Cai, Tao et al. (2021) International Worm Meeting "FRKsrc-2 is a Novel Candidate as a Hemifacial Microsomia and Mandibular Dysplasia Gene that Exhibits Developmental Defects in Zebrafish (D. rerio) and C. elegans"

Green, Rebecca, Oegema, Karen, Fan, Chunxin, Yan, Jizhou, Cai, Tao, Golden, Andy, Bai, Xiaofei, Wang, Xi

[International Worm Meeting, 2021]

Hemifacial microsomia (HFM), also known as lateral facial dysplasia, is a common birth defect involving the first and second branchial arch derivatives. Several chromosomal abnormalities (like trisomy 10p and OTX2 duplication) and gene variants (like PLCD3 and MYT1) were identified in a few cases with HFM; however, the genetic etiologies in a majority of cases with HFM remain unknown. Using whole-exome sequencing, we identified a homozygous missense mutation (c.484G>A; p.V162I) in a SH2 domain) of the FRK gene in an affected individual with HFM. FRK encodes the Fyn-related Src family Tyrosine Kinase and is highly expressed in the Meckel's cartilage during embryonic development in zebrafish and mouse. Knockdown of FRK in zebrafish with the fynrk-specific morpholinos showed several defects, including a shorter ratio of length and width of Meckel's cartilage, a larger angle of ceratohyal, disorganized ceratobranchial, and smaller otoliths. Using CRISPR/Cas9, we generated the patient-specific allele (p.V162I) in the C. elegans ortholog gene src-2, which is named src-2(V170I). The homozygous src-2(V170I) mutant as well as a null mutant src-2(ok819) were found to have ~ 25% reduction in the brood size when compared with wildtype (P < 0.0001). Given that src-2 has a paralogous gene src-1, which is an essential kinase during embryogenesis, we tested the genetic interactions between the src-2 mutants and src-1with RNAi interference (RNAi). Indeed, the embryonic lethality and the defective morphogenesis in pharyngeal and intestinal tissues in the src-1 RNAi treated worms were synergistically enhanced in either src-2(V170I) or src-2(ok819) (P£0.0001). Using a 4-D high-content imaging approach, irregular neuronal and hypodermal patterning as well as protrusive rupture phenotypes were observed in the src-1(RNAi) arrested embryos. These defective embryonic phenotypes were increased by 23%, 23%, and 30%, respectively, when the src-2(V170I) mutants were combined with the src-1(RNAi) (P £ 0.0296 by one tailed chi-square test), suggesting a synergistically interaction between these paralogous genes. Taken together, we identified the Fyn-related Src family Tyrosine Kinase as a novel candidate gene for human hemifacial microsomia or lateral facial dysplasia and revealed its effects on brood size and craniofacial development in the models of zebrafish and C. elegans.

Tao Cai et al. (2003) International Worm Meeting "IA-2, a Vesicle Membrane-Associated Protein, Regulates Synaptic Transmission in C. elegans"

Michael Krause, Abner L Notkins, Tao Cai, Tetsunari Fukushige

[International Worm Meeting, 2003]

IA-2, a major autoantigen in type 1 diabetes, is a receptor-tyrosine phosphatase-like protein associated with the membrane of secretory granules of neural and endocrine-specific cells. IA-2 lacks phosphatase activity due to an amino acid substitution within the catalytic domain and the function of IA-2 remains unclear. In order to gain insight into the cellular functions of IA-2, we have studied the C. elegans homolog, CeIA-2 encoded by the ida-1 gene. Using two independent, putative null, deletion alleles of ida-1 we show that animals lacking CeIA-2 are viable with only subtle visible phenotypes. Pharmacological studies show loss of CeIA-2 results in presynaptic neuronal defects involving neurotransmitter release. Furthermore, ida-1 interacts genetically with unc-31 and unc-64, two genes encoding factors required for neuronal vesicle function. These results suggest that CeIA-2 is a novel regulator of synaptic transmission that regulates vesicle release.

Tao Cai et al. (2008) C.elegans Neuronal Development Meeting "Loss of the Transcriptional Repressor PAG-3 Results in Enhanced Neurosecretion that is Dependent on the Dense-Core Vesicle Membrane Protein IA-2"

Ping Yu, Guofeng Zhang, Tao Cai, Michael Krause, Hiroki Hirai, Tetsunari Fukushige, Abner Notkins

[C.elegans Neuronal Development Meeting, 2008]

It is generally accepted that neuroendocrine cells regulate dense core vesicle (DCV) biogenesis and cargo packaging in response to secretory demands, although the molecular mechanisms of this process are poorly understood. One factor that has previously been implicated in DCV regulation is IA-2, a catalytically inactive protein phosphatase present in DCV membranes. Our ability to visualize a functional GFP-tagged version of IA-2 directly in live C. elegans animals has allowed us to capitalize on the genetics of the system to screen for mutations that disrupt DCV regulation. We found that loss of activity in the transcription factor PAG-3, that functions as a repressor in many systems, results in a dramatic up-regulation of IA-2 and other DCV proteins. The up-regulation of DCV components was accompanied by an increase in presynaptic DCV numbers and resulted in phenotypes consistent with increased neuroendocrine secretion. Double mutant combinations revealed that these PAG-3 mutant phenotypes were dependent on wild type IA-2 function. Our results support a model in which IA-2 is a critical element in DCV regulation and reveal a novel genetic link to PAG-3-mediated transcriptional regulation.

Werner, Kristen et al. (2013) International Worm Meeting "Small Molecule Communication: C. elegans and bacterial chemical signals."

Perez, Lark, Werner, Kristen, Semmelhack, Martin, Bassler, Bonnie

[International Worm Meeting, 2013]

Bacterial group behaviors are governed by a process called quorum sensing, in which bacteria produce, secrete, and detect extracellular signal molecules called autoinducers (AIs). Vibrios produce multiple AIs, some enable intra-species communication and others that promote inter-species communication. Vibrio cholerae produces an intra-species AI called CAI-1 that is a 13 carbon long fatty acyl molecule and the interspecies signal called AI-2 that is a boron-containing furanone. The information contained in the AIs is funneled into a shared phosphorelay signaling cascade that controls virulence, biofilm formation, and other traits. The bacteriovorous nematode, Caenorhabditis elegans, also uses small molecules to interpret its environment. A class of C. elegans-derived molecules called ascarosides influence nematode behaviors including attraction, repulsion, and mating. The presence of bacteria stimulates chemotaxis, egg-laying, and feeding in C. elegans, however, the bacteria-produced molecules that the nematode detects to control these phenotypes are largely unknown. We demonstrate that in addition to playing a vital role in quorum-sensing-regulated behaviors in V. cholerae, CAI-1 also influences behavior in C. elegans. C. elegans is more strongly attracted to V. cholerae than to its food source E. coli HB101 and C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant for CAI-1 production. Consistent with this finding, robust chemoattraction occurs to synthetic CAI-1. CAI-1 is detected by the sensory neuron AWCON. Laser ablation of this cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. To define which moieties of CAI-1 are crucial for recognition by C. elegans, we synthesized CAI-1 analogs and tested whether they promote chemoattraction. The fatty-acid chain length as and the precise position of the CAI-1 ketone group are the key features required for mediating CAI-1-directed nematode behavior. Together, these analyses define a bacteria-produced signal and the nematode detection apparatus that permit interkingdom communication.

Arun Samidurai et al. (2008) Development & Evolution Meeting "Identification and Characterisation of a Human PDE3 Homolog in C.Elegans"

Cai Tao, Arun Samidurai, Vincent Manganiello, Faiyaz Khan

[Development & Evolution Meeting, 2008]

Cyclic Nucleotide Phosphodiesterases (PDEs) regulate intracellular concentrations of the second messengers, cAMP and cGMP, by controlling their rate of hydrolysis.Of the 11 PDE gene families, the mammalian PDE3 family is known to play an important role in insulin signaling pathways, and in cardiovascular tissues, oocytes and adipose tissues. Caenorhabditis elegans represents a unique model for genetic manipulation, as well as for identification and functional analysis of genes that regulate signal transduction. The insulin-signaling pathway is well characterized in C.elegans, and is thought to be functionally similar to the mammalian cascade. We report here the identification and characterization of the C.elegans Phosphodiesterase3 gene family, a homolog of the mammalian PDE3 family. In contrast to the mammalian PDE3 family, which consists of two closely related subfamilies, PDE3A and PDE3B, that are generated from separate genes located on human chromosomes 12 and 11, respectively, the single nematode PDE3 gene is present on chromosome II, spaning about 20.2kb, and encodes two different PDE3 isoforms. The CEPDE3 long form consists of 11 exons and codes for a 63.5 kda protein, and the short form has 8 exons and codes for a 51.9 kda protein. Both isoforms have Pfam and phosphodiester domains and also contain the HD metal binding motif, which is characteristic of all PDE gene families. The CEPDE3 long form and short form show an overall 97 % homology between each other and 100 % homology among their catalytic domains, similar to the ~80 % similarity found in the catalytic regions of mammalian PDE3A and 3B isoforms. We also compared the unique signature insert present in the mammalian PDE3 catalytic domain and found 11 out of 44 amino acids matched with CEPDE3. We have cloned both long and short CEPDE3 isoforms, and expressed them in Sf21 insect cells. Although recombinant mouse PDE3B and the C.elegans long and short forms are all inhibited by cilostamide (a specific inhibitor of mammalian PDE3 isoforms), C.elegans PDE3 is also inhibited by rolipram (a specific inhibitor of mammalian PDE4 isoforms). The molecular mechanisms underlying catalysis by PDE3 and its inhibitor selectivity still remain unsolved. Further characterization of CEPDE3 may enhance our understanding of these properties of PDE3 enzymes, as well as their roles in cellular physiology.

Redemann, Stefanie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Codon adaptation-based control of protein expression levels in C. elegans and its application to GPR-1 allows control of microtubule-based pulling force"

Redemann, Stefanie, Ernst, Susanne, Ayloo, Swathi, Bringmann, Henrik, Schloissnig, Siegfried, Pozniakowski, Andrej, Hyman, Anthony A

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The variation of the expression level of a protein could provide a powerful tool to study protein function. However, there is no method that allows the precise control of protein levels under a native promotor in eukaryotes. We developed a method, which enables us to fine tune the protein expression levels in C. elegans by using synthetic genes with adapted codons. By modifying the codon usage of a gene, the Codon adaptation index (CAI) can be changed and the level of protein expression can be controlled. We used this method to regulate the expression of the G-protein regulator GPR-1/2, which is involved in force generation during spindle positioning in the first asymmetric cell division in C. elegans. By gradually increasing the amount of GPR-1/2, we found that the amount of force acting on the spindle in C. elegans embryos is directly related to the amount of the G protein regulator GPR1/2 in the cell. In C. elegans GPR-1/2 is found in a complex, the force-generating complex,which is thought to consist of at least three proteins: GPR-1/2, LIN-5 and a G-alpha protein. Since increasing the amount of GPR1/2 is sufficient to increase the force, this suggests that the other proteins are there in excess and that GPR-1/2 is the limiting component. The modification of the CAI is a good example of how the ability to over-express proteins is essential for identifying components that are limiting as opposed to permissive for force generation. This method provides the first way to control the level of protein expression levels in C. elegans, and the first method for overexpression of proteins in the C. elegans germline. With this method the protein levels of a protein of interest can be varied, while maintaining all the wild type genetic regulation and the wild type protein sequence.

Naoki Hisamoto et al. (2002) Japanese Worm Meeting "Survey of components of JNK/p38 MAP kinase cascades in C. elegans"

Naoki Hisamoto, Kunihiro Matsumoto, Kenji Nishide

[Japanese Worm Meeting, 2002]

The c-Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK) pathways are involved in various stress responses and apoptosis. In mammal, JNK is activated by MKK7 and MKK4 MAPK kinases (MAPKKs) and p38 is activated by MKK3, MKK4 and MKK6 MAPKKs. These members of the MAPKK superfamily are activated by members of MAPKKK superfamily such as MEKK, TAK1, MLK, TAO and ASK1. Although these pathways have been implicated in the response to stresses and inflammation, the physiological roles of the JNK and p38 pathways in the normal development and function of the organism is less well understood. In C. elegans, several mutants corresponding to the components of JNK/p38 cascades have been isolated: jnk-1 (JNK homolog), jkk-1 (MKK7 homolog), sek-1 (MAPKK), mek-1 (MAPKK), mom-4 (TAK1 homolog) and nsy-1 (ASK1 homolog). The C.elegans genome project shows that there are at least seven members of the MAPKKK superfamily, five members of the MAPKK superfamily and seven members of the MAPK superfamily in C.elegans. To investigate the roles of these components and the relationships among them, we isolated several deletion mutants of these genes. Analysis of these mutants will be discussed.

Spiga, Fabio et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The TAO kinase KIN-18 regulates contractility and establishment of polarity in the embryo."

Spiga, Fabio, Gotta, Monica, Prouteau, Manoel

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Establishment and maintenance of cell polarity are fundamental processes for many aspects of cell and developmental biology, including asymmetric cell division. In the C. elegans embryo the first axis of polarity, the anterior-posterior axis, is determined by the sperm entry site. Shortly after meiosis, the cortex is uniformly contractile and the conserved PAR-3/PAR-6/PKC-3 complex (PAR-3 complex) is localised all around the cortex. Entry of the sperm triggers posterior smoothening and anterior-directed cortical acto-myosin flows, which are regulated by the small G protein RHO-1. Asymmetric contractions lead to the formation of an anterior domain containing the PAR-3 complex, while PAR-2 occupies the posterior cortex. Although contractility is necessary for the localization of PAR proteins, anterior P AR proteins exert a feedback regulation on contractility. Despite the central role of cortical contractility and of the PAR-3 complex in cell polarity, little is known on how they regulate each other. Here we describe the role of KIN-18, a member of the TAO/Ste20-like kinase family, in the regulation of cortical contractions and cell polarisation. kin-18(RNAi) embryos exhibit increased and prolonged cortical contractions, producing a more persistent pseudocleavage. This hyper-contractility results in a defect in the position of the boundary between the anterior and posterior PAR domains during polarity establishment. We find that KIN-18 binds to the small G protein RHO-1 and acts by modulating its activity in a PAR-3 regulated fashion. Our data reveal that KIN-18 provides a novel link between the cytoskeleton remodelling and the PAR polarity machineries and show that KIN-18 is essential to precisely regulate the establishment of cell polarity.

Lao, R.X. et al. (2019) International Worm Meeting "Understanding Argonaute/Small RNA-based intercellular communication."

Buck, A.H., Claycomb, J.M., Wadi, L., Lao, R.X., Seroussi, U., Maity, T., Blaxter, M., Abreu-Goodger, C.

[International Worm Meeting, 2019]

Cells have many ways to regulate gene expression, one of which is by using small RNAs (sRNAs) and sRNA pathway effectors, called Argonautes (AGOs). Studies in plants and worms point to sRNA mediated communication between cells in a single organism (e.g., systemic RNAi; Winston et al., Science 2002). Perhaps even more exciting is the prospect that sRNA/AGO complexes could be used as a means of communication between organisms (Buck et al., Nat Comm. 2014, Cai et al., Science 2018). The discovery that sRNAs exist extracellularly in conjunction with AGOs is particularly important for host/parasite or pathogen relationships. For example, a parasitic nematode of mouse, H. polygyrus bakeri (Hb), secretes extracellular vesicles, which possess sRNAs and an extracellular AGO protein, exWAGO (extracellular Worm AGO). However, Hb is not genetically tractable, hence we have turned to C. elegans to study the functions of exWAGO. Although exWAGO is not present in the C. elegans genome, three homologs, SAGO-1, SAGO-2, and PPW-1 exist. We have engineered strains of C. elegans expressing exWAGO in various tissues to characterize its molecular function in sRNA pathways. In parallel, we engineered GFP-tagged strains of SAGO-1, -2, and PPW-1 using CRISPR-mediated gene editing. These three C. elegans AGOs share a common localization pattern to the apical gut membrane, where exWAGO is expressed in Hb. To further understand the roles of these intestinal secondary AGOs (iSAGOs), we immunoprecipitated the iSAGOs and performed sRNA sequencing and mass spectrometry analysis to identify sRNA and protein binding partners. Top hits from the IP/MS analysis included trafficking, secretory pathway, and membrane anchoring proteins, highlighting the potential for the iSAGOs to be secreted and act intercellularly in C. elegans. We are now in the midst of identifying the pathways that contribute to apical membrane anchoring of iSAGOs and determining what role the iSAGOs may play in systemic RNAi in C. elegans.