Fischer, Christian et al. (2021) International Worm Meeting "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analysing Mitochondrial Morphology"

Fischer, Christian, Conradt, Barbara, Haeussler, Simon, Besora-Casals, Laura, Singh, Kritarth, Rolland, Stephane, Duchen, Michael, Marr, Carsten

[International Worm Meeting, 2021]

While the analysis of mitochondrial morphology has emerged as an important tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a difficult task and bottleneck for statistically robust conclusions. Here, we present the Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology (Fischer, Besora-Casals et al. 2020). The MitoSegNet was generated by training a modified fully convolutional neural network with fluorescent microscopy, maximum-intensity projection images, depicting mitochondria in body wall muscle cells of adult C. elegans worms. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet for all operating systems as an easy-to-use graphical user interface tool that combines segmentation with morphological analysis. Reference Fischer, C. A., L. Besora-Casals, S. G. Rolland, S. Haeussler, K. Singh, M. Duchen, B. Conradt and C. Marr (2020). "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology." iScience 23(10).

Gerami B et al. (1997) International C. elegans Meeting "MAPPING QUANTITATIVE TRAIT LOCI FOR CHEMOTAXIS: CORRESPONDENCE BETWEEN QTL AND odr CANDIDATE LOCI"

Morphew JJ, Phillips PC, Gerami B

[International C. elegans Meeting, 1997]

Understanding the genetics of complex traits is challenging because it can be difficult to separate out the effects of the numerous genes affecting the trait. C. elegans can serve as a useful model system for studying complex traits because of the wealth of genetic and functional information available. Chemotaxis provides an ideal trait for this type of study because it ecologically important to the nematodes, as well as likely to be influenced by many genes (a number of which are known). In collaboration with Thomas Johnson and David Shook [1] and Robert Shmookler Reis and Robert Ebert [2], who provided genotyped lines generated in a cross between N2 and BO, we have assayed the chemotaxic response of 180 recombinant inbred lines to benzaldehyde at two concentrations. These concentrations, 50 nl and 500 nl, respectively lead to an attraction or repulsion response. Separate analysis of the lines from the two labs yield different results. The SR lines show a QTL mapping to the fourth chromosome, whereas the TJ lines show several QTL, primarily on the X chromosome. The QTL on X map to the same locations as two mutants known to block the response to benzaldehyde, odr-1 and odr-5 [3]. We are in the process of evaluating these candidate loci as the actual QTL using backcrossing and complementation testing approaches. Our hope is to create a model system for studying a variety of quantitative genetic questions. [1] Genetics 142:801 (1996), [2] Genetics 135:1003 (1993), [3] Bargmann et al., Cell 74:515 (1993)

Shroff, Hari et al. (2013) International Worm Meeting "DiSPIM: time to leave your confocal behind."

Winter, Peter, Christensen, Ryan, Colon-Ramos, Daniel, Shroff, Hari, Wawrzusin, Peter, Santella, Anthony, Bao, Zhirong, Senseney, Justin, Fischer, Robert, McAuliffe, Matthew, Waterman, Clare, York, Andrew, Wu, Yicong

[International Worm Meeting, 2013]

Optimal 3D time-lapse imaging requires microscopy that provides high resolution in all spatial dimensions, high speed, and minimal photobleaching and photodamage. By creating algorithms that register and appropriately combine two perpendicular specimen views, we developed a dual-view inverted selective plane illumination microscope (diSPIM) with 330 nm isotropic resolution and 200 Hz acquisition, enabling rapid 3D imaging at 2 volumes/second. Unlike spinning-disk confocal or Bessel beam illumination methods that dose the sample outside the focal plane, leading to significant photobleaching, we maintain this spatiotemporal resolution over hundreds of volumes (hundreds of thousands of images) with negligible photobleaching. DiSPIM enables the study of biological systems that are intractable using other optical microscopes, including 4D microtubule tracking in live cells, improved nuclear imaging over 14 hours of nematode embryogenesis, and visualization of neural wiring during C. elegans brain development.

Christian Frokjar-Jensen et al. (2007) International Worm Meeting "Efficient low copy gene insertion at determined loci."

Soren-Peter Olesen, Christian Frokjar-Jensen, Wayne M Davis, Erik M. Jorgensen, Morten Grunnet, Blake Newman

[International Worm Meeting, 2007]

The ability to insert single copies of genes into the C.elegans genome in a controlled manner is desirable for a number of experiments; e.g. when germ line expression or near endogenous levels of protein expression is important. We have developed a new technique based on the mosTIC technique (Robert & Bessereau, 2006) and previously suggested by (Plasterk & Groenen, 1992) that allows us to insert transgenes at low copy number into two different non-coding loci on chromosome II or IV. The technique relies on the ability to create double-stranded breaks in the chromosomes by excising the Mos1 transposon. The insertion of transgenes then takes place by repairing the double stranded break through homologous recombination off an extrachromosomal array. In order to efficiently recover these events we have developed a system that utilizes a combination of positive and negative selection markers. An unc-119 rescue fragment is co-inserted into the chromosomal location for positive selection while a dominant temperature-sensitive mutant ion channel is used to select against animals still carrying the extragenic array. Animals that have inserted the gene of interest and have lost the extrachromosomal array will therefore be the only wild-type animals after shifting the animals to 25C. After mobilizing the transposon by heat shock we recover one integrant for every 100 heat-shocked adult animals in the F2 generation. For practical purposes, this is equivalent to 1 integrated line in every 5 large plates. In initial experiments we have inserted 4.2 kb of DNA. PCR confirms that the insertion occurred at the targeted location. Also, only a fraction of the integrated lines contain extraneous vector DNA, indicating mostly low or single copy insertions. Currently, we are: 1) testing if we can express transgenes in the germ line 2) determining the protein expression level from inserted transgenes 3) inserting larger transgenes and 4) testing a modified injection protocols designed to ensure single copy gene insertions. We believe the technique has a number of advantages compared to inserting genes by bombardment; 1) insertions do not disrupt coding regions, 2) different integrated lines are directly comparable, 3) there is no requirement for specialized equipment. The use of positive and negative selection markers makes the screening process easy. To facilitate use of the technique we have developed a Gateway compatible vector set for transgene insertion at the two chromosomal sites. _{Robert, V. & Bessereau, J.-L. (2007) EMBO 26: 170-83. Plasterk R.H., Groenen J.T. (1992) EMBO 11: 287-290}.

Amanda Hughes et al. (2007) International Worm Meeting "C. elegans RNA Helicase A genetically interacts with other proteins involved in RNAi."

Katherine M. Walstrom, Deborah Schmidt, Amanda Hughes

[International Worm Meeting, 2007]

RNA helicase A (RHA) is a conserved protein with roles in transcription regulation and histone modification in numerous organisms such as flies and humans. We are studying the role of C. elegans RHA (RHA-1) in transcription regulation and germline development. A null mutant, rha-1(tm329), has a temperature-dependent defect in repressive histone modifications and in germline transcriptional silencing of transgene arrays (Walstrom, Schmidt, Bean, and Kelly (2005) Mech. Dev. 122, 707). These defects in gene regulation lead to a sterile phenotype in hermaphrodite and male worms. Others have shown that rha-1 is required for RNA interference in the germline (Robert et al. (2005) Genes Dev. 19, 782-787). We found that rha-1 interacts genetically with rde-2 and mut-7. The RDE-2 and MUT-7 proteins are required for RNAi and physically associate with each other (Tops et al. (2005) NAR 33, 347). The precise roles of these two proteins and RHA-1 in RNAi are not well understood. Interestingly, rde-2;rha-1 mutants exhibit mutual repression while mut-7;rha-1 mutant hermaphrodites have an enhanced phenotype. We are further characterizing these double-mutants to determine how the germline histone modifications, germline development, and RNAi phenotypes are affected. We are particularly interested in the histone modifications, to which RNAi has been linked, because transcriptional silencing is an important process required for early germline development in many organisms.

Leyva-Diaz, Eduardo et al. (2017) International Worm Meeting "pals-22, a member of an expanded C. elegans gene family, is involved in repetitive transgene silencing."

Carrera, Ines, Stefanakis, Nikolaos, Leyva-Diaz, Eduardo, Hobert, Oliver

[International Worm Meeting, 2017]

Repetitive DNA sequences are subject to chromatin-based gene silencing in various animal species. Under specific circumstances repetitive DNA sequences can escape such silencing. For example, while exogenously added, extrachromosomal DNA sequences that are stably inherited in multicopy, repetitive arrays in the nematode C. elegans are often silenced in the germline, such silencing often does not occur in the soma. This indicates that somatic cells may utilize factors that prevent repetitive DNA silencing. Such "anti-silencing" factors have been revealed through genetic screens that identified mutant loci in which repetitive transgenic arrays are aberrantly silenced in the soma (Fischer et al. 2008, Grishok et al. 2005, Hsieh et al. 1999, Knight and Bass 2002). We describe here a novel mutant locus, pals-22 (for protein containing ALS2CR12 domain) with a transgene silencing phenotype. We find that pals-22 is a member of a large family of divergent genes, defined by the presence of an ALS2CR12 domain (39 members, including 2 pseudogenes). While family members are highly divergent they show striking patterns of genomic clustering. The family expansion appears C. elegans specific and has not occurred to the same extent in other nematode species (e.g. C. briggsae only codes for 6 pals genes). A PALS-22::GFP fusion protein that is fully functional in rescue assays displays a cytoplasmic subcellular localization, which rule out any direct chromatin-mediated silencing mechanism. This fusion protein is expressed across different tissues: the nervous system (pan-neuronally), muscle (pharyngeal, vulval, anal and body wall muscle), gut and seam cells. Lastly, the transgene silencing phenotype of pals-22 depends on the biogenesis of small RNAs, since silencing is abolished in the RNAi defective mutant rde-4, suggesting that pals-22 might regulate RNAi dependent silencing in the cytoplasm of neurons and other tissues.

Yang, Bing et al. (2013) International Worm Meeting "Expression of MET-2, a H3K9 methyltransferase, in the C. elegans germ line."

Maine, Eleanor, Xu, Xia, Yang, Bing

[International Worm Meeting, 2013]

An important feature of core histones, especially their N-terminal tails, is their subjection to a plethora of epigenetic modifications. One aspect of epigenetic modification involves histone methylation (Liu et al., 2011). Numerous reports have shown the methylation status of certain amino acids correlates with transcriptional status. For example, methylation of histone H3 lysine 4 (H3K4me) serves as a marker of active transcription in euchromatin regions, while methylation of histone H3 lysine 9 (H3K9me) and histone H3 lysine 27 (H3K27me) usually correlates with transcription repression in heterochromatin regions (Fischer et al., 2003, Sims et al., 2006). The C. elegans germ line serves as an ideal system to study the establishment and maintenance of chromatin modifications. Our research focuses on regulation of H3K9me2 deposition. In the adult germ line, H3K9me2 marks are broadly distributed at a relatively low level among all chromosomes as nuclei pass through the various stages of first meiotic prophase. In addition, chromosomes that do not pair and synapse, such as the male X, are highly modified with H3K9me2 (Kelly et al., 2002). MET-2, a member of the SETDB1/ESET family of lysine methyltransferases, is required for germline H3K9me2 (Bessler et al., 2010). We are investigating the mechanism that targets MET-2 activity to unpaired chromosomes. To this end, we generated several single copy, functional met-2::gfp transgenes by using Mos1 single copy insertion (mosSCI), and we also generated anti-MET-2 antibody. These reagents have allowed us to determine that MET-2 preferentially localizes to meiotic nuclei in the adult germ line, an observation consistent with a role for MET-2 as a histone methyltransferase. We are using these reagents to identify MET-2 interactors.

Elisabeth Cox et al. (2006) Development & Evolution Meeting "The C. elegans tropomodulin homolog, TMD-1, regulates adherens junctions during epidermal morphogenesis"

Jeff Hardin, Elisabeth Cox

[Development & Evolution Meeting, 2006]

The adherens junctions of C. elegans contain a highly conserved cadherin-catenin complex that is important for providing cell-cell adhesion during epithelial development. In a screen to discover regulators of the cadherin-catenin complex, we identified a tropomodulin homolog, TMD-1. Tropomodulins cap the slow growing, pointed ends of actin filaments. In vertebrates, tropomodulins regulate various actin networks, including those present in muscle sarcomeres, lamellipodia, and the cortical actin-spectrin cytoskeleton (reviewed in Fischer and Fowler, 2003, Trends Cell Biol., 13, 593-601). Our work on TMD-1 indicates a new role for tropomodulins as regulators of adherens juntions during morphogenesis. Although tmd-1(RNAi) does not yield a phenotype on its own, it enhances the embryonic lethality of worms homozygous for a loss-of-function allele of hmp-1 / α-catenin, hmp-1(fe4) (Pettitt et al., 2003, J. Cell Biol. 162, 15-22). We find that hmp-1(fe4); tmd-1(RNAi) results in 100% embryonic lethality with failed epidermal elongation, a process that requires the junctional anchorage and contraction of an ordered array of actin bundles. Wild-type embryos elongate about 4 fold their initial length before hatching. We find that 95% (n=93) of hmp-1(fe4); tmd-1(RNAi) embryos elongate 1.5 fold or less and then retract to their initial length. Only 7% (n=95) of hmp-(fe4) embryos exhibit a similar phenotype. Additionally, we find that the adherens junctions of hmp-1(fe4); tmd-1(RNAi) embryos do not maintain their strict apical-lateral position, and develop a frayed appearance as the embryos attempt to elongate. These areas of perturbed cadherin-catenin complex distribution correlate with areas of perturbed actin morphology, which likely accounts for the failed elongation of these embryos. Consistent with this, a rabbit polyclonal anti-TMD-1 antibody stains epidermal cell borders in elongating embryos where actin bundles anchor. Together, these results suggest that TMD-1 acts with HMP-1 / α-catenin to maintain anchorage of actin bundles to adherens junctions during epidermal elongation.

Thomas M Ratliff et al. (2002) Mid-west Worm Meeting "EGL-27 MTA-interacting proteins are components of the CCR4-NOT complex and synMuv B pathway"

Duaa H Mohammad, Michael A Herman, Thomas M Ratliff

[Mid-west Worm Meeting, 2002]

Mutations in egl-27 cause defects in cell migrations, cell fate specification, cell fusion and morphogenesis. The amino terminal region of EGL-27 is similiar to a human metastasis-associated factor (MTA), recently identified as a co-purifying member of a human complex having ATP-dependent nucleosome-remodeling and histone deacetylase (NURD) function. Other C. elegans homologs of human NURD components--specifically, let-418 Mi-2, lin-53 RbAp48 and hda-1 HDAC--have been shown to act as synthetic multivulva (synMuv) class B genes. The synMuv class B genes function redundantly with synMuv class A genes to antagonize Ras signaling-mediated vulval induction in the vulval precursor cells. Several class B synMuv members have been identified, including those similar to the human tumor suppressor, pRB, and its human binding partners, DP and E2F. Despite a ubiquitous nuclear expression pattern that overlaps with other NURD and synMuv B members, the apparently obscure role of EGL-27 MTA in vulval induction suggests a lack of functional conservation with its human counterpart. In an effort to better understand the function of EGL-27, a two-hybrid screen was performed in yeast (two-hybrid library kindly provided by Robert Barstead). Multiple library clones were retrieved that corresponded to the mep-1 locus and two genes having similarity to components of the highly conserved CCR4-NOT complex, Cenot-2 and Cenot-3. MEP-1 is a large C2H2 zinc-finger protein with two potential homologs in Drosophila. CeNOT-2 and CeNOT-3 contain a previously unidentified domain of similarity located near their respective carboxy termini. Bacterially-expressed GST::EGL-27 interacts in vitro with CeNOT-2 and CeNOT-3 fragments corresponding to the two-hybrid library clones, thus verifying the two-hybrid interactions.

Krempel, S.J. et al. (2019) International Worm Meeting "Understanding the convergence of ccm-3 and kri-1 signaling in C. elegans."

YU, B., Krempel, S.J., Derry, W.B.

[International Worm Meeting, 2019]

Cerebral cavernous malformations (CCM) is the most common human neurovascular disease and is characterized by the loss of endothelial integrity in blood vessels of the central nervous system, leading to raspberry-like malformations of capillaries. This can cause a variety of neurological symptoms such as migraines, seizures and strokes. CCM is caused by loss-of-function mutations in any of the three CCM genes: KRIT1/CCM1, CCM2 and CCM3. While loss of CCM1 or CCM2 lead to similar clinical presentation, loss of CCM3 causes a more severe form of the disease characterized by an earlier onset and higher lesion burden (Fischer et al 2013). The C. elegans CCM1 protein KRI-1 physically interacts with the CCM2 ortholog CCM-2 to promote germline DNA damage-induced apoptosis through a conserved ERK5 kinase cascade and the Kruppel-like transcription factor KLF-3 (Chapman et al 2019). In contrast, CCM-3 interacts with striatin interacting phosphatase and kinase (STRIPAK) complex components GCK-1 and CASH-1 to promote germline development and fertility as well as excretory canal extension (Pal et al 2017, Lant et al 2015). While loss of CCM1 and CCM3 in humans results in the same vascular anomalies, no known phenotypes or downstream effectors are shared between kri-1 and ccm-3 in the worm. Furthermore, the mechanism by which CCM1/2 and CCM3 converge to regulate vascular integrity are not known. Since kri-1 is synthetic lethal with ccm-3 (Lant et al 2015), we hypothesize that the pathways downstream of these genes converge on an uncharacterized essential process. In order to identify the downstream effectors of kri-1 required for viability, we are be conducting an RNAi screen of genes previously found by RNAseq to be transcriptionally regulated by kri-1 and mpk-2/ERK5 for synthetic interactions with ccm-3. We are also taking advantage of a transgenic strain for the inducible degradation of CCM-3 in a kri-1 mutant background to characterize the developmental and morphological defects arising from loss of kri-1 and ccm-3. It is anticipated the mechanisms by which these genes cooperate to maintain viability in C. elegans will reveal conserved mechanisms by which the CCM1/2 and CCM3 pathways prevent identical vascular malformations in humans.