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In JoVE (1)
- Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans
Other Publications (12)
- Development (Cambridge, England)
- Development (Cambridge, England)
- Genesis (New York, N.Y. : 2000)
- American Journal of Physiology. Cell Physiology
- WormBook : the Online Review of C. Elegans Biology
- Development (Cambridge, England)
- BMC Developmental Biology
- Developmental Biology
- Current Biology : CB
- BMC Molecular Biology
- Genetics Research
- Developmental Biology
Articles by Cathy Savage-Dunn in JoVE
Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans
Jun Liang1, Sheng Xiong2,3, Cathy Savage-Dunn2,3
1Department of Science, Borough of Manhattan Community College, City Universtiy of New York (CUNY), 2Department of Biology, Queens College, The City University of New York (CUNY), 3Biochemistry Program, The Graduate Center, Queens College, The City University of New York (CUNY)
We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-β signaling.
Published February 13, 2013. Keywords: Developmental Biology, Genetics, Cellular Biology, Molecular Biology, Biochemistry, Basic Protocols, RNAi feeding technique, genetic screen, TGF-beta, body size, C. elegans, Caenorhabditis elegans, RNA-mediated Interference, RNAi, RNA, DNA, gene expression knock down, animal model
Other articles by Cathy Savage-Dunn on PubMed
Development (Cambridge, England). Nov, 2002 | Pubmed ID: 12397107
In C. elegans, a TGFbeta-related signaling pathway regulates body size. Loss of function of the signaling ligand (dbl-1), receptors (daf-4 and sma-6) or Smads (sma-2, sma-3 and sma-4) results in viable, but smaller animals because of a reduction in postembryonic growth. We have investigated the tissue specificity of this pathway in body size regulation. We show that different tissues are reduced in size by different proportions, with hypodermal blast cell size most closely proportional to body size. We show that SMA-3 Smad is expressed in pharynx, intestine and hypodermis, as has been previously reported for the type I receptor SMA-6. Furthermore, we find that SMA-3::GFP is nuclear localized in all of these tissues, and that nuclear localization is enhanced by SMA-6 activity. Interestingly, SMA-3 protein accumulation was found to be negatively regulated by the level of Sma/Mab pathway activity. Using genetic mosaic analysis and directed expression of SMA-3, we find that SMA-3 activity in the hypodermis is necessary and sufficient for normal body size. As dbl-1 is expressed primarily in the nervous system, these results suggest a model in which postembryonic growth of hypodermal cells is regulated by TGFbeta-related signaling from the nervous system to the hypodermis.
The Caenorhabditis Elegans Schnurri Homolog Sma-9 Mediates Stage- and Cell Type-specific Responses to DBL-1 BMP-related Signaling
Development (Cambridge, England). Dec, 2003 | Pubmed ID: 14627718
In Caenorhabditis elegans, the DBL-1 pathway, a BMP/TGFbeta-related signaling cascade, regulates body size and male tail development. We have cloned a new gene, sma-9, that encodes the C. elegans homolog of Schnurri, a large zinc finger transcription factor that regulates dpp target genes in Drosophila. Genetic interactions, the sma-9 loss-of-function phenotype, and the expression pattern suggest that sma-9 acts as a downstream component and is required in the DBL-1 signaling pathway, and thus provide the first evidence of a conserved role for Schnurri proteins in BMP signaling. Analysis of sma-9 mutant phenotypes demonstrates that SMA-9 activity is temporally and spatially restricted relative to known DBL-1 pathway components. In contrast with Drosophila schnurri, the presence of multiple alternatively spliced sma-9 transcripts suggests protein isoforms with potentially different cell sublocalization and molecular functions. We propose that SMA-9 isoforms function as transcriptional cofactors that confer specific responses to DBL-1 pathway activation.
Genesis (New York, N.Y. : 2000). Apr, 2003 | Pubmed ID: 12717735
In the nematode Caenorhabditis elegans, a TGFbeta-related signaling pathway regulates body size and male tail morphogenesis. We sought to identify genes encoding components or modifiers of this pathway in a large-scale genetic screen. Remarkably, this screen was able to identify essentially all core components of the TGFbeta signaling pathway. Among 34 Small mutants, many mutations disrupt genes encoding recognizable components of the TGFbeta pathway: DBL-1 ligand, DAF-4 type II receptor, SMA-6 type I receptor, and SMA-2, SMA-3, and SMA-4 Smads. Moreover, we find that at least 11 additional complementation groups can mutate to the Small phenotype. Four of these 11 genes, sma-9, sma-14, sma-16, and sma-20 affect male tail morphogenesis as well as body size. Two genes, sma-11 and sma-20, also influence regulation of the developmentally arrested dauer larval stage, suggesting a role in a second characterized TGFbeta pathway in C. elegans. Other genes may represent tissue-specific factors or parallel pathways for body size control. Because of the conservation of TGFbeta signaling pathways, homologs of these genes may be involved in tissue specificity and/or crosstalk of TGFbeta pathways in other animals.
American Journal of Physiology. Cell Physiology. Dec, 2004 | Pubmed ID: 15282194
Intracellular iron regulates gene expression by inhibiting the interaction of iron regulatory proteins (IRPs) with RNA motifs called iron-responsive elements (IREs). To assay this interaction in living cells we have developed two fluorescent IRE-based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity. The reporters were also sufficiently sensitive to distinguish apo- from holotransferrin in the medium, to detect the effect of modifiers of the transferrin pathway such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin neutrophil gelatinase-associated lipocalin (Ngal). In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron-dependent response. These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo.
WormBook : the Online Review of C. Elegans Biology. 2005 | Pubmed ID: 18050404
TGF-beta superfamily ligands play fundamental roles in the development and physiology of diverse animal species. Genetic and genomic analyses in the model organism Caenorhabditis elegans have contributed to the understanding of TGF-beta-related signal transduction mechanisms. In this chapter, I describe the currently characterized TGF-beta-related signals and signal transduction cassettes in C. elegans. Homology searches of the genome identify five TGF-beta-related genes, for which functions have been identified for three. Two of the TGF-beta-related genes, daf-7 and dbl-1, function through conventional signaling pathways. These signaling pathways are comprised of ser/thr kinase receptors, Smads, and transcription co-factors. A third TGF-beta-related gene, unc-129, functions in axonal guidance using novel signaling mechanisms. Thus, TGF-beta-related signaling in C. elegans proceeds via both conserved and novel paradigms that can inform studies in other animal systems.
C-terminal Mutants of C. Elegans Smads Reveal Tissue-specific Requirements for Protein Activation by TGF-beta Signaling
Development (Cambridge, England). Aug, 2005 | Pubmed ID: 16000380
TGF-beta signaling in the nematode Caenorhabditis elegans plays multiple roles in the development of the animal. The Sma/Mab pathway controls body size, male tail sensory ray identity and spicule formation. Three Smad genes, sma-2, sma-3 and sma-4, are all required for signal transduction, suggesting that the functional complex could be a heterotrimer. Because the C termini of Smads play important roles in receptor-mediated activation and heteromeric complex formation, we generated C-terminal mutations in the C. elegans Smad genes and tested their activities in vivo in each of their distinct developmental roles. We show that pseudophosphorylated SMA-3 is dominant negative in body size, but functional in sensory ray and spicule development. Somewhat differently, pseudophosphorylated SMA-2 is active in any tissue. The C-terminal mutants of SMA-4 function like wild type, suggesting that the SMA-4 C terminus is dispensable. Using a combination of different C-terminal mutations in SMA-2 and SMA-3, we found a complex set of requirements for Smad-phosphorylation state that are specific to each outcome. Finally, we detected a physical interaction of SMA-3 with the forkhead transcription factor LIN-31, which is enhanced by SMA-3 pseudophosphorylation and reduced in an unphosphorylatable mutant. We conclude that the tissue-specific requirements for Smad phosphorylation may result, in part, from the need to interact with tissue-specific transcription co-factors that have different affinities for phosphorylated and unphosphorylated Smad protein.
BMC Developmental Biology. 2007 | Pubmed ID: 17472754
In insects and in mammals, male sperm and seminal fluid provide signaling factors that influence various aspects of female physiology and behavior to promote reproductive success and to compete with other males. It is less apparent how important such signaling is in the context of a self-fertile hermaphrodite species. We have addressed this question in the nematode Caenorhabditis elegans, which can reproduce either by hermaphrodite self-fertilization or by male-hermaphrodite mating.
Transcriptional Repressor and Activator Activities of SMA-9 Contribute Differentially to BMP-related Signaling Outputs
Developmental Biology. May, 2007 | Pubmed ID: 17397820
In the nematode Caenorhabditis elegans, the BMP-related growth factor DBL-1 regulates body size and male tail morphogenesis via a conserved receptor/Smad signaling pathway. Smads are transcription factors, but rely on transcription cofactors for appropriate regulation of target genes in response to TGF-beta- and BMP-related signals. In the DBL-1 pathway, sma-9 encodes multiple zinc finger transcription factors homologous to Drosophila Schnurri, which functions in Dpp/BMP signaling. We have studied the molecular functions of SMA-9 as a model for transcription cofactor-dependent regulation of gene expression. Using SMA-9 fusions to known transcriptional activators and repressors, we demonstrate that SMA-9 acts primarily as a transcriptional repressor in body size regulation in vivo. In contrast, both activator and repressor functions contribute to male tail patterning. We further show that different SMA-9 regions have intrinsic repressor and activator activities using a yeast transcription assay. We use microarray analysis to identify transcriptional target genes in body size regulation. Consistent with the importance of repression in mediating body size regulation, we find more repressed genes than activated genes in this pool. Finally, we identify five transcriptional targets with body size and/or male tail patterning phenotypes, including transcription factors related to Runx and fos and signaling molecules related to hedgehog and patched. Our results thus suggest that SMA-9 products function differentially as transcriptional repressors and activators in DBL-1/BMP pathway regulated body size and male tail morphogenesis.
Current Biology : CB. Sep, 2008 | Pubmed ID: 18786372
Proper growth and development of multicellular organisms require the tight regulation of cell growth, cell division and cell death. A recent study has identified a novel regulatory link between two of these processes: cell growth and cell death.
Alternative Trans-splicing of Caenorhabditis Elegans Sma-9/schnurri Generates a Short Transcript That Provides Tissue-specific Function in BMP Signaling
BMC Molecular Biology. 2010 | Pubmed ID: 20565799
Transcription cofactors related to Drosophila Schnurri facilitate the transcriptional programs regulated by BMP signaling in C. elegans, Drosophila, Xenopus, and mouse. In different systems, Schnurri homologs have been shown to act as either agonists or antagonists of Smad function, and as either positive or negative regulators of transcription. How Schnurri proteins achieve this diversity of activities is not clear. The C. elegans sma-9/schnurri locus undergoes alternative splicing, including an unusual trans-splicing event that could generate two non-overlapping shorter transcripts.
Non-stringent Tissue-source Requirements for BMP Ligand Expression in Regulation of Body Size in Caenorhabditis Elegans
Genetics Research. Dec, 2011 | Pubmed ID: 22189608
In Caenorhabditis elegans, the Bone Morphogenetic Protein (BMP)-related ligand Dpp- and BMP-like-1 (DBL-1) regulates body size by promoting the larval and adult growth of the large epidermal syncytium hyp7 without affecting cell division. This system provides an excellent model for dissecting the growth-promoting activities of BMP ligands, since in this context the growth and differentiation functions of DBL-1 are naturally uncoupled. dbl-1 is expressed primarily in neurons and the DBL-1 ligand signals to its receptors and Smad signal transducers in the target tissue of the epidermis. The requirements constraining the source(s) of DBL-1, however, have not previously been investigated. We show here that dbl-1 expression requirements are strikingly relaxed. Expression in non-overlapping subsets of the endogenous expression pattern, as well as ectopic expression, can provide sufficient levels of activity for rescue of the small body size of dbl-1 mutants. By analysing dbl-1 expression levels in transgenic strains with different degrees of rescue, we corroborate the model that DBL-1 is a dose-dependent regulator of growth. We conclude that, for body size regulation, the site of expression of dbl-1 is less important than the level of expression.
C. Elegans ADAMTS ADT-2 Regulates Body Size by Modulating TGFÎ² Signaling and Cuticle Collagen Organization
Developmental Biology. Apr, 2011 | Pubmed ID: 21256840
Organismal growth and body size are influenced by both genetic and environmental factors. We have utilized the strong molecular genetic techniques available in the nematode Caenorhabditis elegans to identify genetic determinants of body size. In C. elegans, DBL-1, a member of the conserved family of secreted growth factors known as the Transforming Growth Factor Î² superfamily, is known to play a major role in growth control. The mechanisms by which other determinants of body size function, however, is less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma-21. We now demonstrate that sma-21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with or in multiple size regulatory pathways. We demonstrate that ADT-2 is required for normal levels of expression of a DBL-1-responsive transcriptional reporter. We further demonstrate that adt-2 regulatory sequences drive expression in glial-like and vulval cells, and that ADT-2 activity is required for normal cuticle collagen fibril organization. We therefore propose that ADT-2 regulates body size both by modulating TGFÎ² signaling activity and by maintaining normal cuticle structure.