Saenz-Narciso, Beatriz et al. (2011) International Worm Meeting "Characterizing the adrenomedullin homologue in C. elegans."

Martinez, Alfredo, Cabello, Juan, Gomez-Orte, Eva, Saenz-Narciso, Beatriz

[International Worm Meeting, 2011]

Adrenomedullin (AM) is a peptide hormone that shows multiple physiological functions in vertebrates, such as bronchodilation, neurotransmission, hormone regulation, antimicrobial activity or growth regulation. Deregulation of AM has been shown in different human pathologies such as diabetes, cancer, hypertension, heart failure, and sepsis. Research on molecules capable of modulating AM and its effects may lead to the discovery of new pharmacological agents to treat these important human diseases. Highly interesting is the role of AM in cancer. It has been proven that AM acts as an autocrine/paracrine tumor cell survival factor and its elevated expression in cancer cells can promote angiogenesis leading to tumor progression. While AM has been well characterized in other research models, little is known about this molecule in C. elegans. We have found and characterized a mutant in the AM homologue of C.elegans, here called wam-1. 4D microscopy analysis of wam-1 mutants shows defects in the migration of the hermaphrodite gonad. There are also several defects in embryonic development. Some embryos have cells that are excluded out of the worm during development while fate specification, as well as proliferation is normal. This suggests a function of wam-1 in cell adhesion. To localize the expression of the worm AM homologue protein, we performed inmunostaining using mouse anti-AM antibodies. The AM protein was localized in the hypodermis. In contrast, we also generated transgenic lines expressing GFP under the control of the wam-1 promoter. GFP expression was detected in the mouth of the worm, although due to its homology with secreted hormones, the protein could play a role far away from the secretory cells. At the moment, we are performing a detailed molecular and genetic characterization, in order to understand the function of this molecule.

Cristina Nieto et al. (2008) European Worm Meeting "Analysis of the adrenomedullin homologue in C.elegans."

Cristina Nieto, Alfredo Martinez, Sergio Moreno, Juan Cabello

[European Worm Meeting, 2008]

In vertebrates, adrenomedullin (AM) is a peptide hormone that shows. multiple physiological functions, such as bronchodilation,. neurotransmission, hormone regulation, antimicrobial activity or growth. regulation. Deregulation of AM has been shown in different human. pathologies such as diabetes, cancer, hypertension, heart failure, and. sepsis.. Research on molecules capable of modulating AM and its effects may lead to. the discovery of new pharmacological agents to treat these important human. diseases. Thus, the injection of a monoclonal antibody against AM produces. an antidiabetic effect, by reducting the glycemic level in diabetic. experimental animals, since AM has been shown to reduce insulin secretion.. Highly interesting is the role of AM in cancer. It has been proven that AM. acts as an autocrine/paracrine tumor cell survival factor and its elevated. expression in cancer cells can promote angiogenesis leading to tumor. progression.. While AM has been well characterized in other research models, little is. known about this molecule in C. elegans. We have found and characterized a. mutant in the AM homologue of C.elegans, here called wam-1. 4D microscopy. analysis of wam-1 mutants shows defects in the migration of the. hermaphrodite gonad. There are also several defects in embryonic. development. Some embryos have cells that are excluded out of the worm. during development while fate specification, as well as proliferation are. normal. This suggests a function of wam-1 in cell adhesion.. To localize the expression of the worm AM homologue protein, we generated. transgenic lines expressing GFP under the control of the wam-1 promoter.. GFP expression is detected in the mouth of the worm although due to its. homology with secreted hormones, the protein could play a role far away. from the secretory cells.. At present, we are performing a detailed molecular and genetic. characterization, in order to understand the function of this molecule.

Waring DA (1996) West Coast Worm Meeting "A C. ELEGANS HOMOLOG OF NEUROD"

Waring DA

[West Coast Worm Meeting, 1996]

The neuroD gene was identified in our lab as a bHLH transcription factor controlling neuronal differentiation in mouse and Xenopus. Extopic expression in Xenopus embryos leads to production of ectopic neurons within the epidermis. It is proposed that neuroD acts downstream of the proneural genes and negative regulators of the Notch/Delta pathway. I am characterizing a C. elegans homolog of neuroD cnd-1. In order understand the function of the gene I am in the process of knocking the gene out. I have identified an insertion of a transposable element within an intron of the gene. This has no phenotype and is apparently spliced out without effecting the gene. I am presently screening for imprecise excision of the transposable element that will knock out cnd-1. In addition I am characterizing larger deficiencies and lethal mutation in the region. I have generated antibodies to cnd-1 and I am beginning to characterize the expression pattern. Neurogenesis in C. elegans is still poorly characterized. Several bHLH proteins related to the Achaete/Scute family, have been found in C. elegans, but as yet there is functional data for only one of these, lin-32 which was identified by mutations lacking a subset of neuroblasts, suggesting that as in other species bHLH proteins may regulate neurogenesis. If cnd-1 functions in C. elegans as its homologs do in vertebrates, the single cell resolution that C. elegans affords should be very useful in understanding its role in neurogenesis, and its relationship to the proneural genes.

Carson CC et al. (1995) International C. elegans Meeting "BIOCHEMICAL AND GENETIC ANALYSIS OF A C. ELEGANS ELAV-LIKE PROTEIN IMPLICATED IN GROWTH AND DIFFERENTATION"

Carson CC, Keene JD

[International C. elegans Meeting, 1995]

ELAV is a Drosophila protein that has been linked genetically to the differentiation of neuroblasts to neurons and is also responsible for the maintenance of terminally differentiated neurons. It has been shown that ELAV like proteins bind to the 3' UTR of selected transcription factors and cytokines. It is hypothesized that the expression of the ELAV family of proteins help take proliferating cells out of the cell cycle and enhance differentiation. I am using both biochemical approaches and genetic analysis to study the mechanism and function of the ELAV family of proteins in C. elegans. Using antibodies to an ELAV like protein, I have detected a protein in C. elegans mixed-stage extracts that migrates at 45 kDa on an SDS-PAGE gel. By screening of lambda zap libraries, I have obtained clones of Cel-1(C. elegans ELAV Like protein 1) that correspond to a putative ORF found by the genomic sequencing project, and appears to correspond to the 45kDa protein detected by immunoblotting. Given the striking homology among the vertebrate and invertebrate forms of the ELAV family within the RRM regions, it is clear that these are important cellular RNA-binding proteins that likely target homologous, key mRNAs regulated in growth and differentiation from worms to humans. I am in the process of expressing the Cel-1 clone in E. coli to determine if the protein is cross reactive to the Hel-N1 antibodies, to perform in vitro RNA binding experiments, make rabbit polyclonal sera, and to utilize random RNA selection. I am also attempting to determine if the Cel-1 protein is developmentally regulated, if there are pre existing mutants, where the protein is localized, and am collaborating with Dr. Plasterk, to isolate Tc1 insertions in the Cel -1 gene for genetic analysis.

Heon S Kim (2004) East Coast Worm Meeting "Roles of PAR-3 and PKC-3 in establishment and maintenance of epithelial cell polarity in C. elegans"

Heon S Kim

[East Coast Worm Meeting, 2004]

Cell polarity is required at many different stages of development, including early embryogenesis and organogenesis. Intercellular junctions are believed to be required to polarize epithelial cells. PAR-3/PKC-3/PAR-6 complex, which serves as one of the earlist polarity cues during the early embryogenesis in C. elegans , has been found to be localized at apical junctions of gut epithelium in C. elegans , Drosophila , and mammals. However, the function of PAR-3/PKC-3/PAR-6 in establishment and maintenance of epithelial cell polarity in C. elegans has not been studied, mainly because the loss of function of this protein complex perturbs early embryogenesis. I am trying to overcome this barrier in two different ways. Heat shock-induced par-3 RNAi, right after early embryogenesis will deplete par-3 mRNA and hopefully will result in par-3 knock-out phenotypes in late embryos or L1~L2 larvae. Alternatively, I am examining the effect of a mutation in pkc-3 on epitheial polarity. I have a pkc-3 mutant, which is newly generated by Vancouver Gene Knockout Lab. This mutant carries a 1.5kb deletion ( ok544 ) that removes kinase domain. Homozygotes undergo normal embryogenesis due to maternal contribution, but they are arrested as L2 larvae. Currently I am trying to optimize the heat shock conditions for par-3 RNAi, and to identify the phenotypes of arrested pkc-3 mutants.

Anne B Allison et al. (2007) International Worm Meeting "A Meiotic Nuclear Envelope Bridge Complex in C. elegans."

Anne B Allison, Abby F Dernburg

[International Worm Meeting, 2007]

Events in meiotic cell division, such as homologous chromosome pairing, synapsis, and recombination, are required for proper chromosome segregation. I am investigating how Pairing Centers, cis-acting sites required for homologous chromosome pairing during meiosis in C. elegans, attach to the nuclear envelope via a Nuclear Envelope Bridge Complex (NEBC). Recently, the Dernburg Lab identified a family of related zinc-finger proteins required for Pairing Center function. These proteins form patches at the nuclear periphery with nuclear envelope proteins, such as ZYG-12 and SUN-1. These two factors a part of the NEBC that bridges the inner and outer nuclear membranes thereby connecting chromosomes with microtubules external to the nucleus. I am identifying additional components of the NEBC through suppressor screens of mutations in zyg-12 and sun-1. This work will address fundamental mechanisms of interaction between chromosomes, the nuclear envelope, and the cytoskeleton to better understand their contribution to normal cell division.

Nicola Tremain et al. (2002) European Worm Meeting "Suppressors of social feeding behaviour"

Mario De Bono, Nicola Tremain

[European Worm Meeting, 2002]

npr-1 encodes a putative G-protein coupled receptor similar to neuropeptide Y receptors. Social or solitary feeding behaviour in wild isolates of C. elegans is associated with a single residue change in this receptor1. Null mutants of npr-1 exhibit strong social feeding behaviour, suggesting that this gene acts to suppress social feeding. I am characterizing and mapping mutations that disrupt social feeding to delineate molecular pathways and neuronal networks that regulate this behaviour.

Johnathan W. Edmonds et al. (2007) International Worm Meeting "RNAi screen for oocyte genes that control directional sperm motility."

Johnathan W. Edmonds, Michael A. Miller

[International Worm Meeting, 2007]

The mechanism(s) by which motile sperm find oocytes in the female reproductive tract is not understood. To investigate this mechanism, we have developed an in vivo assay for tracking the movement of sperm in the uterus of C. elegans hermaphrodites. Previous results support the hypothesis that oocytes generate sperm-recruiting signals derived from polyunsaturated fatty acids (PUFAs). These signals direct sperm migration to the spermatheca, the site of fertilization. In mammals, PUFAs are synthesized into eicosanoids, which direct T-cell migration to sites of inflammation. I am using RNA-mediated interference (RNAi) to identify genes that function in oocytes to control directional sperm motility. Genome-wide DNA microarray (Reinke et al. 2004) and in situ hybridization (Kohara 2001) databases were screened for genes expressed in oocytes. I have identified eight genes that are required in hermaphrodites for the directional movement of wild-type sperm. Several of these genes are related to human genes implicated in eicosanoid synthesis and transport. I am currently focusing on the genes T28F3.1 and C01F6.1, which encode proteins called copines. Copines are conserved calcium-dependent membrane binding proteins with unknown functions. They are characterized by having two Ca(2+)-binding C2-domains at the N-terminal region and an A-domain at the C-terminal region. We hypothesize that C01F6.1 and T28F3.1 function in oocytes to target PUFA-modifying enzymes to the plasma membrane where they can generate sperm-recruiting signals. I am currently conducting phenotypic analysis of loss of function mutations in C01F6.1 and T28F3.1. Results from this analysis and site of action studies will be presented.

Jin Li et al. (2003) International Worm Meeting "A Search for Proteins Complexed with PAR-1 in vivo"

Jin Li, Kenneth J Kemphues

[International Worm Meeting, 2003]

In C. elegans, asymmetric divisions are a prominent feature of early embryogenesis. The par genes (partitioning defective) are a class of maternal genes that are required for establishment of embryonic polarity. Among them, PAR-1 is dependent upon all the other par genes for its posterior localization, and loss of par-1 activity severely affects the cytoplasmic localization of polarity determinants. PAR-1 is a highly conserved Serine/Threonine kinase. Some substrates of PAR-1 homologs have been identified in other organisms. Two mammalian homologs phosphorylate microtubule associated proteins. In Drosophila, dPAR-1 can phosphorylate Oskar, Dsh, 14-3-3 binding proteins and LKB1. However, the substrate of PAR-1 in C. elegans for embryonic polarity remains unknown. In order to identify the substrate and other interacting proteins of PAR-1, I am doing co-immunoprecipitation followed by MALDI-TOF. I am also trying to purify 6His tagged PAR-1 protein. I will use this purified PAR-1 protein to test whether it can phosphorylate several substrate candidates in vitro.

Johnathan Edmonds et al. (2008) Development & Evolution Meeting "Prostaglandins in Sperm Chemotaxis"

Michael Miller, Johnathan Edmonds

[Development & Evolution Meeting, 2008]

The eicosanoids, which include prostaglandins, leukotrienes and thromboxanes, are signaling molecules derived from polyunsaturated fatty acids or PUFAs. In mammals, eicosanoids are important mediators of inflammation, immunity, reproduction and central nervous system function. However, the roles of eicosanoids at the cellular level and the molecular mechanisms that control these actions are not well understood. We have shown that C. elegans oocytes generate sperm-attracting signals derived from PUFAs. I conducted an RNAi screen to identify genes that function outside of sperm to regulate their motility. These genes are candidates for mediating the synthesis or secretion of the sperm attractant. I screened approximately forty genes with oocyte-enriched transcription profiles and predicted functions in lipid metabolism and transport. The screen resulted in the identification of five genes encoding proteins implicated in prostaglandin metabolism. I am focusing on the glutathione S-transferase K08F4.7, which has significant sequence similarity to human prostaglandin D synthase. Genetic analysis using a method to track sperm motility in vivo confirms that K08F4.7 is required for sperm guidance. Wild-type sperm in K08F4.7 null mutants move with reduced velocity and severely reduced directional velocity. These results support the hypothesis that prostaglandin D2 or a structurally similar prostaglandin functions as a sperm attractant. Consistent with this hypothesis, the microinjection of human prostaglandin D2 into the uterus of PUFA-deficient hermaphrodites stimulates sperm motility. Currently, we are collaborating with the UAB mass spectrometry facility to directly test whether oocytes synthesize prostaglandins. I am also testing the site of K08F4.7 action and whether recombinant K08F4.7 can function as a prostaglandin D synthase in vitro. In addition to K08F4.7, I am characterizing two other genes identified in the RNAi screen, the copines C01F6.1 and T28F3.1. Copines are evolutionarily conserved proteins with predicted lipid binding domains, but largely unknown functions. Loss of function mutations in C01F6.1 and T28F3.1 do not cause sperm motility defects as severe as those observed with RNAi. The C. elegans genome encodes 5 copine family members and I am currently testing whether these genes exhibit functional redundancy. Triple- and quadruple-mutant strains are being constructed for further phenotypic analysis.