Hajji, Ismail et al. (2021) International Worm Meeting "Cell-fate decisions in dynamically perturbed signaling environments during C. Elegans vulval development"

Siggia, Eric, Corson, Francis, Keil, Wolfgang, Hajji, Ismail

[International Worm Meeting, 2021]

During development, cells take a series of fate decisions to acquire different roles in the future organism. The decisions that make a cell specify into one role and not the others depend on complex and dynamic inter/intracellular signals. However, at each decision point, cells typically only decide between a limited repertoire of fates. This suggests that simple, intuitive regulatory biophysical principles may successfully summarize pathway complexity. Here, we propose to establish and quantitatively test such principles, using the vulval development of C. elegans as a model system. To do so, we propose to combine: (i) Controlled perturbations of in-vivo signaling dynamics using Auxin-Inducible Degradation (AID) System and Temperature-Sensitive strains (Zhang et al., 2015; Martinez et al., 2020) (ii) In-vivo measurements of the perturbations using a real-time biosensor for ERK activity (de la Cova et al., 2017) (iii) Large-scale 4D live imaging to capture cell-fate transformations in response to perturbed signaling in microfluidics (Keil et al., 2017) (iv) Parsimonious mathematical modeling of the underlying cell-fate acquisition dynamics (Corson and Siggia, 2012, 2017) Our preliminary results confirm the counter-intuitive asymmetry between P4/8.p in the degree to which they are induced to 2° fates by an EGF pulse (lin-15(n765)), as predicted by (Corson and Siggia, 2017). We will also compare our model predictions to experimental measurements in a background with weak ectopic Notch activity and no Anchor Cell (lin-12(n302)). Our project promises fundamental insights into stem cell behavior by experimental consolidating a generic mathematical modeling approach, applicable to a wide range of stem-cell paradigms.

Corson, Francis et al. (2011) International Worm Meeting "Geometric modeling of C. elegans vulval patterning."

Siggia, Eric, Corson, Francis

[International Worm Meeting, 2011]

The conventional approach to the modeling of gene networks begins with a list of molecular parts and interactions, which are translated into a description of the dynamics, e.g. a set of differential equations. While the behavior of idealized networks comprising a small number of elements can be analyzed systematically in this way, actual biological networks are far more intricate, making it much less straightforward to relate model structure and observed behavior. The geometrical theory of dynamical systems suggests an approach to gene network modeling that constructs a representation of their "effective dynamics" in terms of a small number of variables. In this minimal description, the many molecular constants collapse onto a few parameters that have a natural relation to the dynamics, and whose effect on the behavior of the system can easily be visualized. We have applied this approach to the development of C. elegans vulva, a well-studied model system for which abundant experimental data are available in the literature. The vulva forms from a row of six vulval precursor cells (VPCs), which adopt an invariant pattern of fates in wild type. The three possible fates of the VPCs are specified by two signaling pathways (inductive signaling from the neighboring anchor cell and lateral signaling between VPCs), suggesting a two-dimensional representation of their dynamics. Qualitatively distinct dynamics, embodying different "scenarios" for vulval patterning, can be enumerated and discriminated by experimental observations. A more quantitative representation is obtained by formulating a generic model of the dynamics (the simplest equations with three stable states corresponding to the three fates), which is then fit to experimental data (proportions of different fate patterns under given conditions). This minimal model is found sufficient to account for several non-intuitive mutant phenotypes, which are given simple interpretations in terms of the geometry of the trajectories taken by the cells, and can be used to generate novel predictions, e.g. double mutant phenotypes. This approach, by its generic nature, could readily be transposed to other systems, and, by providing a representation of dynamics that is abstracted from the details of its implementation on the molecular level, points to the possibility of discussing the organization and evolution of developmental mechanisms on a phenotypic level.

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755

Yasuda H et al. (1987) Worm Breeder's Gazette "screening C elegans expression libraries with nerve specific anit-tubulin antibodies."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1987]

C. elegans, like many other multicellular organisms, has multiple tubulin genes. Linda Gremke has shown that there are at least 4 and 5 alpha tubulin genes in the nematode, based on homology to the chicken tubulin gene probes(1). Similarly, it has been shown that purified tubulin from the nematode can be resolved in to at least 10 isotypes on isoelectric focusing, including 2 major alpha, 2 major , and about 6 minor alpha and isotypes(2). We have screened C. elegans cDNA and genomic expression libraries (kindly provided by Bob Barstead and Bob Waterston), with a set of anti-tubulin monoclonal antibodies, which preferentially stain neurons cytochemically(2). In the present screen 44 clones have been identified, which test positive on rescreening. The insert size of these clones range from 0.6 to 1.7 kb. Among these 29 clones are specific to anti- tubulin, and 14 are specific to anti-alpha tubulin antibodies. Our attention has been drawn to the problem of false positives in screening expression libraries with antibodies(e.g. C. Link and W. Wood, WBG Nov. 1986, and Eric Aamodt, personal communication). These workers have found E. coli DNA in the 'positive' clones, as a major artefact. Affinity purification of antibodies with the fusion protein, may not preclude the inclusion of false positives (Eric Aamodt, personal communication) either. Therefore, we plan to subclone the inserts in an appropriate vector, and determine the nucleotide sequence of the inserts to distinguish the true from the false positives. This is possible for the tubulin clones, as the nucleotide sequence of tubulin variants is available, including that of C. elegans tubulin(1). We plan to use this strategy for other genes for which we have collected 'positives' using expression libraries, including N-CAM like antigen, and N- CADHERIN like antigen of C. elegans, as these genes have conserved sequences, and their nucleotide sequence data are available.

Irene Zelepuhin et al. (2005) International Worm Meeting "Genetics of sex Reversal of C. briggsae::C. remanei XO hybrids."

Scott Baird, Shannon Romer, Irene Zelepuhin

[International Worm Meeting, 2005]

Sterile F1 adult hybrids can be obtained from crosses of C. briggsae males to C. remanei females. Depending on the strains used, all hybrids are females. The absence of male hybrids results from sexual transformation, not male-specific lethality, as both XX and XO females are observed. Variant strains of C. briggsae and C. remanei suppress this hybrid sexual transformation, demonstrating genetic variation within both species the affects sex determination in F1 hybrids. This variation has been used to map hybrid sex reversal loci. In C. remanei, an ill-defined region of the X chromosome is implicated. In C. briggsae, three loci linked to hybrid sex reversal have been identified. The strongest linkage is to allelic variants in Cb-tra-2. A null mutation in Cb-tra-2 (thanks Eric) completely suppresses the transformation of XO hybrids. Other C. briggsae loci linked to sex reversal variants include Cb-daf-5 and Cb-mab-9.

Davidson EH et al. (1999) J Exp Zool "Themes from a NASA workshop on gene regulatory processes in development and evolution."

Davidson EH, Ruvkun GB

[J Exp Zool, 1999]

A memorable workshop, focused on causal mechanisms in metazoan evolution and sponsored by NASA, was held in early June 1998, at MBL. The workshop was organized by Mike Levine and Eric H. Davidson, and it included the PI and associates from 12 different laboratories, a total of about 30 people. Each laboratory had about two and one half hours in which to represent its recent research and cast up its current ideas for an often intense discussion. In the following we have tried to enunciate some of the major themes that emerged, and to reflect on their implications. The opinions voiced are our own. We would like to tender apologies over those contributions we have not been able to include, but this is not, strictly speaking, a meeting review. Rather we have focused on those topics that bear more directly on evolutionary mechanisms, and have therefore slighted some presentations (including some of our own), that were oriented mainly toward developmental processes. J. Exp. Zool. (Mol. Dev. Evol. ) 285:104-115, 1999.

Almeida MV et al. (2018) EMBO J "GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans."

Redl S, Hildebrandt A, Ulrich HD, Dietz S, Renz C, Almeida MV, Konig J, Ketting RF, Butter F, Karaulanov E

[EMBO J, 2018]

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the <i>Caenorhabditis elegans</i> Gtsf1 homolog, named it <i>gtsf-1</i> and characterized it in the context of the sRNA pathways of <i>C.elegans</i> We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, <i>gtsf-1</i> mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying <i>rrf-3</i> mutants. We show, both <i>invivo</i> and <i>invitro</i>, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

Sawh, Ahilya et al. (2015) International Worm Meeting "Regulation of DCR-1-dependent RNAi pathways through DCR-1 phosphorylation."

Duchaine, Thomas, Wohlschlegel, James, Lewis, Alexandra, Sawh, Ahilya

[International Worm Meeting, 2015]

RNA interference (RNAi) pathways are directed by small (18-22nt) non-coding RNAs and orchestrate a variety of evolutionarily-conserved gene-silencing programs. The type III RNase protein Dicer is at the center of most of these pathways, including responses triggered by exogenous sources of long double-stranded RNA (exoRNAi). Other pathways, such as microRNA-mediated silencing and endoRNAi are triggered by endogenous loci, and control developmental and homeotic events. Dicer's role is crucial and three-fold: it cleaves long dsRNA precursors into smaller RNAs that direct silencing events, it loads the small RNAs into Argonaute proteins to form an active RNA-induced silencing complex (RISC), and it acts as a scaffold for several protein co-factors required for substrate recognition and processing. In C. elegans, Dicer (DCR-1) is essential in three small-RNA mediated silencing mechanisms including exoRNAi and ERI endoRNAi, two RNAi pathways that compete for downstream effectors. DCR-1 is part of distinct multi-protein complexes in each of these RNAi pathways: RDE complex in exoRNAi and ERIC in ERI endoRNAi.We discovered and validated several phosphorylated determinants of DCR-1 in the C. elegans. Particular attention was placed on a cluster of phosphorylated sites between the PAZ and RNase III domains, one of which could implicate AMPK signaling. Recent crystal structures of human Dicer have shown that this region could be involved in recognition of dsRNA substrates. We thus hypothesized that phosphorylation in this cluster could impair substrate recognition, or alter its specificity.To study the effect of DCR-1 phosphorylation on the three DCR-1-dependent RNAi pathways in C. elegans, transgenic strains were engineered expressing phosphorylation-deficient, and phosphorylation-mimetic mutants of DCR-1 in this cluster of sites. Functional analysis on these strains revealed that phosphorylation-deficient mutants display an impaired exoRNAi response, and severe developmental defects. Phosphorylation-deficient DCR-1 mutants also enhance interaction with ERI-endoRNAi DCR-1 cofactors. Taken together, these results support a model wherein DCR-1 phosphorylation may promote its shift from the ERIC to the RDE complex. Post-translational regulation of Dicer and RNAi pathways are largely unexplored and could potentially be incredibly impactful in light of their crucial roles in development and disease. The present study will shed light on how RNAi pathways integrate signals through phosphorylation signaling cascades.

Goehring A et al. (2014) Nat Protoc "Screening and large-scale expression of membrane proteins in mammalian cells for structural studies."

Lee CH, Fischer S, Althoff T, Michel JC, Gouaux E, Garcia KC, Baconguis I, Claxton DP, Wang KH, Goehring A

[Nat Protoc, 2014]

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.