Adler, Victoria et al. (2019) International Worm Meeting "Characterization of meiotic prophase I in a natural isolate of Caenorhabditis elegans."

Adler, Victoria, Libuda, Diana, Anderson, Erik, Zhang, Gaotian

[International Worm Meeting, 2019]

In most sexually reproducing organisms, meiosis ensures reproductive success and creates genetic variation within a population. Errors in meiosis can lead to birth defects and infertility. Evaluation of meiotic processes in closely related species and natural populations can reveal the ancestral state of critical meiotic processes as well as new variants of meiotic genes. Here, we characterize the key processes of meiotic prophase I in the Caenorhabditis elegans strain ECA701, which has several traits indicative of defects during meiosis (see abstract by G. Zhang et al.). To determine the changes in the specific meiotic processes that cause the defective meiotic phenotypes observed in ECA701, we used immunofluorescence with antibodies generated to N2 meiotic proteins to characterize the germ lines of ECA701 males and hermaphrodites. Preliminary observations suggest that although many meiotic proteins between N2 and ECA701 are well conserved, several aspects of meiotic prophase I differ between the two strains. In ECA701, the synaptonemal complex (SC), a well conserved meiosis-specific complex that holds together homologous chromosomes, assembles along most chromosome pairs, but some developing oocytes have a set of chromosomes that are unable to pair and form the SC. In contrast to ECA701 oocytes and N2 spermatocytes, spermatocytes in ECA701 males have no unpaired chromosomes and instead, consistently pair and form SC on five sets of chromosomes. Notably, pairing-associated chromosome movement is significantly longer during ECA701 oogenesis than is observed in the N2 strain. Moreover, during diakinesis, when sets of homologous chromosomes are held together by crossover recombination events to form six bivalent structures in N2, 4-5 sets of chromosomes are able to form bivalents but 1-2 chromatids form univalent structures during ECA701 oogenesis. Future experiments will determine if these unpaired chromosomes are the sex chromosomes, which may explain the Him phenotype (40-45% male progeny) of ECA701. Also, we characterized loading of the recombinase RAD-51, which localizes to sites of double strand DNA breaks (DSBs). Notably, the number of RAD-51 foci per nucleus in ECA701 is less than what is observed in N2. This reduction in RAD-51 foci indicates that either fewer DSBs are formed in ECA701 or some DSBs are marked by another meiotic recombinase. Finally, we observed a complete loss of sperm in the spermatheca of day-three adult hermaphrodites of ECA701. Overall, our preliminary results and future experiments may reveal the possible ancestral state of Caenorhabditis elegans meiosis, the evolution of an androdioecious species, or natural alleles of key meiotic genes that can underlie recombination variation.

Toraason, Erik et al. (2021) International Worm Meeting "DNA repair is altered during C. elegans germline aging"

Libuda, Diana, Toraason, Erik, Adler, Victoria

[International Worm Meeting, 2021]

Reproductive aging leads to a decline in oocyte quality and female fertility. Previous studies in multiple organisms have found altered expression of DNA repair genes during oocyte aging, however it is largely unknown whether DNA repair is defective in aged oocytes. To determine whether DNA break formation and repair changes in oocytes upon aging, we used C. elegans to examine DNA repair in aged germlines of both wild-type hermaphrodites, which continuously produce new oocytes, and fog-2 mutant females, which can hold and age their oocytes in specific stages of meiotic prophase I. Our results found that RAD-51 foci, which mark DNA double-strand breaks (DSBs), were elevated in both aged wild-type and fog-2 germlines, indicating that oocytes accumulate DSBs in old germlines regardless of oocyte age. We further determined that the elevated DSBs in aged germlines were SPO-11-dependent, indicating that age either increases SPO-11 activity or delays DSB repair. To assess efficiency of meiotic DSB repair upon aging, we introduced exogenous DSBs via irradiation to both young and old germlines and examined the kinetics of DSB repair following irradiation. Our results indicate that old germlines maintained a higher number of DSBs in a subset of nuclei following irradiation, supporting a model in which DSB repair is less efficient upon aging. To determine how DSB repair is regulated during oocyte aging, we examined mutants deficient in the E2 ubiquitin ligase variant UEV-2, which is upregulated in mutants with an extended reproductive lifespan and is suggested to function in DSB repair. Our analyses indicate that both young and aged uev-2 mutant germlines exhibit elevated but similar levels of RAD-51 foci, implicating UEV-2 as a key regulator of DSB repair during germline aging. Taken together, our work reveals defects associated with germline aging and identifies novel players ensuring efficient DSB repair in young oocytes.

Chalfie M et al. (1994) Science "Green fluorescent protein as a marker for gene expression."

Euskirchen G, Tu Y, Chalfie M, Prasher DC, Ward WW

[Science, 1994]

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

Saleel Raut et al. (2008) C.elegans Neuronal Development Meeting "HLH-3, a C. elegans Achaete/Scute protein, has a role in the axonal pathfinding of hermaphrodite-specific motor neuron (HSN) that is independent of UNC-40."

Aixa Alfonso, Saleel Raut

[C.elegans Neuronal Development Meeting, 2008]

The basic helix loop helix protein, HLH-3, is a member of the achaete/scute family in C. elegans hlh-3 mutants have an egg-laying defective (Egl) phenotype. We have shown (Doonan et al., 2008 submitted) that the mutant phenotype results from abnormal axonal pathfinding in hermaphrodite-specific motor neurons (HSNs) that fail to innervate the vulval muscles. The NETRIN/UNC-6 receptor UNC-40/DCC is necessary for the guidance of the HSN axons (Garriga et al.,1993; Chan et al., 1996; Gitai et al., 2003; Adler et al., 2006). We hypothesized that mutant animals are not uncoordinated (Unc). Currently we are looking at SYG-1, a receptor on HSN''s that binds with the guidepost signal SYG-2 for proper synapse formation (Shen et al., 2004), as a possible target of HLH-3.

Miller DM et al. (1999) Biotechniques "Two-color GFP expression system for C. elegans."

Desai NS, Piston DW, Hardin DC, Xu SQ, Miller DM, Fleenor J, Fire A, Patterson GH

[Biotechniques, 1999]

We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.

Galindo-Malagn XA et al. (2022) Zootaxa "New species, synonymies and records in the genus Rhagovelia Mayr, 1865 (Hemiptera: Heteroptera: Veliidae) from Colombia."

Moreira FFF, Morales I, Mondragn-F SP, Galindo-Malagn XA

[Zootaxa, 2022]

Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.

David Alan Wharton (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Panagrolaimus davidi, an Antarctic nematode model for the survival of extreme environmental stress"

David Alan Wharton

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Nematodes are found in almost all environments, including those where they are often exposed to extreme environmental stress. Panagrolaimus davidi is an Antarctic nematode living associated with moss and algae in terrestrial habitats on the Victoria Land coast that are free of snow and ice for part of the year. It has to survive very variable thermal and hydric environments where liquid water and temperatures suitable for growth are only periodically available. P. davidi can survive complete water loss (anhydrobiosis) and is the only organism that has been shown to survive intracellular ice formation throughout its tissues. It has several cold tolerance strategies, including; freeze avoidance, cryoprotective dehydration, freezing tolerance and anhydrobiosis. The mechanisms involved may include the production of trehalose, ice active proteins and the control of ice nucleation. Do the different survival strategies of P. davidi represent the expression of different gene sets or does the production of stress-related compounds provide protection against a variety of environmental challenges? Other nematodes, including Caenorhabditis elegans, are not so resistant to desiccation and freezing. Comparing the genomes of P. davidi and C. elegans may thus highlight the adaptations that are necessary for the survival of extreme environmental stress.

Thomas BJ et al. (2019) PLoS One "CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans."

Cole FS, Silverman GA, Thomas BJ, Chou WYY, Wambach JA, Kim H, Buland JR, Jia H, Homayouni A, Moreno M, Luke CJ, Pak SC, Huang H, Wight IE, Dawson Z

[PLoS One, 2019]

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

Millan MI et al. (2009) Medicina (B Aires) "[The green fluorescent protein that glows in bioscience]."

Millan MI, Becu-Villalobos D

[Medicina (B Aires), 2009]

Green fluorescent protein (GFP) is a protein produced by the jellyfish Aequorea victoria, that emits bioluminescence in the green zone of the visible spectrum. The GFP gene has been cloned and is used in molecular biology as a marker. The three researchers that participated independently in elucidating the structure and function of this and its related proteins, Drs. Shimomura, Chalfie and Tsien were awarded the Nobel Prize in Chemistry 2008. Dr. Shimomura discovered and studied the properties of GFP. Using molecular biological techniques, Chalfie succeeded in introducing the GFP gene into the DNA of the small, almost transparent worm C. elegans, and initiated an era in which GFP would be used as a glowing marker for cellular biology. Finally, Dr.Tsien found precisely how GFP's structure produces the observed green fluorescence, and succeeded in modifying the structure to generate molecules that emit light at slightly different wavelengths, which gave tags of different colors. Fluorescent proteins are very versatile and are being used in many areas, such as microbiology, biotechnology, physiology, environmental engineering, development, etc. They can, for example, illuminate growing cancer tumours; show the development of Alzheimer's disease, or detect arsenic traces in water. Finding the key to how a marine organism produces light unexpectedly ended up providing researchers with a powerful array of tools with which to visualize cell biology in action.

Sawa H et al. (1996) East Coast Worm Meeting "Molecular and genetic analyses of lin-17, which encodes a putative receptor required for asymmetric cell divisions"

Horvitz HR, Sawa H

[East Coast Worm Meeting, 1996]

Asymmetric cell division is a fundamental mechanism responsible for increasing cellular diversity during development. lin-17 is required for the asymmetric divisions of a number of cells in C. elegans (1). lin-17 encodes a putative seven-transmembrane protein similar to the frizzled gene product of Drosophila. frizzled is required for the correct polarities of a number of cells and tissues (2). Using a reporter construct, we have found that lin-17 is expressed in cells prior to their asymmetric cell divisions and also in both daughter cells after the divisions. Our results suggest that lin-17 encodes a receptor that regulates the polarities of cells undergoing asymmetric cell divisions. We have identified the molecular lesions of twelve lin-17 alleles. One, n3091, has a stop codon close to the N-terminus of the coding sequence, suggesting that it is a null allele. n669 causes a Gly-to-Arg change in the middle of the seventh transmembrane domain. rh71 causes a Gly-to-Asp change in the third transmembrane domain close to a presumptive cytoplasmic region. The residues affected in these mutants may define interaction sites of the LIN-17 receptor with its ligands or effector molecules. We are screening for suppressors of these missense mutations in an attempt to identify genes in the lin-17 signaling pathway. (1) Sternberg, P.W. & Horvitz, H.R. Dev. Biol. 130, 67-73 (1988). (2) Adler, P.N. BioEssays 14, 735-741 (1992).