[
International Worm Meeting,
2017]
Transforming Growth Factor-beta (TGF- beta ) is a family of secreted cell signaling ligands. DBL-1, TGF- beta superfamily member in the roundworm C. elegans, is secreted from nervous tissue, but must be received by receptors in neighboring cells to regulate body size, secretion of specialized extracellular matrix (surface barrier), and other processes. The control of TGF- beta within the secreting cells is not well known in any system. We are using the C. elegans model system to identify new regulators involved in this conserved signaling pathway. For that, we are using two approaches to identify TGF- beta regulators. First, we used a C. elegans strain expressing GFP-tagged DBL-1 in an RNA interference (RNAi) screen to determine candidates involved in the regulation for this pathway. We knocked down candidate genes by RNAi and assayed changes in GFP-tagged DBL-1 fluorescence intensity within the ventral nerve cord. We then asked if the candidate gene products found with this screening co-localized with GFP-tagged DBL-1. Our results show in vivo the implication of a novel protein secretory pathway for DBL-1 transport. Second, we are performing co-immunoprecipitation using GFP-tagged DBL-1 as a bait to identify the different proteins that directly bind DBL-1. Together, this work is expected to identify proteins that interact with DBL-1 and regulate it within the secreting cell and in the extracellular milieu between DBL-1-secreting and receiving cells.
[
MicroPubl Biol,
2019]
Loss of some cuticle collagens negatively affects DBL-1 pathway signaling in a stage-dependent manner (Lakdawala et al. 2019; Madaan et al. 2019). We previously observed that in one-day old adult animals, loss of
dpy-2 or
dpy-9 had no effect on GFP::DBL-1 expressed from the
dbl-1 promoter (Beifuss and Gumienny 2012; Lakdawala et al. 2019). We also observed that expression of
spp-9p::gfp, a reporter that is negatively regulated by the DBL-1 pathway, was not affected in one-day old adult animals (Roberts et al. 2010; Lakdawala et al. 2019). Post-embryonic expression of
dpy-2 and
dpy-9 is highest in L2 and L3, but low in L4 and even lower in young adults (Gerstein et al. 2010). Because cuticle secreted in one stage creates the cuticle in the next stage, this is consistent with the observation that loss of
dpy-2 and
dpy-9 has no effect on DBL-1 signaling in the adult (Hall and Altun 2008; Lakdawala et al. 2019). However, the DPY-2 and DPY-9 expression patterns led us to ask if DBL-1 signaling is affected at L4 by loss of
dpy-2 or
dpy-9. To our surprise, we found that
dpy-2(
e8) or
dpy-9(
e12) resulted in significant increases of GFP::DBL-1 fluorescence within DBL-1-secreting cells in L4 animals compared to control populations (Figure 1, Table 1). We also tested DBL-1 pathway reporter activity in these
dpy-2 and
dpy-9 mutants. Consistent with the increased GFP::DBL-1 fluorescence at L4, we observed significantly decreased fluorescence from the
spp-9p::gfp reporter at L4 (Figure 1, Table 1). These results are consistent with DPY-2 and DPY-9 affecting DBL-1 signaling at the L4 stage but not at the adult stage. This suggests that these two collagens have a stage-specific effect on DBL-1 signaling, but this effect is normally inhibitory, as loss of
dpy-2 or
dpy-9 increased GFP::DBL-1 fluorescence and decreased
spp-9p::GFP fluorescence.
Gilleland, Cody, Norton, Stephanie, Haggarty, Stephen, Samara, Chrysanthi, Yanik, Mehmet, Rohde, Christopher
[
International Worm Meeting,
2009]
Therapeutic treatment of central nervous system pathologies, such as spinal cord injuries, brain trauma, stroke, and neurodegenerative disorders, will greatly benefit from the discovery of small molecules that enhance neuronal growth after injury. Identification of a diverse repertoire of such molecules and of their cellular targets can also provide important tools for fundamental investigations of the mechanisms involved in the regeneration process. Currently, small-molecule screens for factors affecting neuronal regrowth can only be performed using simple in vitro cell culture systems. However, these systems do not truly represent in vivo environment. Importantly, off-target, toxic or lethal effects of chemical compounds can only manifest in model organisms with multiple tissue types. Thus, the thorough investigation of neuronal regeneration mechanisms requires in vivo neuronal injury models. In vivo neuronal regeneration studies have been performed mainly in mice and rats. However, their long developmental periods, complicated genetics and biology, and expensive maintenance limit large-scale studies in these animals. We previously demonstrated femtosecond laser microsurgery as a highly precise and reproducible injury method for studying axonal regeneration mechanisms in Caenorhabditis elegans1,2. Wild type nematodes move constantly, and to perform precise laser axotomy or imaging at the cellular level, animals must be immobilized. Therefore, we developed microfluidic on-chip technologies that allow automated and rapid manipulation, orientation, and non-invasive immobilization of C. elegans for sub-cellular resolution imaging and femtosecond-laser microsurgery3,4. These technologies can be used for high-throughput genetic and compound assays. We report here the results of the in vivo small-molecule screens for compounds affecting axonal regeneration after laser-induced axotomy in C. elegans. Using the technologies described above, we screened a library of small molecules. A number of compounds with a wide variety of cellular targets, such as cytoskeletal components, vesicle trafficking, and protein kinases were found to affect mechanosensory neuron regeneration following laser axotomy. REFERENCES: 1.Yanik M.F. et al., (2004). Nature. 432, 822 2.Yanik M.F. et al., (2006). IEEE J Sel Top Quant Electron. 12, 1283-91 3.Rohde C.B. et al., (2007). Proc Natl Acad Sci U S A. 104, 13891-5 4.Zeng F. et al., (2008). Lab Chip. 8, 653-6.
[
International Worm Meeting,
2011]
Traumatic neuronal damage triggers large intracellular calcium transients that are associated both with subsequent neuronal degeneration and cell death, and alternatively with regenerative repair and outgrowth [1, 2]. Combining femtosecond laser ablation with the use of genetically encoded calcium sensitive fluorophores, we can optically measure intracellular calcium signaling within a specific target neuron of an intact adult C. elegans in response to precision laser damage [3-5]. Here we characterize the damage induced calcium signal across a variety of laser ablation experiments. This includes variations in the proximity of the damage point to the cell soma, targeting of distinct morphological structures and different neuronal types, modulation of laser power, different animal ages, and multiple surgeries to the same neuron. Results are revealing complex subcellular calcium dynamics that are precisely tuned to control cell fate. We find that in general large, extended calcium transients correlate with cell degeneration, while particularly small transients are associated with reduced regenerative outgrowth. This suggests an optimum window in which elevation of cytoplasmic calcium successfully facilitates neuronal repair and outgrowth without initiating cell death. Our results are helping to pinpoint the critical aspects of this signaling pathway that dictate neuronal survival and regeneration following traumatic damage.
1.Coleman, M., Axon degeneration mechanisms: commonality amid diversity. Nat Rev Neurosci, 2005. 6(11): p. 889-98.
2.Kamber, D., H. Erez, and M.E. Spira, Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone. Exp Neurol, 2009. 219(1): p. 112-25.
3.Yanik, M.F., et al., Neurosurgery: functional regeneration after laser axotomy. Nature, 2004. 432(7019): p. 822.
4.Gabel, C.V., et al., Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans. Development, 2008. 135(6): p. 1129-36.
5.Ghosh-Roy, A., et al., Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase. Journal of Neuroscience, 2010. 30(9): p. 3175-3183.
[
International Worm Meeting,
2005]
The
med-1 and
med-2 genes encode a pair of essentially identical GATA factor- related transcription factors that have been proposed to be necessary for specification of the entire C. elegans endoderm (intestine or E lineage) as well as MS-derived mesoderm [1].
med-1 and
med-2 are proposed to be the direct downstream targets and the principal effectors of the maternally provided SKN-1 transcription factor;
med-1 and
med-2 would thus occupy the pivotal interface between maternal and zygotic control of gene expression. The conclusion that
med-1 and
med-2 are essential for C. elegans endoderm specification was based on a partially-penetrant (~50%) loss of endoderm markers produced by RNA-mediated interference (RNAi) [1]. To determine whether this partial penetrance reflects: (i) inefficient RNAi against early zygotic transcripts; (ii) approximately the expected level of endoderm loss in
skn-1 nulls, or; (iii) a further redundancy in the pathway of endoderm specification, we constructed worm strains that segregate embryos lacking both the
med-1 gene (because of a gene-specific deletion) and the
med-2 gene (using either of two overlapping chromosomal deficiencies). Contrary to expectations, we observe that the large majority (80 - 97 percent) of
med-2(-);
med-1(-) embryos still express markers of endoderm differentiation. Thus, we conclude that zygotic expression of the
med-1 and
med-2 genes are not necessary to specify the C. elegans endoderm. Our results could possibly suggest that the C. elegans endoderm can be efficiently specified by a new pathway involving a maternal contribution from the med genes and that (presumably) does not require SKN-1. However, any maternal contribution of the med genes has so far resisted conventional RNAi (maternal injection), under conditions where parallel injections with control dsRNAs (e.g.
glp-1,
let-413) are highly effective. 1.Maduro, M.F., Meneghini, M.D., Bowerman, B., Broitman-Maduro, G., and Rothman, J.H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol Cell 7, 475-485.
[
International Worm Meeting,
2009]
A deletion screen (1) has developed a resource of mutants carrying deletions in most C. elegans microRNAs (miRNAs), short regulatory RNAs that act to repress gene expression post-transcriptionally. The function of most miRNAs in C. elegans is unknown and the majority of mutants show no gross abnormal phenotype (1). The miR-51 family is a redundant and conserved family of six miRNAs whose putative promoters drive GFP in overlapping but not identical spatial patterns. Mutants lacking multiple members of the miR-51 family show gradually more severe defects in growth and animals lacking all members of the family arrest as larvae with an unattached pharynx (Pun) phenotype. These mutant phenotypes can be rescued by short genomic regions encoding any of the miR-51 family miRNAs, demonstrating their redundancy. The pharynx of C. elegans forms from a ball of cells specified in the interior of the developing embryo and extends anteriorly to attach to the hypodermis (2). This attachment requires the arcade cells, which become polarised during pharyngeal attachment and form a continuous epithelium between the pharyngeal and hypodermal cells (2). In the early development of
mir-51 family mutant larvae, this epithelium is labelled with a DLG-1-mCHERRY fusion protein indicating that the arcade cells have polarised. However, the attachment of the cells to the pharynx is not maintained and arcade cells separate from the anterior pharynx. We have identified the first target mRNA of the miR-51 family of microRNAs as the cadherin,
cdh-3. A reporter for
cdh-3 drives GFP expression in the arcade cells and the 3''UTR of
cdh-3 mRNA confers direct regulation by the miR-51 family of miRNAs. However, mutations in
cdh-3 or
cdh-3(RNAi) fail to suppress the Pun phenotype, suggesting the regulation of a network of miR-51 family target mRNAs is important in the maintenance of pharyngeal attachment. (1) Miska, E. A., Alvarez-Saavedra, E. A., Abbott, A. L., Lau, N. P., Hellman, A. B., McGonagle, S., Bartel, D. P., Ambros, V. R., Horvitz, H. R. - PLoS Genetics, 2007) (2) Portereiko, M.F., Mango, S.E. - Developmental Biology (2001).
[
West Coast Worm Meeting,
2004]
Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .