage repeatedly occur on the same axis, due to a 90° rotation of the nuclear-centrosome complex. Hermaphrodites homozygous for mutations in ooc-5 and ooc-3 (for abnormal oocyte formation), produce reduced sized embryos that fail to undergo nuclear rotation in the P1 cell. As an initial step in understanding the ooc-5 phenotype, we have compared the distribution of cytoskeletal elements in wild-type and ooc-5 embryos. In the P1 cell of wild-type embryos, both actin and actin capping protein (CP) accumulate in an anterior cortical "dot" which correlates strongly with nuclear rotation (Waddle et al., 1994). The percentage of ooc-5 embryos with a cortical accumulation of CP in the P1 cell is comparable to what we observe in wild-type embryos. This suggests that failure of rotation is not due to a lack of CP accumulation. The microtubule cytoskeleton appears normal in ooc-5 embryos as observed using standard immunofluorescence microscopy. Specifically, astral microtubules are seen projecting toward the cell periphery and appear long enough to interact with the cortex, indicating that failure of rotation is not due to microtubules being too short. To determine whether ooc-5 mutations are specific for spindle orientation or affect general aspects of emryonic polarity, we examined embryos for PAR-3 and P granule localization. In the majority of one-cell ooc-5 embryos examined, P granules and PAR-3 localization appeared normal. Specifically, P granules were localized to the posterior of the embryo and PAR-3 was restricted to the anterior periphery. However, at the 2-cell stage the majority of ooc-5 embryos examined had PAR-3 mislocalized and contained abnormal looking clumps of P granules that were localized more laterally than posteriorly in P1. In these 2-cell embryos, PAR-3 staining was observed extending very far posteriorly in P1, sometimes being present around the entire periphery of the cell. This observation indicates that mutations in ooc-5 result in the mislocalization of PAR-3 at the 2-cell stage. PAR-3 localization has been shown to inhibit nuclear rotation (Etemad-Moghadan et al., 1995) and our results are thus consistent with the idea that PAR-3 mislocalization in ooc-5 embryos prevents nuclear rotation from occurring in the P1 cell. This view is supported by the observation that rotation can occur in both the AB and the P1 cell in ooc-5;par-3 and ooc-3;par-3 double mutant embryos. We have begun a molecular analysis of both the ooc-5 and ooc-3 genes. Using germ-line transformation, we have identified a single cosmid that rescues the ooc-5 phenotype. We are taking the same approach to clone the ooc-3 gene. These studies will help determine the wild-type function of ooc-5 and ooc-3 and will provide additional information on how spindle orientation is controlled in early embryos.

Basham SE et al. (1997) International C. elegans Meeting "ooc-5 MUTATIONS DISRUPT OOCYTE FORMATION AND DIVISION ORIENTATION IN C. ELEGANS."

Basham SE, Rose LS

[International C. elegans Meeting, 1997]

Orientation of cell division plays a critical role in the development of both plants and animals. In early C. elegans embryos, two basic patterns of division occur. The AB lineage displays a typical orthogonal pattern of cleavage. In the P lineage, divisions repeatedly occur on the same axis due to a 90 degree rotation of the nuclear-centrosome complex. Rotation depends on an interaction between astral microtubules and an anterior site on the cortex of P1. Embryos from animals homozygous for mutations in ooc-5 (for abnormal oocyte formation) fail to undergo nuclear rotation in the P1 cell. As an initial step to understanding the ooc-5 phenotype, we are comparing the distribution of cytoskeletal elements in ooc-5 mutant and wild-type embryos. In the P1 cell of wild-type embryos, both actin and actin capping protein accumulate in an anterior cortical "dot" which correlates strongly with nuclear rotation. We are examining ooc-5 mutant embryos for the cortical accumulation of these proteins. We are also interested in determining whether astral microtubules in ooc-5 mutant embryos are comparable to wild-type embryos. Mutations in ooc-5 also affect oogenesis as the germlines of ooc-5 mutants contain a double row of oocytes that are smaller than normal. Embryos produced by ooc-5 mutants are approximately half the size of a wild-type embryo while their nuclei are approximately 80 percent wild-type size. This observation raises the possibility that failure of nuclear rotation in these embryos is due to the steric effect of having a large nucleus in a small cell. We are currently examining another strain which produces small embryos to see if reduced embryo size generally correlates with a failure of nuclear rotation. These studies will lead to a better understanding of the ooc-5 phenotype and will provide insight into the function of wild-type ooc-5 product.

Basham SE et al. (2000) West Coast Worm Meeting "ooc-5 encodes a putative ATPase required for the reestablishment of asymmetric PAR protein localization in two-cell embryos"

Basham SE, Rose LS

[West Coast Worm Meeting, 2000]

C. elegans embryos display two distinct patterns of spindle orientation. Divisions in the AB lineage occur in an orthogonal pattern while some division in the P lineage repeatedly occur on the same axis, due to a rotation of the nuclear-centrosome complex. Genetic and molecular analyses suggests that PAR-3 protein plays an important role in preventing nuclear rotation in the AB cell, while PAR-2 protein functions to allow nuclear rotation in P1 by restricting PAR-3 localization within this cell. We have screened maternal-effect lethal mutants for alterations in cleavage pattern. Hermaphrodites homozygous for mutations in the ooc-5 or ooc-3 gene produce reduced sized embryos that fail to undergo P1 nuclear rotation. Our phenotypic characterization of ooc-5 and ooc-3 mutants indicates that polarity is perturbed in the P1 cell of 2-cell mutant embryos. In particular, we have found that in most ooc-5 and ooc-3 mutant embryos, PAR-3 localization appears normal at the 1-cell stage but is no longer restricted to the anterior of the P1 cell in 2-cell embryos. PAR-2 is also normal in 1-cell mutant embryos, but is absent from the periphery of the P1 cell in most 2-cell ooc mutant embryos. Finally, P granules are localized normally in 1-cell mutant embryos, but often fail to localize normally to the posterior of the P1 cell in 2-cell mutant embryos. Thus, ooc-5 and ooc-3 may function (directly or indirectly) in polarizing the P1 cell. One model for OOC-5 and OOC-3 is that they function in P1 to maintain PAR-2 at the posterior of P1, thus restricting PAR-3 localization in this cell. We have cloned ooc-5 and it is predicted to encode a 350 amino acid protein containing an amino-terminal signal sequence, a transmembrane spanning region and an ATP binding domain. A BLAST search indicates that ooc-5 is related to two other predicted genes in the C. elegans genome and is a member of a family of predicted ATPases that have been identified in a variety of species. A mutation in one of the human orthologs of this gene results in a neuromuscular disease, but the biological basis for this disease is not well understood. We are currently generating GFP reporter constructs and anti-OOC-5 antibodies to examine OOC-5 expression and localization.

SE Basham et al. (1999) International C. elegans Meeting "Polarized Localization of PAR-3 and PAR-2 in C. elegansEmbryos Requires ooc-5 and ooc-3"

LS Rose, SE Basham

[International C. elegans Meeting, 1999]

Division of a cell containing a polar distribution of fate determinants can produce daughters with distinct cell fates. For this process to work efficiently, polarity and spindle orientation must be coordinated. In C. elegans embryos, divisions in the AB lineage are symmetric and occur in an orthogonal pattern of cleavage. Polarized cells in the P lineage undergo asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear-centrosome complex. The par genes are required for both normal polarity of the 1-cell embryo and division pattern in the 2-cell embryo. Genetic and molecular analyses suggests that PAR-3 protein plays a role in preventing nuclear rotation in the AB cell while PAR-2 functions to allow nuclear rotation in P1 by restricting PAR-3 localization in this cell (reviewed in Rose and Kemphues, Ann. Rev. Genet. 1998. 32). Hermaphrodites homozygous for mutations in ooc-5 or ooc-3 produce reduced sized embryos that fail to undergo nuclear rotation in the P1 cell. We examined the distribution of PAR proteins in ooc mutant embryos. In almost all 1-cell ooc-5 and ooc-3 embryos examined, localization of PAR-1, PAR-2 and PAR-3 was normal. However, in the majority of 2-cell mutant embryos examined, PAR-3 was present around the entire P1 periphery or extended abnormally far posteriorly. PAR-2 localization was reciprocally reduced or absent from the P1 periphery. In addition, nuclear rotation can occur in ooc-5;par-3 and ooc-3;par-3 double mutant embryos, suggesting that failure of P1 nuclear rotation in ooc embryos is due, at least in part, to the mislocalization of PAR-3 in this cell. PAR-1 is asymmetrically localized in wild-type embryos in a pattern similar to PAR-2. PAR-1 localization in the P1 cell of ooc-5 embryos parallels that of PAR-2, being reduced or absent from the periphery. To our knowledge, these are the first mutations described that specifically affect re-establishment of PAR domains in the P1 cell. We have begun a molecular analysis of both the ooc-5 and ooc-3 genes. Using germline transformation, we have identified a single cosmid that rescues the ooc-5 phenotype and a single cosmid that rescues the ooc-3 phenotype. We are currently using RNAi and injection of cosmid fragments to identify the ooc-5 and ooc-3 genes.

Kathleen Morgan et al. (2007) International Worm Meeting "Modeling environmentally-induced sporadic ALS."

Kathleen Morgan, Annette Estevez, Miguel Estevez

[International Worm Meeting, 2007]

Exposure to high levels of selenium (Se) has been associated with increased risk of developing the motor neuron disease Amyotrophic Lateral Sclerosis (ALS) in human epidemiologic studies and motor neuron degeneration resembling ALS in livestock animals exposed to high levels of Se under laboratory conditions. Similarly, in C. elegans supplementation of NGM plates with sodium selenite (NaSe) induces a progressive paralysis involving motor neuron degeneration. Assessment of motor neuron morphology in Se-treated animals expressing a pan-neuronal GFP (Punc-119::gfp, Maduro and Pilgrim 1995), demonstrated classical signs of neuronal injury including: neuronal swelling, loss of the nucleus cytoplasm boundary, loss of chromatin staining, axonal blebbing/beading along the ventral cord, and altered mitochondrial morphology. The morphologic changes observed do not phenocopy the changes described in the mec neurodegeneration pathway since it involves much less swelling and is not genetically suppressible by cad-1(rf), vha-2(rf), or crt-1(rf) (Bianchi, et al. 2004). In addition, the Se-induced neurodegenerative changes do not phenocopy hypoxic changes in that pharyngeal and body wall muscles seem to be spared (Scott, et al., 2003). In order to determine what signaling pathways might be involved in this process we have screened the major stress-activated growth factor pathways in C. elegans for effects on Se-induced neurodegeneration. These studies have found that reduced signaling through the TGF-<font face=symbol>b</font> cascade powerfully sensitizes animals to Se-neurotoxicity. In contrast, decreased signaling through the insulin signaling pathway (Tissenbaum and Ruvkun, 1998), in daf-2(rf) and age-1(rf), resulted in significant resistance to Se-neurotoxicity. These insulin-pathway effects on Se-neurotoxicity appear to involve alterations in neuroprotective gene expression since reduction-of-function in daf-16(m26 and mgDf50), a gene encoding a transcriptional effector of insulin signaling, conferred increased sensitivity to Se exposure. Since the insulin cascade regulates expression of various antioxidant enzymes these results suggested that high environmental Se induces neuronal injury through increasing oxidative stress. Consistent with this hypothesis both glutathione and quercetin significantly reduced the emergence of paralysis under conditions of high environmental Se. Collectively, these results show that Se-neurotoxicity in C. elegans involves features associated with ALS pathology including: motor neuron degeneration, increased oxidative stress, mitochondrial injury, and DNA damage.

Kathleen L Morgan et al. (2005) International Worm Meeting "Developing a model of Amyotrophic Lateral Sclerosis (ALS) using Selenium toxicity in C. elegans"

Kathleen L Morgan, Annette Estevez, Miguel Estevez, Robin H Cowie

[International Worm Meeting, 2005]

Selenium (Se) is an essential nutrient which is utilized by glutathione peroxidase in the scavenging of free radicals. In humans, excess Se is toxic causing nausea, weakness, diarrhea, damage to the nervous system, and rarely death. Environmental exposure to high Se levels in a small Italian city lead to a 40-fold increase of amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disorder that affects motor neurons in the spinal cord. We are using Se toxicity in the worm to study and develop a model of ALS. Previous studies in our lab have shown that Se toxicity in worms causes embryonic lethality, larval arrest, and in adults, reduced fertility, paralysis and death. Using the adult endpoint of paralysis and death, various concentrations of seleno-L-methionine (SeMet) were used to generate a toxicity curve for N2s exposed to SeMet. Adult animals were plated on a SeMet concentration that caused paralysis and death within 48-72 hours. Before paralysis and death, animals expressing the pan-neuronal marker unc-119::GFP treated with SeMet were examined and shown to have blebbing along the ventral cord, neuronal swelling, and altered nuclear staining that was not present in untreated controls. Examination of muscle fibers using myo-3::GFP animals showed that muscle fibers were unaffected by SeMet treatment. Taken together, these data support using Se toxicity as a model for ALS. Currently, we are screening known growth factor and signal transduction mutants for resistance to selenium toxicity. In addition, we have also generated a number of selenium resistant mutants by EMS mutagenesis and are in the process of genetically characterizing them.

Robin H Cowie et al. (2003) International Worm Meeting "Selenium toxicity in C. elegans leads to paralysis, developmental arrest, and reduced fertility"

Annette Estevez, Miguel Estevez, Robin H Cowie

[International Worm Meeting, 2003]

Although selenium(Se) is an essential micronutrient required for normal antioxidant activity, endocrine function, and immune system function, the difference between healthy levels of selenium intake and toxic levels is quite narrow. At high concentrations Se can lead to diverse pathologic consequences including motor neuron degeneration indistinguishable from polio and Amyotrophic Lateral Sclerosis (ALS), reproductive failure, and congenital abnormalities (especially of the CNS). Although the toxic effects of Se have been known for many years its role in processes other than free radical scavenging and thyroid hormone synthesis remain unclear despite the large number of identified selenoproteins. Because of the association of high environmental Se with ALS, and reproductive failure in wildlife we have begun to develop a genetic approach to identify the pathological processes it causes. The effects of selenium exposure on C. elegans have not previously been characterized. A baseline selenium concentration generating observable toxicity in populations of N2 was defined using a range of seleno-L-methionine (SeMet) concentrations in the growth media. (Note: Sodium selenite was originally used and was also toxic, but bacterial metabolism of the compound led to emission of a volatile sulfurous smelling compound that caused mucosal irritation in lab members. Dont try this at home!). To develop an appropriate screening tool with which to select selenium resistant strains we standardized four classes of toxicity assays which measure acute toxicity, developmental arrest/egg viability, and fertility. Our studies have shown that SeMet is directly toxic to neurons and leads to axonal blebbing, loss of neuronal cell body morphology, and paralysis over a 48-hour period. In embryos, SeMet induces congenital malformations and developmental arrest. Embryos that do manage to hatch often have bizarre malformations of the head. SeMet also reduces the amount of eggs produced per animal. These toxic responses are similar to those seen in wildlife populations exposed to Se contaminated water. Several mutations that affect sensitivity to Se toxicity have been isolated and are being characterized.

Jianghua Yin et al. (2007) International Worm Meeting "Regualtion and function of a sma-9 target gene."

Cathy SavageDunn, Ling Yu, Jianghua Yin

[International Worm Meeting, 2007]

BMP/TGF<font face=symbol>b</font> signaling cascades regulate many aspects of development and differentiation in both vertebrates and invertebrates. In the well-characterized Drosophila Dpp signaling cascade, upon Dpp ligand binding, type I receptor phosphorylates the intracellular signal transducer Mad (R-Smads), which then forms a complex with the Co-Smad Medea (Med). The Smad protein complex will translocate into the nucleus, recruiting transcription cofactor Schnurri (Shn) and inhibiting the brinker gene transcription, which in turn negatively regulates the expression of most Dpp target genes. In Caenorhabditis elegans, the Schnurri homolog SMA-9 is also found to function as a transcription cofactor in the DBL-1 pathway, a BMP/TGF<font face=symbol>b</font>-related signaling cascade, regulating body size and male tail development. The molecular mechanisms of SMA-9 function are still unknown. Study of the sma-9 target genes will help us to illustrate the conserved role of Schnurri/SMA-9 protein function in invertebrates. The T27F2.4 gene, a bZip transcription factor, is found to be highly upregulated in sma-9 mutant background, according to the microarray analysis and real-time PCR data. Meanwhile, RNAi experiment show that in vivo, knockdown of T27F2.4 gene will cause long body size phenotype. Recently, we found that the downstream region of this gene contains a putative silencer element (SE) that is essential for mediating the Dpp-dependent repression of brinker gene transcription in Drosophila via the Shn/Mad/Med complex. This SE also functions similarly in vertebrates. We hypothesize that the regulation of T27F2.4 gene expression and tissue specificity are also mediated through this conserved mechanism. Now we are using GFP reporter constructs containing the SE element to test our hypothesis. Meanwhile, examining how T27F2.4 gene functions in body size development is another goal of our studies.

Robert K Andrews et al. (2005) International Worm Meeting "The role of ER proteins in C.elegans spindle orientation"

Julie A Ahringer, Robert K Andrews

[International Worm Meeting, 2005]

ooc-3 and ooc-5 mutant embryos have abnormal spindle orientations at the 1- and 2-cell stages (Pichler et al. 2000; Basham et al. 2001). Both genes encode ER-resident proteins, indicating a link between the ER and spindle positioning. However, the mechanism through which the ER affects the spindle remains unexplained. We have observed that RNAi disruption of the C.elegans Oligosacchryl Transferase complex (OST) results in spindle misorientation in the early embryo, in addition to a near-identical set of secondary phenotypes to those recorded in ooc mutants. The OST and the oocs are apparently involved in different ER processes. The OST complex carries out the first step of N-linked glycosylation and OOC-5 is predicted to be a chaperone. The similarity of the OST RNAi and ooc mutant phenotypes suggests a general role for the ER in spindle orientation. We have carried out assays of the ER and found that it is stressed in both ooc and OST RNAi embryos. Furthermore, using a GFP ER marker, we have observed a marked redistribution of the embryonic ER away from the mitotic spindle and onto the cellular cortex in OST RNAi embryos. We are currently investigating interactions between the ER and spindle microtubules to assess how this contributes to spindle dynamics in the C.elegans embryo.

Yang, Ya-Luen et al. (2013) International Worm Meeting "Sestrin confers the regulations of muscle aging and lifespan in Caenorhabditis elegans."

Yang, Ya-Luen, Chu, I-Hua, Liou, Bang-Yu, Loh, Kah-Sin, Chen, Huan-Da, Chen, Chang-Shi, Kuo, Cheng-Ju

[International Worm Meeting, 2013]

Aging is associated with the progressive decline in body function and physiology that is shared by all multicellular organisms. The free radical theory argues that the accumulation of damage caused by reactive oxygen species (ROS) over the time is the cause of aging. Sestrins are evolutionarily conserved in metazoans and are required for ROS clearance. However, whether sestrins per se regulate longevity in multicellular organisms is still unclear. Here, we report that SESN-1, the only Sestrin ortholog in Caenorhabditis elegans, is a positive regulator of lifespan. C. elegans sesn-1 mutants exhibit shorter lifespans, hypersensitivity to oxidative stress, and premature aging in muscle. When sesn-1 gene was overexpressed and rescued, the lifespan of sesn-1 mutant worms could be extended. These findings imply that SESN-1 is required to protect against general life stressors, is important in regulation of lifespan and healthspan late in life, and might play a key role in muscle integrity.