Jiang N et al. (2015) Dev Cell "Muscles get dendrites into shape."

Jiang N, Parrish JZ

[Dev Cell, 2015]

Sensory neurons interact with muscles in many contexts, but muscle-derived signals that pattern sensory dendrites have not been extensively characterized. In this issue of Developmental Cell, Liang etal. (2015) report a signaling system in which positional cues from muscle are transduced to hypodermal cells to direct sensory dendrite outgrowth.

Calovich-Benne, Canyon et al. (2021) International Worm Meeting "Alternative mec-2 isoforms exhibit neuron type-specific expression and function"

Calovich-Benne, Canyon, Norris, Adam, Liang, Xiaoyu

[International Worm Meeting, 2021]

Alternative splicing can contribute to functional diversity by creating multiple isoforms of a single gene. Alternative splicing has been widely observed but it is often unknown whether specific isoforms are expressed in unique cell types, and whether they are functionally relevant. Neuron-specific RNA sequencing (RNA-seq) of C. elegans in our lab showed that the conserved membrane protein mec-2 exhibits unique splicing patterns in specific neurons. We found that the canonical long mec-2A isoform is expressed in mechanosensory neurons and was regulated by the mec-8 RNA binding protein, but the non-canonical short mec-2B isoform was detected in neurons other than mechanosensory neurons. We have recently validated our RNA-Seq results using isoform-specific endogenous fluorescent tags, and determined that the mec-2B isoform is present in chemosensory neurons as well as mechanosensory neurons. We then tested whether there was a functional role for the mec-2B isoform in chemosensory neurons. Chemotaxis assays show that mec-2 mutants have a significant deficiency in chemotaxis to various organic volatile odorants. Using the CRISPR-Cas9 system we forced the expression of one or the other isoform of mec-2 to see whether there is a difference in behaviors. We found worms expressing only the short mec-2B isoform can sense odors but not soft touch, while worms expressing long mec-2 can sense both odors and soft touch. When we re-introduced the long mec-2 isoform into mec-8 mutant, the mec-8 touch insensitivity phenotype was rescued. These experiments therefore demonstrate regulation of mec-2 alternative splicing resulting in isoforms with differential expression and function in different neuron types.

Edlira Yzeiraj et al. (2007) International Worm Meeting "Regulation and expression of collagens and fat metabolism genes in the dbl-1 pathway."

Dr. Cathy Savage-Dunn, Edlira Yzeiraj, Mariya Fabisevich

[International Worm Meeting, 2007]

In C. elegans DBL-1 is a member of the TGF-<font face=symbol>b</font> superfamily closely related to Drosophila Dpp and vertebrate BMP-2/BMP-4 (Suzuki et al., 1999, Development 126, 241-250). The DBL-1 signaling pathway regulates body size and patterning of male-specific copulatory structures. In regulating body size DBL-1 acts as a dosage-dependent regulator (Suzuki et al., 1999). Loss of dbl-1 activity results in smaller animals, while overexpression of dbl-1 results in longer animals. From microarray analysis 242 genes regulated by dbl-1 were identified. From these, 159 genes are repressed but only 83 genes are activated (Liang et al., 2007, Developmental Biology, In Press). We are interested in determining the biological function of some of these putative target genes. We are focusing on collagens and genes involved in fat metabolism. Collagen genes are involved in body size regulation. Cuticle collagens are synthesized by the hypodermis and secreted on the surface of the worm body as an exoskeleton. In the C. elegans genome, about 175 genes encode collagen-like polypeptides. Mutation of these genes causes defects in body morphogenesis, e.g. dumpy, roller, blister, and embryonic lethality (Myllyharju and Kivirikko, 2004, Trends Genet. 20, 33-43). From the microarray analyses three collagen genes were identified as dbl-1-regulated target gene, col-141, rol-6 and col-41. It is believed that dbl-1 represses col-141 and activates rol-6 and col-41 (Liang et. al., 2007). We plan to confirm the gene regulation of these genes by initially performing RT-PCR and then do RNAi to test their function. We are also focusing on genes involved in fat metabolism and transport, specifically vit-5, vit-6, fat-6, fat-7, and ins-7 (Liang et. al., 2007). We are interested in whether the dbl-1 pathway regulates body size in part by regulation of fat storage and metabolism. In addition these genes are believed to play a key role in C. elegans aging. vit-5, vit-6, and ins-7 are upregulated by the dbl-1 pathway, and fat-6 and fat-7 are downregulated by it, and this regulation has been confirmed by RT-PCR for some of these genes. To examine these genes we will use fat staining, GFP-reporters, and RNAi feeding. During the fat staining procedure, worms ingest fluorescent particles, which are temporarily stored in the intestinal cells. The cells can then be seen under a fluorescent microscope, and analyzed for number and degree of fat content. GFP reporters will be used on fat-6, fat-7 and ins-7 to locate the physical expression of the gene.

Alicia Melendez et al. (2001) International C. elegans Meeting "Analysis of the C. elegans homolog of the mammalian autophagy gene beclin 1"

Alicia Melendez, David H Hall, Beth Levine

[International C. elegans Meeting, 2001]

Autophagy is the bulk degradation of cytoplasmic components through an autophagosomic-lysosomal pathway. Under stress or nutrient starvation, mammalian cells as well as yeast cells undergo major degradation of proteins and remodelling of cellular compartments. Autophagy is essential for differentiation, survival during nutrient deprivation, and normal growth control; however, nothing is known about autophagy in the development of multicellular organisms. We are analyzing the role of evolutionarily conserved autophagy genes in C. elegans development, by studying the function of the worm beclin1 homolog. The human beclin 1 gene is the first identified mammalian gene to mediate autophagy and also has tumour suppresor and antiviral function (Liang et al., 1999). In yeast, the beclin1 gene was isolated as an autophagy defective mutation, Apg6 , or as a vacuolar protein sorting mutation, Vps11 . Mammalian Beclin 1 promotes autophagy, but not vacuolar protein sorting in autophagy-defective yeast with a targeted disruption of the yeast ortholog, APG6/VPS30 (Liang et al., 1999). Human Beclin1 shares 28% amino acid with the C. elegans Beclin1 (T19E7.3; C. elegans genome sequencing project). We have inactivated the C. elegans beclin1 homologue by injecting T19E7.3 dsRNA or by soaking N2 worms with T19E7.3 dsRNA. beclin1 RNAi-soaked N2 worms arrested at the L1 stage, whereas, the progeny of the injected beclin1 RNAi N2worms reached adulthood with varying defects in the intestine, including the accumulation of large vacuolar structures in the intestine. Ce beclin1 RNAi also decreases the endocytosis of a YP170(yolk protein) ::GFP fusion by oocytes, an assay that has been used to isolate mutants in receptor mediated endocytosis (Grant and Hirsh, 1999). As autophagy is required for survival under stress or nutrient starvation conditions, we hypothesized that autophagy might be required for dauer formation or survival of the dauer larva. To investigate this hypothesis, we injected daf-2 mutants with Ce beclin1 dsRNA. daf-2 mutants are dauer constitutive when raised at the restricted temperature and have an increased lifespan at the permissive temperature (Riddle, 1997). Ce beclin 1 (RNAi); daf-2 double mutants arrested at different stages of dauer formation when grown at the restrictive temperature. Survival of the daf-2 dauers was also affected after beclin1 RNAi. We are now using electron microscopy to determine if autophagy is increased upon dauer formation, and if autophagy is affected in the Ce beclin 1 RNAi mutants. In summary, Ce beclin 1 RNAi-soaked N2 worms arrest at the L1 stage and display intestinal defects, and the Ce beclin 1 RNAi-injected daf-2 worms arrest at different stages of dauer formation. While the molecular basis of these phenotypes is not yet known, these observations suggest a possible role for beclin 1 -mediated autophagy in C. elegans development and in the survival or formation of the C. elegans dauer stage larvae. B. Grant and D. Hirsh, 1999. Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 1999 Dec;10(12):4311-26. X. H. Liang et al., 1999. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999 Dec 9;402(6762):672-6. D. L. Riddle, in Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997), pp. 739-768.

MJ Munoz et al. (1999) International C. elegans Meeting "Isolating genes asociated with longevity"

MJ Munoz, DL Riddle

[International C. elegans Meeting, 1999]

Dauer larvae can live for as long as six months, whereas the life span of animals that develop continuosly to the adult is only about two weeks. Temperature-sensitive daf-2 mutants enter the dauer stage constitutively at 25degC, and when grown at permissive temperature their adult life span is doubled (Kenyon et al. 1993). Mutations in daf-16 , encoding a forkhead transcription factor (Ogg et al. 1997, Lin et al. 1997), suppress the increased daf-2 longevity. Thus, expression of some of the genes that are targets of DAF-16 activation may be responsible for increased longevity. We are using the differential display PCR method (Liang and Pardee 1997) to look for differences in gene expression between a daf-2 mutant (long life span) and a daf-2; daf-16 double mutant (wild-type life span). We have identified two differentially expressed genes that have a higher level of expression in the daf-2 mutants. The sequence of these clones predicted two different proteins with unknown function. We have also looked for differences in gene expression between a daf-2; daf-12 double mutant (four times wild-type life span) and a daf-2; daf-12; daf-16 triple mutant (wild-type life span) (Larsen et al. 1995). In this case we have found eleven differentially expressed genes. Seven of them are expressed at higher levels in the triple mutant. We have sequenced two of these and both encode proteins homologous to lectins. The other four genes are expressed at higher levels in the daf-2; daf-12 double mutant. We have cloned and sequenced one, which encodes a protein with unknown function. We are cloning and sequencing the remaining genes. We plan to integrate this information with that obtained from other methods (SAGE analysis, subtraction libraries and microarrays) to select candidates for genes that enhance longevity. We then plan test the effect of their (1) overexpression and (2) loss of function on life span. Kenyon et al. (1993) Nature 366:461-464 Larsen P. et al. (1995) Genetics 139:1567-1583 Liang P. and Pardee, A. B. (1997) Science 257:967-971 Lin K et al. (1997) Science 278:1319-1322 Ogg et al. (1997) Nature 389:994-999

Marisa L Foehr et al. (2004) East Coast Worm Meeting "SMA-9, a Protein Involved in Patterning of the C.elegans M Lineage"

Ming Xu, Jun Liu, Marisa L Foehr

[East Coast Worm Meeting, 2004]

We are interested in understanding mesodermal patterning and cell fate specification using the C. elegans postembryonic mesodermal lineage, the M lineage, as a model system. The M lineage is derived from a single precursor cell (M), which undergoes a series of asymmetric divisions to generate a number of differentiated cell types. In particular, the dorsal daughter of M produces two coelomocytes (CCs), while the ventral daughter produces two sex myoblasts (SMs). The mechanisms underlying this asymmetry are not clear. We are interested in a molecule, SMA-9, that functions in establishing this polarity and in regulating cell fate specification in the M lineage. sma-9 mutants generate a duplication of the ventral descendants of the M lineage. This results in an end phenotype in which the M derived CCs are missing and extra SMs are generated. In addition, sma-9 mutant animals are smaller than wild type and males have tail patterning defects (Liang et al, 2003 1 ). In order to understand how sma-9 functions in regulating the dorsal/ventral asymmetry of the M lineage, we set out to determine the expression pattern of sma-9 . sma-9 is a highly complex molecule that has multiple splicing isoforms. We are interested in dissecting the expression patterns of the different isoforms, as well as understanding which isoforms are specifically functioning in the M lineage. To this end, we have generated isoform specific antibodies to two of the three SMA-9 C-terminal isoforms and examined the expression pattern of SMA-9 using these antibodies. Additionally, GFP fusion constructs linking the genomic N terminus to the different C terminal ends have been made. We are currently generating transgenic lines carrying these fusion constructs. sma-9 encodes the C. elegans homolog of the Drosophila protein Schnurri, a known regulator of the TGF-beta signaling pathway. While sma-9 functions in the TGF-beta signaling pathway in regulating body size and male tail patterning (Liang et al, 2003), it does not appear to function in the TGF-beta signaling pathway in patterning the M lineage. Instead, we have found some intriguing evidence that sma-9 genetically interacts with the lin-12/Notch signaling pathway in patterning of the M lineage. 1. J.Liang et al. Development 130, 6453-6464. 2003.

Ji Sze (2008) "Serotonin targets the DAF-16/FOXO signaling pathway to modulate stress responses"

Ji Sze

[2008]

"Stress response is a fundamental form of behavioral and physiological plasticity reflecting neuronal and endocrine modifications that allow organisms to respond and adapt to changes in the environment. Using C. elegans as a model organism, we have shown that serotonin (5HT) signaling governs stress behavior by regulating the neuroendocrine pathway from the DAF-2 insulin/IGF-1 receptor to the DAF-16/FOXO transcription factor at the nexuses of development, metabolism, immunity and stress responses [Liang et al. Cell Metabolism 4, 429-440, 2006]. Serotonin deficient mutant tph-1, like daf-2 mutants, exhibits DAF-16 nuclear accumulation, constitutive stress physiological states, and excessive fat accumulation. Conversely, exogenous 5HT and fluoxetine (Prozac) prevent DAF-16 nuclear accumulation in wild-type animals under stresses. Our recent DNA microarrays profiling tph-1 mutants revealed that serotonin deficiency affects the expression of many mitochondrial components and fourteen antioxidant genes. We will present in vivo analyses of these candidate genes indicating that serotonin regulates the expression of particular antioxidant genes in particular cells and tissues. Our studies demonstrate that the C. elegans is a genetically tractable model that can be used to delineate the molecular mechanisms and drug actions linking 5HT, neuroendocrine signaling, immunity, and energy metabolism. "

Alicia Melndez et al. (2002) East Coast Worm Meeting "The role of C. elegans autophagy genes in development"

Beth Levine, Alicia Melndez, David H Hall

[East Coast Worm Meeting, 2002]

Autophagy is the bulk degradation of cytoplasmic components through an autophagosomic-lysosomal pathway. Under nutrient starvation, or some other forms of stress, mammalian cells as well as yeast cells, undergo bulk degradation of proteins and remodelling of cellular compartments. Autophagy is the major route of delivery of cytoplasmic proteins into lysosomes; it is important for cell survival, in growth control, and may be defective in tumour cells. However, little is known about the role of autophagy in the development of multicellular organisms. We are analyzing the role of evolutionarily conserved autophagy genes in C. elegans development, by studying the function of the worm beclin 1 homolog (Ce bec-1). The human beclin 1 gene is the first identified mammalian gene to mediate autophagy and also has tumour suppresor and antiviral function (Liang et al. Nature. 1999; 402:672-6). In yeast, the beclin 1 gene was isolated as an autophagy defective mutation, apg6, or as a vacuolar protein sorting mutation, vps30. Mammalian Beclin 1 promotes autophagy, but not vacuolar protein sorting in autophagy-defective yeast with a targeted disruption of the yeast ortholog, APG6/VPS30 (Liang et al., 1999). C. elegans BEC-1 shares 28% amino acid homology with the human Beclin 1 (T19E7.3;C. elegans genome sequencing project). We have inactivated the Ce bec-1 homologue by injecting wild-type (N2) worms with bec-1 dsRNA or by soaking worms with bec-1 dsRNA. bec-1 RNAi-soaked worms arrested at the L1 stage, with pronounced defects in the intestine and in the head. In contrast, the progeny of injected bec-1 RNAi worms reached adulthood with varying defects in the intestine, characterized by the accumulation of large vacuolar structures. Ce bec-1 RNAi also decreases the endocytosis of a YP170(yolk protein) ::GFP fusion by oocytes, an assay that has been used to isolate mutants in receptor mediated endocytosis (Grant and Hirsh Mol. Biol. Cell. 1999;10:4311-26). In C. elegans, a developmental arrest, the dauer larva, occurs in response to high population density and limited food (Golden and Riddle Devel. Biol.. 1984; 102:368-78). As autophagy is induced by both of these stimuli in yeast and mammalian cells, we hypothesized that autophagy might be required for dauer formation or survival of the dauer larvae. To investigate this hypothesis, we injected daf-2 mutants with Ce bec-1 dsRNA. Mutations in the daf-2 gene, encoding a member of the insulin receptor family, result in constitutive formation of dauer larvae at the restricted temperature and increased lifespan at the permissive temperature. Ce bec-1 (RNAi); daf-2 double mutants arrest at different stages and do not complete dauer formation when grown at the restrictive temperature. Dauer formation is also affected in a daf-2; unc-51 double mutant, when grown at the restrictive temperature (unc-51 is the C. elegans orthologue of another autophagy gene in yeast, apg1). We are now using electron microscopy to determine if autophagy is increased upon dauer formation, and if autophagy is affected in the Ce bec-1 RNAi mutants. In summary, Ce bec-1 RNAi-soaked worms have developmental defects and arrest at the L1 stage. Furthermore, inactivation of homologs of two different yeast apg genes in C. elegans has an effect on the daf-2 constitutive dauer formation. While the molecular basis of these phenotypes is not yet known, these observations suggest a possible role for autophagy genes in C. elegans non-dauer development, as well as in the formation and/or survival of the C. elegans developmentally arrested dauer larva.

He, Liu et al. (2021) International Worm Meeting "PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) redundantly mediate MTOC localization and microtubule polarity in dendrites"

Hoogenraad, Casper, He, Liu, Harterink, Martin

[International Worm Meeting, 2021]

In neurons the microtubule cytoskeleton is key for proper axon and dendrite deployment. In axons microtubules are arranged with their plus ends distal to the cell body, whereas dendritic microtubules are predominantly arranged with their minus ends distal to the cell body (invertebrates) or with mixed orientation (vertebrates). This difference in microtubule organization allows for selective transport into axons or dendrites to differentiate these cell extensions. It was recently found in C. elegans that during dendrite development of the PVD neuron, RAB-11 positive vesicles function as a non-centrosomal microtubule organizing center (MTOC). These vesicles localize to the growing dendritic tip to nucleate microtubules with their characteristic minus ends distal organization (Liang et al. eLife, 2020). However, the mechanism that localizes these MTOC vesicles to the dendrite tip is poorly understood. Looking for proteins that may act at the microtubule minus ends, we found that PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) act redundantly for distal MTOC localization during dendrite development; their loss of function leads to defects in microtubule polarity establishment. We found that NOCA-2 colocalizes to the MTOC vesicles, and its depletion partially disrupted the recruitment of gamma-Tubulin to the MTOC vesicles. CAMSAP proteins are well described microtubule minus-end binding proteins that can stabilize/protect the minus-end from depolymerization. Not surprisingly we observed a punctate PTRN-1 distribution throughout the dendrite but also an accumulation around the MTOC vesicles. Surprisingly we found that during dendrite tip growth the MTOC vesicles and the surrounding Camsap puncta co-migrate with the growing tip, suggesting that these structures are connected. We are currently probing whether sliding of the distal microtubule skeleton is responsible for pushing the MTOC vesicles forward to form and maintain the dendritic minus end distal microtubes.

Jun Liu et al. (2002) East Coast Worm Meeting "The C. elegans Schnurri homolog SMA-9 in postembryonic mesodermal cell fate specification"

Jun Liu, Andrew Fire, Jun Liang, Cathy Savage-Dunn, Judith Yanowitz

[East Coast Worm Meeting, 2002]

We are interested in understanding how mesodermal cell fates are specified. The postembryonic mesodermal lineage, the M lineage, provides a valuable system for these studies. During postembryonic development, the single blast cell M divides characteristically and reproducibly to generate 14 striated bodywall muscles, 2 un-differentiated sex myoblasts that give rise to 16 non-striated sex muscles (vulval and uterine muscles), and two non-muscle cells (coelomocytes). Previous work from us and others have shown that correct patterning of the M lineage involves both lineage specific intrinsic factors and cell-cell signaling. We are interested in identifying additional factors in M lineage fate specification and characterizing functional interactions among these different factors. Using GFP-based mutagenesis screens, we have isolated a number of mutants that have defects in M lineage patterning. Three of these mutants were allelic to sma-9, a mutation affecting the body size of the animal. sma-9 mutations caused a coelomocyte to sex myoblast fate transformation in the M lineage. We have cloned sma-9 and shown that its product contains multiple C2H2 type zinc fingers and is the C. elegans homolog of the Drosophila Schnurri protein (see abstract by J. Liang and C. Savage-Dunn). Schnurri (Shn) in flies has been shown to play critical roles in the TGF-beta signaling pathway. The TGF-beta signaling pathway, however, does not appear to play an important role in M lineage cell fate specification. Instead, we observed intriguing genetic interactions between sma-9 and lin-12. Our current efforts are directed towards a better understanding of how SMA-9 functions in M lineage fate specification.