Du H-P et al. (1997) International C. elegans Meeting "A hox-7-like GENE IS NEEDED FOR TOUCH CELL DEVELOPMENT"

Chalfie M, Du H-P

[International C. elegans Meeting, 1997]

We conducted an EMS mutagenesis with a mec-2gfp transgenic line (uIs9) to identify genes involved in touch cell development. mec-2gfp is expressed in the cell bodies and processes of the touch receptor neurons. From 2638 mutagenized haploid genomes, we obtained 11 mutations in as many genes that affect outgrowth (3), migration (3) or cell fate(5) of the touch cells. Eight mutations are in known genes (unc-73, unc-51, a mec gene on X, mig-1, unc-40, mec-3, lin-32, unc-86). The three remaining mutations define three new genes. Recessive lethal and dominant ts mutation u788 (I) causes the generation of extra PVM or AVM cells, and u787 (III) causes PVM to migrate anteriorly instead of posteriorly, and u781 (X) gives a complex developmental phenotype. u781 animals are Mec in both the head and tail and only the ALM cells express mec-2gfp. One of ALMs often migrates more anteriorly than in WT. Only FLP cells and ALM cells express mec-3gfp in u781 animals (this result suggests that u781 acts upstream of mec-3). Consistent with this activity which affects the posterior body, u781 larvae and adult males have extremely deformed tails. These data suggest a more severe posterior phenotype. u781 is also partially lethal, and the animals coil, kink and move backward with greater difficulty. u781 mutants are fully rescued by a 9.5kb SalI-HpaI fragment from cosmid R07B1. This fragment encodes a homeobox gene whose homeobox is 82% amino acid identical to that of the mouse hox-7 or hox-8 genes. Homologues of these genes exist in various organisms, and have been implicated to function in many aspects of development, including embryonic and mesoderm development (Xenopus), limb development (chicken and mouse) and inner ear and sensory hair cell development (zebrafish).

Du H-P et al. (1996) East Coast Worm Meeting "Looking For Mutations Affecting the Outgrowth of the Touch Cells"

Du H-P, Chalfie M

[East Coast Worm Meeting, 1996]

We have conducted an EMS screen to look for more mutations affecting the touch cells by directly examining a mec-2gfp transgenic line uIs9. The mec-2gfp and rol-6 transgenes are integrated onto Chr.V. The processes of the touch cells can be seen very clearly in the uIs9 animals. Eleven mutations (from over 2000 mutated haploid genomes) affect the differentiation, migration and outgrowth of the touch cells. Five of the six mapped mutations are alleles of the known genes: mec-3, unc-86, lin-32, mig-1 and unc-73. u781 maps close to and to the right of lon-2. u781 animals have ALMs but lack the other touch cells, and one of the two ALMs is often anteriorly displaced. The remaining five mutations include two mutations in which the touch cell processes are much shorter than usual and three mutations which affect the migration of AVM and PVM. We are continuing this screen and the genetic characterization.

Du H-P et al. (1995) International C. elegans Meeting "MEC-9 ENCODES A POTENTIAL SECRETED PROTEIN NEEDED FOR TOUCH FUNCTION"

Gu GQ, Du H-P, Chalfie M

[International C. elegans Meeting, 1995]

mec-9 mutants are touch insensitive but their touch cells have no morphological defects. mec-9 has two overlapping transcripts. A 2 kb transcript encodes a protein of 502 aa that is expressed in over 30 ventral cord motor neurons and 14 cells in the head. A 3 kb transcript encodes a 834 aa polypeptide that is only expressed in the touch cells and the PVD cells in a mec-3 dependent manner. The larger protein has two Kunitz-type serine protease inhibitor domains, three EGF-like repeats (two of the calcium-binding type), three more Kunitz domains, three non-calcium binding EGF-like repeats, and a glutamic acid-rich domain. The shorter protein has the last two Kunitz domains and downstream sequences. These proteins may be secreted since the Kunitz and the EGF-like repeats are only found in extracellular proteins. Fifteen of the sixteen mec-9 mutations we sequenced are specific to the larger protein. Seven missense mutations affect either the EGF-like repeats or the Kunitz domains, an indication that these regions are functionally important. The sixteenth mutation was an 11 bp deletion of a region common to both transcripts; mutants with this defect were only touch insensitive (i.e., not Unc). Thus the shorter transcript may be either nonessential or redundant. mec-9 mutations are dominant enhancers of mec-5 temperature-sensitive mutations. Since mec-5 encodes a collagen needed for the touch response, this non-allelic-specific enhancement may suggest an interaction between these proteins.

Duggan A et al. (1997) International C. elegans Meeting "CLONING OF THE EGL-46 GENE"

Duggan A, Chalfie M

[International C. elegans Meeting, 1997]

The egl-46 gene is required for the correct differentiation of the HSN neurons, a pair of motor neurons necessary for egg-laying (1,2). In addition, an extra pair of touch receptor-like cells is present in egl-46 mutant animals (3). This finding led to the hypothesis that egl-46 represses touch cell fate in these cells. We have cloned the egl-46 gene using transformation rescue and we have confirmed the assignment by identifying the mutations present in the four known egl-46 alleles. A 5.5kb fragment of CO9E6 partially rescues the Egl phenotype of egl-46 animals. The egl-46 gene (K11G9.4) encodes a C2H2 zinc finger protein that contains three putative zinc fingers. We have confirmed the exon-intron structure of egl-46 by sequencing a cDNA and by 5' RACE analysis. egl-46::lacZ and egl-46::GFP reporter constructs are expressed in a complex pattern which includes cells in the head, tail and ventral cord. We are now trying to determine the identity of the staining cells. Future work will address the mechanism by which the egl-46 gene controls neuronal fate and/or differentiation. (1) Desai, C., Garriga, G., McIntire, S.L. and H.R.Horvitz (1988) Nature 336, 638-646. (2) Desai, C. and H.R.Horvitz (1989) Genetics 121, 703-721. (3) Mitani, S., Du, H., Hall, D.H., Driscoll, M. and M. Chalfie (1993) Development 119, 773-783.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Dana L. Miller, Mark B. Roth

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

De Vore, Deanna Michele et al. (2013) International Worm Meeting "Exploring the role of mechanosensory extracellular matrix components in the structure and function of primary cilia."

Knobel, Karla, Barr, Maureen, De Vore, Deanna Michele

[International Worm Meeting, 2013]

PKD2 encodes a transient receptor potential polycystin (TRPP) channel receptor protein found in non-motile, primary cilia of mammalian cells 1. In humans, PKD2 mutations result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). In the nematode Caenorhabditis elegans, the polycystin-2 homolog, PKD-2, localizes to the primary cilia of male-specific sensory neurons (CEMs and RnBs in the head and tail respectively) where it is required for male mating behaviors. Given the ancient and evolutionarily conserved role for polycystin-2 in cilia, I am using C. elegans as a model to identify new genes required for ciliary receptor localization. Our laboratory performed a forward genetic screen for PKD-2::GFP ciliary localization (Cil) defective mutants (Bae et al 2008). The cil-2(my2) mutants exhibit excess unusual PKD-2 accumulation at the ciliary base, cilium proper, and around the distal dendrite. We performed three-factor and deficiency mapping, whole genome sequencing, and single gene rescue experiments and determined that the cil-2(my2) allele is a mutation in the gene, mec-5. MEC-5 is a unique collagen protein found in the extracellular matrix (ECM) surrounding touch receptor neurons (Du et al 1996). In touch receptor neurons (TRNs), MEC-1, MEC-5, and MEC-9 are found in the ECM, play a role in mechanosensation, and may localize degenerin/epithelial sodium channels (DEG/ENaCs) along the TRN processes (Du et al 1996, Emtage et al 2004). Our preliminary data indicates that mec-1 and mec-9 regulate PKD-2::GFP localization in male-specific sensory neurons. We are currently determining how these ECM proteins regulate localization and channel properties of the PKD-2 TRP polycystin channel. These studies will shed light on how sensory receptors like PKD-2 are targeted to cilia, and may advance the understanding and treatment of ADPKD. References: 1) Bae YK et al, Dev Dyn. 2008 Aug;237(8):2021-9. 2) Du H. et al, Neuron. 1996 Jan;16(1):183-94. 3) Emtage L et al, Neuron. 2004 Dec 2;44(5):795-807.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zelmanovich, V., Livshits, L., Zuckerman, B., Smith, Y., Gross, E., Abergel, R., Romero, L., Abergel, Z.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Neri C et al. (1998) European Worm Meeting "A C. elegans model of neuron dysfunction in polyglutamine expansion diseases: suggestive evidence for polyglutamine-dependent intracellular aggregation and touch receptor neuron dysfunction"

Moriniere S, Chalfie M, Du H-P, Neri C

[European Worm Meeting, 1998]

Polyglutamine expansion (PGE) underlies several neurodegenerative diseases including the Huntington!s disease (HD). An inverse correlation between the age-of-onset and the length of the polyglutamine (polyQ) tract is observed in PGE disease families. The mechanisms which cause neurodegeneration in PGE diseases is unknown. Data from human patients, transgenic mice studies, and mammalian cell-based models have suggested that a gain of toxic properties is likely to be induced by PGE in target neurons, resulting in the formation of intracellular aggregates and cell death. Studies of HD in mammalian cell-based models have suggested that both the length of the polyQ tract and the length of the disease protein fragment carrying the expanded polyQ tract influence the localization of intracellular aggregates and the progression of neurodegeneration. The aim of our study is to develop models of PGE-induced neurodegeneration in C. elegans since the genetics, well-characterized development, and the known genomic sequence make this a useful organism in which to study these problems. We used the mec-3 promoter (mec-3p) to express GFP fusions which contain a short N-terminal fragment of huntingtin with a normal (17 units) or expanded (84 units) polyQ tract. The mec-3 gene is expressed in 10 neurons including the six touch receptor neurons AVM, ALML, ALMR, PVM, PLML and PLMR. Transgenic worms were analyzed for intracellular pattern of GFP expression, touch sensitivity, and occurrence of cell death in touch receptor neurons. Mec-3p driven GFP expression typically shows a diffuse expression pattern in the cell body and process. In transgenic animals expressing the long polyQ construct, obvious protein aggregation was observed, particularly as two large and bright perinuclear GFP aggregates were detected. A diffuse GFP expression was never observed in the processes. In transgenic animals expressing the short polyQ construct, a less severe modification of GFP localization was observed, resulting in a pattern of expression closer to that of wild type animals with GFP expression driven by mec-3p. Touch tests were performed over a period of seven days starting from the young adult stage. In worms expressing the long polyQ construct, less than 30% were Mec at the tail at Day 1, and more than 50% were Mec at the tail at Day 7. In worms expressing a short polyQ construct or in animals with GFP expression driven by mec-3p, less than 7% and 30% were Mec at the tail at Day 1 and Day 7, respectively. Cell death was not observed in these experiments. Other studies have suggested that huntingtin fragments with an expanded polyQ tract (150 Glns) induce a neuronal phenotype in transgenic animals (P. Faber, J. Alter, M. MacDonald, and A. Hart, personal communication). Our preliminary data suggest that GFP fusions comprising a short N-terminal huntingtin fragment with 84 consecutive Glns induce perinuclear aggregation and touch insensitivity when expressed in touch receptor neurons. Our data also suggest that the transgenic animals obtained might be useful for studying the biochemical mechanisms involved in polyQ-induced neuron dysfunction.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Rasika Kalamegham, Wendy Hanna-Rose

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Sternberg, Paul, Schwartz, Hillel

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.