Liu, Jun et al. (2021) International Worm Meeting "Constructing a tool box for imaging and stimulating pharyngeal neurons to understand foraging behavior in C. elegans"

Liu, Jun, Scholz, Monika

[International Worm Meeting, 2021]

To understand the interplay between neural circuit structure, activity, and behavior requires the ability to observe the action of a set of neurons underlying that behavior while the animal is behaving in a well-known circuit. Work in other organisms, such as the lobster has already yielded insight into variability of neural activity, expression and yet remarkable stability in behavioral output. However, these studies often are limited to dissected preparations and behaviors inferred from muscle or motor-neuron activity. In C. elegans, the circuit controlling feeding comprises only 20 neurons and is separate from the 282 somatic neurons, yet it controls food intake and modulates feeding rate effectively. The small pharyngeal circuit is ideally suited to understand the function of a contained, nearly isolated circuit in an intact, behaving animal. Specifically, we want to understand: I) What is the function of individual pharyngeal neurons during foraging? II) How do pharyngeal neurons communicate with the extra-pharyngeal neurons to coordinate foraging behavior? III) What is the activity of individual pharyngeal neurons during foraging? To these ends, we aim to create a toolbox that targets individual and subsets of pharyngeal neurons for expressing a range of optogenetic tools and the calcium indicator GCaMP. We will use the cGAL and split cGAL system developed by the Sternberg Lab which is adapted from the GAL4-UAS system for the C. elegans community. We create driver strains by selecting a single promoter, cGAL, or two intersecting promoters (forming an AND gate), split cGAL, to achieve specific expression in targeted neuron(s). We then cross these drivers with their UAS effector strains, such as GFP, optogenetic activator/inhibitor and GCaMP, to achieve neuron-specific expression of fluorophores or other desired proteins. To search for the promoters that drive unique expression in pharyngeal neurons, we have curated information from the literature and transcription databases. We will generate different "promoter::cGAL" driver lines and then verify the expression using the GFP effector lines and the neuroPAL multicolor Atlas. Successful candidates will then be crossed to other optogenetic and GCaMP effector lines for targeted neuronal manipulations and calcium imaging of foraging animals. We will present our progress in creating such a toolbox. These collectively will pave the way to understand the neuronal basis of foraging behavior in C. elegans.

Jun Liu et al. (2001) International C. elegans Meeting "Mesodermal cell fate specification in the postembryonic M lineage"

Jun Liu, Andrew Fire

[International C. elegans Meeting, 2001]

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, 16 non-striated sex muscles (vulval and uterine muscles), and two non-muscle cells (coelomocytes). Previous work from our lab and others has shown that correct patterning of the M lineage involves both lineage specific intrinsic factors and cell-cell signaling. We are interested in identifying and characterizing the functions of these factors in M lineage fate specification. At the start of the M lineage, two Hox cluster genes lin-39 and mab-5 , as well as their cofactor, a homeodomain protein CEH-20 specify cellular identity and myogenic potential of M. We have found an overlapping function for lin-39 and mab-5 in diversification of different cell fates within the lineage, and have shown that the C. elegans twist ortholog hlh-8 is a direct target of these factors in the M lineage. Using gfp-based mutagenesis screens, we have isolated 26 additional mutants that have defects in M lineage patterning (12 of them came from a screen carried out by Judy Yanowitz in the lab). Three of these mutants turned out to be allelic to sma-9 , and showed intriguing genetic interactions with lin-12(0) . We are in the process of characterizing these and other mutants isolated from the screens.

Jun Liu et al. (2005) International Worm Meeting "Cell culture experiments to characterize a putative VAB-1/Eph RTK and SAX-3/Robo neuronal receptor complex"

Jun Liu, Ian D Chin-Sang

[International Worm Meeting, 2005]

The C. elegans SAX-3/Robo receptor is involved in various aspects of neuronal development, including anterior-posterior, dorsal-ventral, and midline commissural axon guidance decisions. The predicted ligand for SAX-3 is SLT-1 (Slit) and sax-3 mutants share similar axon guidance defects as slt-1 mutants. However sax-3 mutants display a notched head phenotype and other axon guidance defects not observed in slt-1 mutants. This suggests that SAX-3/Robo has Slit-dependent and Slit-independent roles in development. Furthermore, SAX-3/Robo has been shown to bind UNC-34/Ena and UNC-40/DCC in vitro, and to act in parallel to UNC-6/Netrin and UNC-40/DCC axon guidance systems (1-4). Genetic and in vitro results in our lab also suggest that SAX-3/Robo and VAB-1 Eph Receptor Tyrosine Kinase may function together as a receptor complex during C. elegans embryonic morphogenesis (See Ghenea et al., this meeting). To test the significance of the putative VAB-1/SAX-3 receptor complex, we have expressed SAX-3 and VAB-1 in HEK293 mammalian cells. We will confirm the interaction of SAX-3 and VAB-1, and test for the effects of the SAX-3/VAB-1 receptor complex. Specific questions we will address are: Does SAX-3 affect VAB-1's binding affinity to EFN-1? Does EFN-1 bind SAX-3? Does SLT-1 bind SAX-3 or VAB-1 or both? We will also monitor VAB-1's tyrosine kinase activity using Phosphotyrosine antibody (4G10). To search for new candidates that potentially interact with SAX-3/Robo in vitro we have conducted yeast two hybrid screens. Three baits of different intracellular domains of SAX-3/Robo were used: 1) full-length; 2) domain containing the CC1 (Conserved Cytoplasmic region 1) and CC3; and 3) domain containing the CC2. In a preliminary small-scale screen, we isolated three interesting cDNA clones that encode proteins that potentially interact with the intracellular region of SAX-3/Robo: 1) JAC-1 (Juxtamembrane domain-Associated Catenin) a p120 catenin homolog; 2) F07C6.4 a FERM domain (band F ezrin-radixin-moesin homology domains)-FERM domains occur in a wide variety of membrane-associated signaling and cytoskeletal proteins; and 3) FKB-5 a homolog of the human FK506 binding proteins that interact with the mammalian insulin pathway. We are currently confirming these interactions in different protein binding assays and testing whether these genes show genetic interactions with genes known to function with sax-3/Robo such as the vab-1/Eph. We are also conducting large-scale screens. We will further report our results in the poster. 1. Zallen et al., (1998) Cell 92, 217-227; 2. Hao et al., (2001) Neuron 32, 25-38; 3. Yu et al., (2002) Nature Neuroscience 5, 1147-1154; 4. Zallen et al., (1999) Development 126, 3679-3692

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.

Jun Liu et al. (2007) International Worm Meeting "Identifying intracellular partners of the SAX-3/Robo signaling pathway and the introduction of a novel assay to study membrane protein-protein interactions."

Ian Chin-Sang, Jun Liu

[International Worm Meeting, 2007]

SAX-3 is a C. elegans neuronal receptor belonging to the conserved Roundabout (Robo) family of proteins that mediate repulsion in axon guidance decisions (Zallen et al., 1998). The proposed ligand for SAX-3 is SLT-1/Slit, and both sax-3 and slt-1 mutants exhibit axon guidance defects (Hao et al., 2001). However, the downstream signaling pathway is largely unknown, i.e., what signaling cascade does SAX-3 trigger upon binding to its ligand? The only identified downstream effector so far is UNC-34/Enabled, together with two other putative upstream regulators being VAB-8L and UNC-73/Trio (Watari-Goshima et al., 2007; Yu et al., 2002). Our lab has previously shown that SAX-3 may form co-receptor complexes with the VAB-1/Eph receptor (Ghenea et al., 2005). Considering that VAB-1 is a receptor tyrosine kinase and can potentially phosphorylate SAX-3, we modified the traditional yeast two hybrid screen by using VAB-1 kinase region as a bridge to phosphorylate the SAX-3 intracellular region. From this screen, we successfully isolated genes encoding the small isoform of nck-1 twice. nck-1 has been implicated to be involved in the VAB-1 signaling pathway for axon guidance decisions (see Mohamed and Chin-Sang this meeting). We have been conducting research on NCK-1 and its role in the SAX-3 signaling pathway and discovered that SAX-3 not only binds to the SH3 domain of NCK-1, but also binds to its SH2 domain in a VAB-1 kinase-dependent manner. We will further report our result in the meeting. There is an emerging tendency that axon guidance receptors can form co-receptor complexes for guidance decisions, however, the current available assays to study membrane protein-protein interactions are very limited. We describe a novel strategy to show receptor-receptor interaction at the membrane. In principle, we fuse membrane protein A to TEV (tobacco etch virus) protease, and fuse membrane protein B to a TEV protease recognition site followed by a GFP construct tagged with a nuclear localization signal (NLS). Upon interaction, TEV protease will be brought in close proximity to the TEV recognition site and cleave it, thereby releasing the NLS-GFP which will eventually enter the nuclei. On the other hand, if no interaction occurs, the GFP signal will remain on the membrane. We successfully used this assay to confirm the self-association of myristoylated VAB-1 intracellular region, which serves as a positive control. We are using this assay to confirm the putative VAB-1/SAX-3 co-receptor complexes, and will report our result in the meeting.

Jun Liu et al. (2001) International C. elegans Meeting "Essential roles for C. elegans lamin gene in nuclear organization, cell cycle progression and spatial organization of nuclear pore complexes"

Perah Spann, Jun Liu, Klaus Weber, Tom Rolef Ben-Shahar, Millet Treinin, Yosef Gruenbaum, Dieter Riemer, Andrew Fire

[International C. elegans Meeting, 2001]

We are interested in using C. elegans as a model system to study the function of nuclear lamin and lamin-associated proteins during development. C. elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64 kD nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans , and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.

Wang, Lin et al. (2017) International Worm Meeting "Dissecting the roles of the ADAM10 metalloprotease SUP-17 in the BMP signaling pathway in Caenorhabditis elegans."

Liu, Jun, Liu, Zhiyu, Shi, Herong, Wang, Lin

[International Worm Meeting, 2017]

BMPs (Bone Morphogenetic Proteins) belong to the TGFbeta superfamily of ligands, and mediate a highly conserved signal transduction cascade. Upon ligand binding, type II receptors phosphorylate type I receptors, which in turns phosphorylate R-Smads (receptor regulated Smads). Phosphorylated R-Smads then complex with co-Smad (common mediator smad) and enter the nucleus to regulate gene transcription with different co-factors. BMPs play important roles in developmental and physiological processes. Malfunction of the pathway in humans can cause various disorders, such as skeleton diseases, heart diseases and cancers. So it is critical to strictly regulate the level of BMP signaling spatiotemporally. Using a highly specific genetic screen, we have identified several modulators of the BMP pathway in C. elegans. These include the Neogenin homolog UNC-40 [1], two paralogous tetraspanin proteins, TSP-12 and TSP-14, and an ADAM10 metalloproteinase (A Disintegrin and Metalloproteinase 10) SUP-17 [2]. We use the CRISPR/Cas9 system and tagged the endogenous TSP-12, SUP-17 and UNC-40 proteins with different fluorescence tags. We found that TSP-12 is localized to the cell surface and intracellular vesicles, and that TSP-12 can bind to SUP-17 and promote its cell surface localization. We have genetic evidence and preliminary biochemical evidence showing that UNC-40 is one, but not the only, substrate of SUP-17 in BMP signaling. We are currently identifying additional substrate(s) of SUP-17 in BMP signaling. Our work highlights the importance of intracellular signaling and protease processing in the regulation of BMP signaling. [1] Tian C, Shi H, Xiong S, Hu F, Xiong WC, Liu J. The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans. Development. 2013;140(19):4070-80. doi: 10.1242/dev.099838. PubMed PMID: 24004951; PubMed Central PMCID: PMCPMC3775419. [2] Wang L, Liu Z, Shi H, Liu J. Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans. PLoS Genet. 2017;13(1):e1006568. doi: 10.1371/journal.pgen.1006568. PubMed PMID: 28068334; PubMed Central PMCID: PMCPMC5261805.

Gissendanner CR et al. (2014) Genesis "C. elegans nuclear receptor NHR-6 functionally interacts with the jun-1 transcription factor during spermatheca development."

Praslicka B, El Sayed M, Cardin D, Harmson JS, Rowan BG, Gissendanner CR, Dubose CJ

[Genesis, 2014]

The NR4A nuclear receptor NHR-6 is an essential regulator of spermatheca organogenesis in C. elegans. In this study, we perform a focused, RNAi-based screen to identify modifiers of partial nhr-6 loss of function. Ninety-eight genes that encode signaling proteins expressed in the spermatheca were screened for enhancement of the nhr-6 RNAi phenotype. We identify the C. elegans gene jun-1, which encodes the homolog of the Jun transcription factor, as a strong enhancer of nhr-6 partial loss of function. We show that nhr-6 and jun-1 function together to regulate development of the spermatheca and are necessary for generating an organ with the normal number of cells. jun-1 is expressed in all cells of the developing spermatheca. We also provide evidence that NHR-6 and JUN-1 can physically interact in a yeast two-hybrid assay. Our results provide in vivo evidence that NR4A nuclear receptor and Jun transcription factor interactions are essential in regulating developmental processes in metazoans.

Hiatt SM et al. (2009) Mol Biol Cell "Caenorhabditis elegans FOS-1 and JUN-1 regulate plc-1 expression in the spermatheca to control ovulation."

Matsumoto K, Kerppola TK, Ellis RE, Shyu YJ, Hiatt SM, Hisamoto N, Duren HM, Hu CD, Kariya KI

[Mol Biol Cell, 2009]

Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.

Liu, Zhiyu et al. (2021) International Worm Meeting "The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function"

Liu, Zhiyu, Liu, Jun

[International Worm Meeting, 2021]

Tetraspanin proteins are a unique family of four-pass transmembrane proteins that are highly conserved in metazoans. There are 33 tetraspanins in mammals, and 21 in C. elegans. Tetraspanins can be further classified by the number of cysteine residues in their second extracellular loops. While much work has been done to characterize the biochemical properties of tetraspanins, less is known about the in vivo functions of different tetraspanins. We have recently shown that two paralogous tetraspanins, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling by regulating the trafficking of the type II BMP receptor DAF-4. TSP-14 is the sole member of the TspanC8 subfamily of tetraspanins that in mammals include six proteins. We have noticed that there are two isoforms of TSP-14, TSP-14A, and TSP-14B, with TSP-14B having 24 additional amino acids at its N-terminus compared to TSP-14A. By generating isoform-specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B exhibit distinct subcellular localization patterns and functions. While TSP-14A is localized apically and on early, late, and recycling endosomes, TSP-14B is localized to the basolateral side and on the cell surface. We further identified a di-leucine motif within the N-terminal 24 amino acids of TSP-14B that serves as a basolateral and cell surface targeting sequence for TSP-14B. Using isoform-specific knockout and transgenic rescue approaches, we found that TSP-14A plays a broader and predominant role compared to TSP-14B. Specifically, TSP-14A functions redundantly with TSP-12 to regulate body size, embryonic and vulva development, while TSP-14B functions redundantly with TSP-12 to primarily regulate postembryonic mesoderm development. Moreover, proper postembryonic mesoderm development appears to require the functions of both TSP-14A and TSP-14B, as well as TSP-12. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in an intact living organism. Future work will be directed towards a molecular understanding of how TSP-14A and TSP-14B exert their distinct functions in different subcellular compartments.