Choi, Hailey et al. (2015) International Worm Meeting "Towards an understanding of how embryos cope with stochastic gene expression."

Doan, Kollan, Choi, Hailey, Broitman-Maduro, Gina, Maduro, Morris, Carranza, Francisco

[International Worm Meeting, 2015]

Robust gene expression is crucial for proper embryonic development, and yet gene expression is subject to both extrinsic and intrinsic sources of noise. We are investigating mechanisms by which the C. elegans embryo buffers stochastic variations in gene expression in endoderm specification. Perturbation of the regulators that act in gene regulatory network that specifies E, the endoderm progenitor, causes gut specification to become highly variable. This variability includes both the proportion of embryos making gut, and among those with gut, variations in the number of E descendants that adopt a gut fate. Using existing mutations and single-copy transgenes in which critical transcription factor binding sites have been mutated, we have created a set of strains that experience stochastic E specification. These strains cover a range of probabilities of making gut, from 0% to 100%. Using these strains, we are investigating E lineage phenotypes in the embryo, elucidating phenotypes in adults that survive hypomorphic gut specification, and screening for factors that modulate gut specification in these strains.First, we have found that E can make both gut and non-gut cells in the same embryo, using strains in which we can follow E descendants in the presence of reporter transgenes to mark tissue fates.Second, we have found that surviving adults display phenotypes consistent with caloric restriction. Animals store excess lipid, have defects in morphology and fertility, and exhibit developmental delay.Last, we are performing RNAi screens for maternal factors that modulate gut specification. Using the Vidal feeding library, we have selected ~3000 clones that are known to be expressed in the germline. Thus far, we have found that several diverse maternal pathways, including transcription regulation, metabolism, and apoptosis, influence the ability of progeny embryos to make gut in our partial specification-compromised strains. This suggests that embryonic gene expression is sensitive to many sources of variation.Collectively our data show that gut specification is not an "all-or-none" event, and that if specification does not occur properly, specification defects can persist into the adult intestine. Our results have implications in other developmental pathways in C. elegans, and in any metazoan system in which gene regulatory networks drive developmentally important events.

Maduro, Morris F. et al. (2013) International Worm Meeting "Life at the edge of robustness: Partial guts suggest that endoderm specification is not all-or-none."

Ghamsari, Farhad, Maduro, Morris F., Broitman-Maduro, Gina, Carranza, Francisco, Walia, Gurjot

[International Worm Meeting, 2013]

The C. elegans intestine is derived from the embryonic blastomere E. The paradigm for E specification is that maternal SKN-1- and POP-1-dependent input cause activation of the E-specific gut specification factors end-1 and end-3. After reaching a threshold of expression, these activate elt-2, which maintains its expression by autoregulation and drives the commitment to gut differentiation. Prior work suggests that gut specification may not be all-or-none, and that some of the descendants of E are capable of adopting a gut fate independently of others. We have created strains in which E is specified by single-copy transgene forms of end-1 and/or end-3 that are mutated for binding sites for the MED-1,2 GATA factors, themselves direct targets of SKN-1. In such strains, different embryos make variable numbers of apparently normal-sized gut cells, suggesting that specification has become subject to stochastic variations among embryos, and that commitment to a gut fate can occur later in the E lineage. Counting of embryonic elt-2 transcripts by single-molecule FISH suggests that activation of elt-2 is more graded in these strains, as opposed to an all-or-none mode as was previously reported for SKN-1-depleted embryos (Raj et al., 2010). As these effects are confined to the E lineage due to the nature of the strains constructed, we are able to evaluate adults derived from embryos in which functional guts were made. We find that such adults store lipids at significantly higher levels and display other variable pleiotropic phenotypes, suggestive of primary defects in gut function. Together, these results build a picture in which specification of gut is not an all-or-none event, and that in animals that do make an intestine, the endoderm differentiation network is not fully self-correcting for partially compromised specification. We will present these results, including the outcome of an attempt to computationally model the stochasticity of elt-2 activation.

Broitman-Maduro, Gina et al. (2013) International Worm Meeting "Enhanced RNA virus susceptibility results from defects in lipid metabolism."

Ding, Shou-wei, Coffman, Stephanie, Guo, YuanYuan, Carranza, Francisco, Maduro, Morris, Pompura, Saige, Broitman-Maduro, Gina

[International Worm Meeting, 2013]

Prior work has shown that C. elegans can support replication of (+) strand RNA viruses, including the Flock House Virus (FHV), and the Orsay virus. We have previously reported that transgene expression of FHV RNA1, which encodes the viral RdRP and a viral suppressor of RNAi (B2), can direct its own replication (Lu et al., 2005). By replacing the coding region of B2 with that of GFP (FR1gfp) we can visualize viral replication in backgrounds that are virus-susceptible. In an RNAi screen for factors that influence replication of FR1gfp (see abstract by Stephanie Coffman), we identified genes that are known to affect storage or metabolism of lipids, such as nhr-68 (Ashrafi et al., 2003). To evaluate the relationship between lipid storage and RNA virus replication, we examined fat stores using Oil Red O in chromosomal mutants for other genes identified by the screen, and directly assessed viral replication in strains that are known to have fat storage defects. Our results confirm that conditions known to cause increases in fat stores, such as the germlineless glp-4(bn2) background, result in a significant increase in viral replication. As lipid storage defects have been recently shown to be correlated with autophagy (Hansen et al., 2013), we have tested the role of autophagy in lipid storage-mediated viral susceptibility. Overexpression of the lipase lipl-4 appears to be sufficient to increase viral susceptibility, in a manner that is partially dependent on the autophagy gene atg-1. The connection between lipid storage and susceptibility to RNA virus replication is not immediately obvious, and our data suggest that the interactions are complex, though one possibility is that autophagy regulates some components of the RNAi machinery (Gibbings et al., 2012). Results of characterization of viral replication (both FR1gfp and Orsay virus) in various mutant backgrounds will be presented.

Iyer, Sangeetha et al. (2015) International Worm Meeting "A high-throughput screening approach using model organisms for Niemann-Pick Type C disease."

Hartl, Tom, Singh, Kiran, Ludin, Alec, DiPrimio, Nina, Perlstein, Ethan, Iyer, Sangeetha, Portillo, Tamy

[International Worm Meeting, 2015]

Perlstein Lab, PBC is a San Francisco based startup using model organisms to find small molecule therapies for rare genetic disorders. In this abstract, we describe a parallel model organism platform encompassing yeast, C.elegans, drosophila and mammalian cells for Niemann-Pick type C (NPC) disease. NPC, an autosomal recessive disorder caused by mutations in the NPC1 gene, afflicts 1 in 1,50,000 people. Clinically, this disease manifests during childhood in the form of progressive ataxia, dystonia, liver disease in addition to progressive cognitive decline. Biochemically, this disease is characterized by accumulation of free or unesterified cholesterol in the lysosomes. The NPC1 gene is remarkably conserved across species, with an ortholog present in yeast, nematode and fruit flies. In C.elegans, deletion of the homologous ncr-1 gene leads to mutant animals that develop slowly, have smaller brood sizes and importantly, inappropriately form dauer larvae under favorable conditions. These animals are also hypersensitive to the absence of cholesterol. Building on this, Perlstein Lab generated new lines of ncr-1 null mutants using CRISPR technology. After genotyping and validating that two newly generated strains phenocopied similar to their predecessors, one strain was selected for a rapid drug screening assay. Using slowed growth in cholesterol-deprived animals as an endpoint, a high throughput chemical modifier screen was designed to identify compounds that rescued the effects of cholesterol deprivation to near wildtype levels. Hits arising from this primary screen were compared for duplication and structural similarity against hits obtained from parallel screens in yeast and drosophila model organisms. Multi-organism hits were then tested in mammalian cell models of NPC using a filipin stain assay. Using this approach, we demonstrate that, for genetically conserved diseases, a parallel organism screening platform can be used to rapidly screen through tens of thousands of compounds thus paving the way for translational therapeutics.

Chu, Diana S. et al. (2013) International Worm Meeting "Spectrum: Building Pathways to Biomedical Research Careers for Girls and Women of Color."

Tanner, Kimberly D., Chu, Diana S., Parangan-Smith, Audrey G.

[International Worm Meeting, 2013]

Women of color are largely absent from the biomedical research community. Despite some progress made in encouraging girls to pursue career paths in science, few materials exist that specifically attract girls of color to these careers. To address this need, the Science Education Partnership and Assessment Laboratory (SEPAL) at San Francisco State University (SFSU) developed the Spectrum program. This program partners SFSU biomedical scientists who are women of color (including Biology faculty, undergraduate students, Masters students, and alumni in doctoral and biotechnology positions) with middle and high school girls and teachers. These teams collaborate to: 1) co-sponsor after-school science clubs targeting girls of color in high needs public schools, 2) develop a mentoring community of women of color trainees in biomedical research, 3) create video biographies highlighting the research programs and experiences of women of color biologists, and 4) partner with the local and national Expanding Your Horizons organizations to disseminate Spectrum activities. During its initial four years, Spectrum engaged 456 middle and high school girls (45% Latina, 13% African American, 22% Asian, 11% Unknown, 7% White) across nine clubs providing ~20 hours of academic enrichment for each girl, including two field trips to the laboratories of SFSU women of color biologists. Evaluation data shows increases in the percentage of participating girls who agree with the following statements: 1) I have heard a woman scientist talk about why she likes science (pre: 72%, post: 96%), and 2) I have met a woman scientist who is like me (pre: 36%, post: 65%);. Spectrum has also developed two video resources highlighting women of color from SFSU. 1) Women of Color Doing Biomedical Science: Inspiring Stories from Women of Color Biomedical Researchers and 2) From Us to Us: Advice on Careers in Biomedical Sciences for Girls & Women of Color. These are available on the SEPAL website and have been distributed through partnerships with national organizations supporting girls in science. Spectrum is supported by the National Institutes of Health through award #R25RR024307.

Peichuan Zhang et al. (2007) International Worm Meeting "HOW DOES ACTIVITY OF DAF-16/FOXO IN ONE TISSUE AFFECT THE LIFESPAN OF THE WHOLE ANIMAL?"

Peichuan Zhang, Cynthia Kenyon

[International Worm Meeting, 2007]

Peichuan Zhang AND Cynthia Kenyon, University of California, San Francisco Modulation of signaling activity of the insulin/IGF system, particularly in adipose tissues, has been shown to have strong effects on lifespan in many species. In nematode C. elegans, extending lifespan by down-regulating insulin/IGF signaling requires the activity of DAF-16/FOXO transcription factor. Previously, our lab has shown that activation of DAF-16/FOXO specifically in the intestine, which is also adipose tissue in nematodes, produces a large increase of lifespan of the whole animal [1]. We have proposed that this limited local activation of DAF-16/FOXO in the intestine can promote cellular viability and tissue integrity in other daf-16(-) responding tissues. Specifically, DAF-16/FOXO in the intestine must regulate yet un-identified downstream signaling pathway(s) that affect aging in other tissues. Notably, DAF-16/FOXO is known to regulate certain genes that directly encode or are involved in the production of signaling molecules (e.g., insulin-like peptides [2], secretory protein SCL-1 [3]) in daf-2 mutant animals. To understand the molecular mechanisms by which DAF-16/FOXO controls lifespan, we have analyzed how DAF-16/FOXO regulates the expression of its many downstream target genes at cellular level in the whole animal. First, we have found that many genes are regulated cell autonomously by DAF-16/FOXO, particularly in the intestine, in long-lived daf-2 mutant animals. Our results have further pinpointed the critical role of the adipose tissue intestine in lifespan control. On the other hand, we have also found that DAF-16/FOXO regulates certain genes in a cell non-autonomous fashion. In the latter case, activation of DAF-16/FOXO in a specific tissue (e.g., intestine) is able to induce the expression of downstream genes in another, responding tissue (e.g., hypodermis). To identify the downstream signaling pathway(s), we have performed a genetic screen to look for RNAi clones that may disrupt the cell non-autonomous gene regulation. We have identified a few candidate clones and we are currently re-testing these clones. From this screen, we hope to identify novel pathways that have strong effects on lifespan in animals.Reference:1) Libina, N., Berman, J., and Kenyon, C. (2003) Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. Cell 115:489-502.2) Murphy, C. et al. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 427:277-83.3) Ookuma, S., Fukuda, M., and Nishida, E. (2003) Identification of a DAF-16 transcriptional target gene, scl-1, that regulates longevity and stress resistance in Caenorhabditis elegans. Curr. Biol. 13: 427-31.