Ruf, Vanessa et al. (2013) International Worm Meeting "TORC2 Signaling Antagonizes SKN-1 to Induce C. elegans Mesendodermal Embryonic Development."

Holzem, Christina, Ruf, Vanessa, Walz, Gerd, Peyman, Tobias, Neumann-Haefelin, Elke, Blackwell, T. Keith

[International Worm Meeting, 2013]

The evolutionarily conserved target-of-rapamycin (TOR) kinase controls fundamental metabolic processes to support cell growth. TOR functions within the context of two distinct complexes, TORC1 and TORC2. The TORC2 with its specific component Rictor has been recently implicated in aging and regulation of growth and metabolism. Here, we identify rict-1/Rictor as a new regulator of embryonic development in C. elegans. The transcription factor skn-1 is essential to establish the development of the mesendoderm in embryos and is required for cellular homeostasis and longevity in adult worms. Loss of skn-1 function in the embryo leads to mis-specification of the mesendodermal precursor consequently lacking intestine and pharynx. We found that genetic inactivation of rict-1 re-stored mesendodermal specification in skn-1 deficient embryos and thereby suppressed skn-1-associated lethality. Moreover, inactivation of the TORC2 components but not TORC1 partially rescued skn-1 embryonic lethality. SGK-1 mediated these functions downstream of rict-1/TORC2 as a sgk-1 gain of function mutant suppressed the rict-1 mutant phenotype. These data indicate that TORC2 and SGK-1 antagonize SKN-1 during embryonic development.

Paolo Moroni et al. (2006) European Worm Meeting "Characterization of C.elegans NuRD Complexes"

Karin Brunschwig, Fritz Muller, Myriam Passannante, Vanessa Cerantola, Paolo Moroni, Anne-Laure Chanez

[European Worm Meeting, 2006]

Paolo Moroni, Myriam Passannante, Karin Brunschwig, Anne-Laure Chanez, Vanessa Cerantola and Fritz Mller. The NuRD (nucleosome remodeling and histone deacetylase) complex is thought to be involved in the establishment of repressive chromatin structures during development and in the negative control of gene expression. In vertebrates, this complex comprises at least seven polypeptides, including the SWI2/SNF2 helicase/ATPase Mi-2, the histone deacetylases HDAC1/2, the histone-binding proteins RbAp46/48, the metastasis-associated proteins MTA1/2, the methyl-CpG binding protein MBD3 and the potent transcriptional repressor p66. Orthologues of these NuRD subunits are also encoded by the genome of C.elegans, among them the two Mi-2 homologues LET-418 and CHD-3.. Mutations in let-418 show a pleiotropic phenotype, including sterility and larval arrest (von Zelewsky and al., 2000), whereas chd-3 mutants exhibit no obvious defects. However, a null mutation of chd-3 enhances the let-418 phenotype. This suggests that the two proteins have partially redundant functions during development (von Zelewsky et al., 2000).. To find proteins interacting with LET-418 and CHD-3 and to characterize putative NuRD complexes in C.elegans, we take different approaches. We use standard biochemical techniques, such as co-immunoprecipitation, tandem affinity purification (TAP) and yeast two-hybrid screens.. Our preliminary data suggest that LET-418 and CHD-3 may not be interchangeable members of the same complex, but rather are part of different NuRD or NuRD-like complexes.. von Zelewsky and al., Development 127: 5277-5284 (2000)

Ma C et al. (2000) East Coast Worm Meeting "Candidate genes that govern motorneuron synaptic specificity"

Chalfie M, Ma C

[East Coast Worm Meeting, 2000]

The unc-4 gene of C. elegans encodes a homeodomain protein, which when mutated changes the synaptic inputs to a single type of neuron, the VA motor neurons. We have identified several genes whose expression decrease (und-1-4, kin-8) or increase (ruf-1) in unc-4 mutants. These genes encode several novel secreted and membrane-bound proteins and a receptor tyrosine kinase similar to those implicated in the agrin-induced formation of the neuromuscular junction in vertebrates. We have examined the expression patterns of these genes and showed that at least three of these genes are expressed in the ventral cord motorneurons and the levels of expression decreased in both the unc-4(e120) and unc-37(e262) backgrounds. We also examined the synapses formed by the interneurons(AVA, AVD, AVE) and the A-type motorneurons in the ventral cord using a vamp::gfp construct (provided by M. Nonet) driven by a sek-1 promoter (the sek-1 gene is expressed in AVA,AVD,AVE , VCs and some other tissues, M. Tanaka et al. WBG 15(4):30). The sek-1::vamp::gfp is expressed in a similar pattern as reported by Tanaka et al. and punctated staining can be seen along the ventral cord. In the unc-4(e120) and unc-37(e262) backgrounds, the ventral cord staining become weak and diffuse, indicating loss of synapse formation. Similar results were obtained when kin-8 RNAi was injected into wild-type animals carrying the sek-1::vamp::gfp construct. Initial injection of RNAi for und-1, und-2 and und-3 did not yield mutant progeny. Therefore, we made hairpin constructs of these genes under the control of the heat shock promoter. Such hairpin constructs have been shown by Monica Driscolls lab to be effective in inducing the RNAi effect especially in neurons (Tavernarakis, N. et al. Nat Genet. 24(20):180-183). We co-injected these hairpin constructs with the sek-1::vamp::gfp construct using lin-15 as the selectable marker. In the stable lines generated after injection, we could induce a high percentage of mutant uncoordinated progeny (35-40%) by heat -shocking parent L4 animals. Furthermore, the expression of the sek-1::vamp::gfp construct was decreased and became diffuse similar to the kin-8 RNAi case. We are currently testing the let-858 promoter-driving hairpin constructs to see whether we can induce a similar phenotype.