Lynch AS et al. (1995) International C. elegans Meeting "DEVELOPMENTAL EXPRESSION PATTERNS OF GENES PREDICTED BY THE C.ELEGANS SEQUENCING PROJECT."

Briggs DA, Lynch AS, Hope IA

[International C. elegans Meeting, 1995]

Expression pattern screens in the past have been used both to provide cellular markers of differentiation and to identify novel genes of possible importance during development. Previous approaches have been random in nature, such that genes identified usually require further characterisation before being used as tools to probe mechanisms of development.

Lynch AS et al. (1993) International C. elegans Meeting "A screen to identify patterns of expression of genes predicted by the C. elegans sequencing project."

Lynch AS, Hope IA

[International C. elegans Meeting, 1993]

Lynch AS et al. (1997) International C. elegans Meeting "VAB-2 ENCODES AN EPH RECEPTOR TYROSINE KINASE LIGAND THAT FUNCTIONS IN C. ELEGANS EPIDERMAL MORPHOGENESIS."

Chisholm AD, Lynch AS

[International C. elegans Meeting, 1997]

A key step in morphogenesis of the C. elegans epidermis is ventral closure. vab-1 and vab-2 mutants display similar defects in epidermal ventral closure and morphogenesis. vab-1 encodes a receptor tyrosine kinase (RTK) of the EPH family (see abstract by George and Chisholm). vab-2 is defined by six alleles which cause morphogenesis defects similar to those caused by hypomorphic vab-1 alleles. We have found that vab-2 encodes a member of the family of ligands for EPH RTKs, and that the molecular changes associated with different alleles of vab-2 are consistent with partial or complete loss of gene function. Vertebrate EPH ligands are either transmembrane proteins or membrane-anchored via a glycosylphosphatidylinositol (GPI) motif. At present, we are unsure whether VAB-2 is transmembrane or membrane-anchored; however the VAB-2 predicted receptor binding domain is 40% identical to those of transmembrane EPH ligands, and ~30% identical to GPI-anchored ligands. EPH RTKs and their ligands function in vertebrates to control the growth of axons, and are also implicated in morphogenetic pathways. Given the similarity of the vab-1 and vab-2 phenotypes, VAB-1 and VAB-2 might interact as receptor and ligand in a signalling pathway involved in the control of epidermal morphogenesis.

Allison Lynch et al. (2008) Development & Evolution Meeting "MAGI-1 interacts with the C. elegans cadherin-catenin complex during morphogenesis"

Allison Lynch, Jeff Hardin

[Development & Evolution Meeting, 2008]

In multicellular organisms, cell-cell junctions are involved in all aspects of tissue morphogenesis, including regulation of cell shape, movement, and sorting. To establish junctions, adhesion molecules must be stabilized by cytoplasmic proteins that interact with the actin cytoskeleton. For example, intracellular catenins connect cadherins to the cytoskeleton and mutations in catenin proteins significantly impact morphogenesis. A previously isolated hypomorphic hmp-1/alpha-catenin allele, fe4, exhibits ~70% lethality and body shape defects due to a perturbed actin cytoskeleton. Worms homozygous for the fe4 mutation provided a sensitized genetic background that enabled the isolation and characterization of molecules that contribute to the function of the cadherin-catenin complex (Pettitt, et al., 2003, J. Cell Biol. 162, 15-22). In continuing efforts to identify other regulators, we employed the same rationale and conducted a genome-wide RNAi screen to identify genes whose loss of function enhances the fe4 phenotype. MAGI-1, a vertebrate tight junction protein that interacts with actin binding proteins in tissue culture, was identified as an enhancer. MAGI-1 is a MAGUK protein that is highly conserved across species. It has been previously shown to interact with HMP-2/beta-catenin (Stetak, et al. European Worm Meeting Abstract. 2006) and vertebrate actin-binding proteins (Patrie, et al., 2002, J. Biol Chem. 277, 30183-90). We have shown MAGI-1 localizes to adherens junctions in various epithelial tissues and that it genetically interacts with hmp-1 and hmp-2. All hmp-1(fe4);magi-1(RNAi) and hmp-2(qm39);magi-1(RNAi) embryos arrest with a characteristic humpback phenotype, suggesting MAGI-1 may function with the cadherin-catenin complex during morphogenesis. We are currently characterizing how MAGI-1 localizes in vivo and its function at cell junctions. We are also examining how MAGI-1 modulates actin dynamics and we are examining other potential MAGI-1 interacting proteins to further analyze how MAGI-1 functions in a living organism.

Lynch, Tina R. et al. (2015) International Worm Meeting "Visualization of regulated transcription using single-molecule FISH."

Lynch, Tina R., Lee, ChangHwan, Kimble, Judith

[International Worm Meeting, 2015]

Transcriptional regulation controls many aspects of germ cell development, including maintenance of stem cells and sex determination. Our lab is particularly interested in Notch signaling and its transcriptional regulation of downstream genes required for germline stem cell (GSC) maintenance. We recently identified two genes, lst-1 and sygl-1, that respond directly to Notch signaling and are essential for GSC maintenance, albeit redundantly (Kershner et al., 2014). Transcriptional reporters are widely used to visualize the readout of signaling. However, here we use single-molecule RNA Fluorescent In Situ Hybridization (smFISH) as a more direct readout of transcriptional regulation and compare endogenous transcription to more common transcriptional reporters. Specifically, we visualized transcripts from the endogenous sygl-1 gene and also from transcriptional reporter transgenes, Psygl-1::SYGL-1::GFP and Psygl-1::H2B::GFP. The sygl-1 transcripts were detected using both sygl-1 intron- and exon-specific probe sets whereas the reporter transcripts were seen using a gfp exon-specific probe set. Each probe set consisted of more than 30 20nt-long probes designed against the sequence of interest, and all probes within a probe set were labeled with the same fluorophore. Using these probes we saw many relatively dim cytoplasmic spots and a few bright nuclear spots in the distal germline. The cytoplasmic spots disappeared from N2 and Psygl-1::SYGL-1::GFP worms after treatment with sygl-1 siRNA, as expected for mature mRNAs. By contrast, the bright nuclear foci disappeared after alpha-amanitin treatment, as expected for primary transcripts. Importantly, the nuclear foci disappear in a glp-1(ts) mutant soon after shifting to restrictive temperature. We are currently quantifying both primary and cytoplasmic transcripts as a function of distance from the distal end for both endogenous and reporter transcripts. We propose that the smFISH method to visualize both primary and cytoplasmic Notch-regulated transcripts will provide new insights into the dynamics of the Notch signaling response and provide a glimpse of how transgenes are expressed compared to endogenous genes.Kershner, A.M., Shin, H., Hansen, T.J., and Kimble, J. (2014). Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance. Proc. Natl. Acad. Sci. U. S. A. 111, 3739-3744.

Allison Lynch et al. (2009) International Worm Meeting "MAGI-1 modulates the function of the C. elegans cadherin-catenin complex during morphogenesis."

Allison Lynch, Jeff Hardin

[International Worm Meeting, 2009]

In multicellular organisms, cell-cell junctions are involved in all aspects of tissue morphogenesis, including regulation of cell shape, movement, and sorting. To establish junctions, cytoplasmic proteins that interact with the actin cytoskeleton must stabilize adhesion molecules. For example, intracellular catenins connect cadherins to the cytoskeleton and mutations in catenin proteins significantly impact morphogenesis. A previously isolated hypomorphic allele of the C. elegans alpha-catenin homolog, hmp-1(fe4), exhibits ~70% lethality and body shape defects due to a perturbed actin cytoskeleton (Pettitt, et al., 2003, J. Cell Biol. 162, 15-22). In continuing efforts to identify molecules that contribute to the function of the cadherin-catenin complex during morphogenesis, we conducted a genome-wide RNAi screen to identify genes whose loss of function enhances the hmp-1(fe4) phenotype and uncovered MAGI-1. MAGI-1 is a MAGUK protein that is highly conserved across species. Vertebrate MAGI-1 is found at tight junctions where it interacts with actin-binding proteins (Patrie, et al., 2002, J. Biol Chem. 277, 30183-90). We have shown C. elegans MAGI-1 genetically interacts with hmp-1/alpha-catenin and hmp-2/beta-catenin and all magi-1(RNAi);hmp-1(fe4) and magi-1(RNAi);hmp-2(qm39) embryos arrest with a characteristic humpback phenotype, suggesting MAGI-1 may modulate cadherin-catenin function during morphogenesis. Surprisingly, we have shown MAGI-1 localizes at the C. elegans apical junction, but basal to the cadherin-catenin complex. In addition, MAGI-1 junction localization is not dependent on beta-catenin, in contrast to what has been shown in tissue culture (Dobrosotskaya, et al., 2000, Biochem Biophys Res Commun. 270, 903-903). We are currently examining whether MAGI-1 modulates actin dynamics at junctions and we are trying to identify and characterize proteins that interact with MAGI-1 to further assess how MAGI-1 functions in a living organism. 1178B The role of mitochondrial diameter in outer membrane division. Brian Head and Alex van der Bliek. Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA. Mitochondria divide and fuse in response to a variety of cellular and mitochondrial-"intrinsic" signaling events. Current research has elucidated proteins essential for two key steps in mitochondrial division -- mechanical severing of the outer membrane (drp-1, fis-1, mff-1) and regulation of protein recruitment (phosphorylation, nitrosylation, sumolyation) to the surface of mitochondria. It is certain that other processes contribute to ensure proper and efficient division of mitochondria. Using both reverse and molecular genetic analyses, we have identified several mitochondrially-localized proteins that control mitochondrial diameter. These proteins, MOMA-1 (K02F3.10), CHD-1 (M176.3), and MFL-1 (T14G11.3), reside in the mitochondrial outer membrane, intermembrane space, and inner membrane, respectively. Loss of function alleles and RNAi constructs targeting these genes cause abnormal swelling of mitochondria in multiple cell types. These swollen mitochondria are functional in many aspects of normal metabolism and are distributed appropriately in both mitosis and meiosis. However, in situations where meiotic division is sensitized, swollen mitochondria aggregate in the gonad; this ultimately leads to a lethal sterile defect. Additionally, we performed an RNAi screen to identify mitochondrial resident proteins that genetically interact with moma-1. This led to the identification of a subset of the mitochondrial protein import machinery as specifically sensitive to moma-1 gene deletion. In the future we hope to determine the manner in which mitochondrial diameter influences the known division machinery.

Allison M Lynch et al. (2007) International Worm Meeting "A Feeding RNAi Screen Reveals Putative Interactors of the C. elegans Cadherin-Catenin Complex."

Allison M Lynch, Jeff Hardin, Christina Tuskey

[International Worm Meeting, 2007]

Morphogenesis is a fundamental process to the development of an organism that requires cells to be able to dynamically alter cell shapes and positions. The cadherin-catenin complex in worms, consisting of HMR-1/cadherin, HMP-1/alpha-catenin, and HMP-2/beta-catenin, was discovered through its role in regulating the morphogenesis of the hypodermis during ventral enclosure. A previously isolated hypomorphic hmp-1 allele, fe4, exhibits ~70% lethality and body shape defects due to a perturbed actin cytoskeleton. Worms homozygous for the fe4 mutation provided a sensitized genetic background that allowed for the isolation and characterization of molecules that contribute to the function of the cadherin-catenin complex (Pettitt, et al., 2003, J. Cell Biol. 162, 15-22). In continuing efforts to identify other regulators, we have employed the same rationale and conducted a genome-wide RNAi screen to identify genes whose loss of function enhances the fe4 phenotype. We have recently completed a screen of chromosome IV, and identified several genes that when knocked down by RNAi exhibit no phenotype in wild-type worms, but enhance the penetrance and severity of the fe4 mutation. We have focused our attention on MAGI-1, a MAGUK protein that is highly conserved across species. MAGI-1 localizes to adherens junctions and can interact with HMP-2 (Stetak, et al. European Worm Meeting Abstract. 2006) and actin-binding proteins (Patrie, et al., 2002, J. Biol Chem. 277, 30183-90). magi-1(RNAi) also enhances the severity of a hmp-2 hypomorph (qm39) that has not been well characterized to date. All magi-1(RNAi);hmp-1(fe4) and magi-1(RNAi);hmp-2(qm39) embryos arrest with a characteristic humpback phenotype. This suggests MAGI-1 may function with the cadherin-catenin complex and our current goal is to explore the role of MAGI-1 during epidermal morphogenesis of C. elegans.

Lynch, Allison et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "MAGI-1 modulates the function of the C. elegans cadherin-catenin complex during morphogenesis"

Hardin, Jeff, Lynch, Allison

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

In multicellular organisms, cell-cell junctions are involved in all aspects of tissue morphogenesis, including regulation of cell shape and movement. To establish junctions, cytoplasmic proteins that interact with the actin cytoskeleton must stabilize adhesion molecules. For example, catenins connect cadherins to the cytoskeleton and mutations in catenin proteins significantly impact morphogenesis. A previously isolated hypomorphic allele of the C. elegans ?-catenin homolog, hmp-1(fe4) , exhibits ~70% lethality and body shape defects (Pettitt, et al., 2003, JCB 162, 15-22). In continuing efforts to identify regulators of the cadherin-catenin complex, we conducted a genome-wide RNAi screen to identify genes whose loss of function enhances the hmp-1(fe4) phenotype. We uncovered MAGI-1, a vert ebrate tight junction protein known to interact with actin-binding proteins (Patrie, et al., 2002, JBC 277, 30183-90). We have shown C. elegans MAGI-1 genetically interacts with hmp-1 /?-catenin and hmp-2 /?-catenin, suggesting MAGI-1 may modulate cadherin-catenin function during morphogenesis. MAGI-1 localizes at the C. elegans apical junction, but is surprisingly basal to the cadherin-catenin complex. In addition, MAGI-1 junction localization is not dependent on ?-catenin, in contrast to what has been shown in tissue culture (Dobrosotskaya, et al., 2000, BBRC 270, 903-09). We are currently examining if MAGI-1 modulates actin dynamics during morphogenesis and we are characterizing proteins that potentially interact with MAGI-1 to further assess how MAGI-1 functions in a living organism.

Dee R Denver et al. (2001) International C. elegans Meeting "Rates and Patterns of Mutation in the Nuclear and Mitochondrial Genomes of Caenorhabditis elegans"

Larissa Vassilieva, Katherine Harris, Dee R Denver, Suzanne Randall Estes, W Kelley Thomas, Krystalynne Morris, Michael Lynch

[International C. elegans Meeting, 2001]

Mutations are the ultimate source of genetic disorders and offer important data for phylogenetic, forensic and population genetic studies. We directly assayed the rates and patterns of mutation in a set of 74 Caenorhabditis elegans mutation accumulation (MA) lines, where mutations accumulate over time in an effectively neutral manner as each MA line is propagated across generations as a single, random hermaphrodite (Vassilieva and Lynch, 1999). We performed a uniquely direct assay for the underlying rates and patterns of mutation in the MA lines with large-scale DNA sequence collection and analysis from both the nuclear and mitochondrial genomes. The mitochondrial DNA assays revealed a mutation rate that is two orders of magnitude higher than previous indirect estimates, a highly biased mutational spectrum, multiple mutations affecting coding function and mutational hotspots at homopolymeric nucleotide runs (Denver et.al ., 2000). Mutational hotspots have also been identified in the nuclear DNA sequence assays. Furthermore, we have analyzed the same mitochondrial and nuclear loci in C. elegans natural isolates to evaluate the relative roles of mutation and natural selection in shaping molecular variation observed in natural populations. References: L.L. Vassilieva and M. Lynch , Genetics 151 , 119 (1999). D.R. Denver, K. Morris, M. Lynch, L.L. Vassilieva and W.K. Thomas, Science 289 , 2342 (2000).

MacGregor D et al. (1995) International C. elegans Meeting "CONTROL OF GENE T23G5.5 EXPRESSION BY UPSTREAM SEQUENCES."

Hope IA, MacGregor D, Lynch AS, Isaac RE, Coates D

[International C. elegans Meeting, 1995]

The gene T23G5.5 shares homology with the family of neurotransmitter transporters which possess 12 trans-membrane regions. This family of genes is highly conserved throughout the animal kingdom. We have created a fusion between a lacZ reporter gene and 6.4kb of DNA upstream of T23G5.5. The fusion occurs in the middle of the ninth predicted exon of T23G5.5. The 6.4kb upstream region also contains the gene T23G5.6 and its upstream region. When injected into C. elegans this construct shows nuclear localized expression in 6 cells of the head ganglia from the L1 stage onwards. A further component manifests itself during L2, when a bilaterally symmetric pair of nuclei in the posterior lateral ganglia begin expression. This pair of neurons can be identified as being either PDEL/R, PDEsoL/R or PVDL/R. Sequential deletion of the upstream region has shown that a 640bp region directly upstream of the initiation codon is sufficient and necessary for the expression pattern. We are currently studying this region in greater depth.