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In JoVE (2)
- Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI
- RNAi Screening to Identify Postembryonic Phenotypes in C. elegans
Other Publications (6)
Articles by Tina L. Gumienny in JoVE
JoVE General
Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI
We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
JoVE General
RNAi Screening to Identify Postembryonic Phenotypes in C. elegans
Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center
We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.
Other articles by Tina L. Gumienny on PubMed
Lon-1 Regulates Caenorhabditis Elegans Body Size Downstream of the Dbl-1 TGF Beta Signaling Pathway
In Caenorhabditis elegans, two well-characterized TGF beta signaling cascades have been identified: the Small/Male tail abnormal (Sma/Mab) and Dauer formation (Daf) pathways. The Sma/Mab pathway regulates body size morphogenesis and male tail development. The ligand of the pathway, dbl-1, transmits its signal through two receptor serine threonine kinases, daf-4 and sma-6, which in turn regulate the activity of the Smads, sma-2, sma-3, and sma-4. In general, Smads have been shown to both positively and negatively regulate the transcriptional activity of downstream target genes in various organisms. In C. elegans, however, target genes have remained elusive. We have cloned and characterized lon-1, a gene with homology to the cysteine-rich secretory protein (CRISP) family of proteins. lon-1 regulates body size morphogenesis, but does not affect male tail development. lon-1 is expressed in hypodermal tissues, which is the focus of body size determination, similar to sma-2, sma-4, and sma-6. Using genetic methods, we show that lon-1 lies downstream of the Sma/Mab signaling cascade and demonstrate that lon-1 mRNA levels are up-regulated in sma-6-null mutant animals. This provides evidence that lon-1 is negatively regulated by Sma/Mab pathway signaling. Taken together, these data identify lon-1 as a novel downstream target gene of the dbl-1 TGF beta-like signaling pathway.
The Other Side of TGF-beta Superfamily Signal Regulation: Thinking Outside the Cell
The transforming growth factor beta (TGF-beta) superfamily of paracrine and autocrine signaling molecules regulates a vast array of developmental and homeostatic processes and is itself exquisitely regulated. The misregulation of these molecules often results in cancer and other diseases. Here, we focus on new research that explores how TGF-beta superfamily signaling is controlled between the secreting cell and the target cell. Regulation can occur upon ligand secretion (in a latent protein complex) and in the creation of signaling gradients. Proteins in the extracellular milieu sequester ligand away from or facilitate ligand binding to receptor serine kinases. Ligands even positively regulate their own negative regulators. Studies of how TGF-beta signaling is regulated extracellularly have broadened our understanding of TGF-beta pathways, and could provide clues to our understanding and treatment of diseases resulting from misregulation of these pathways.
A Small Issue Addressed
Cell size is an important determinant of body size. While the genetic mechanisms of cell size regulation have been well studied in yeast, this process has only recently been addressed in multicellular organisms. One recent report by Wang et al. (2002) shows that in the nematode C. elegans, the TGFbeta-like pathway acts in the hypodermis to regulate cell size and consequently body size.1 This finding is an exciting step in discovering the molecular mechanisms that control cell and body size.
Glypican LON-2 is a Conserved Negative Regulator of BMP-like Signaling in Caenorhabditis Elegans
Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.
Caenorhabditis Elegans SMA-10/LRIG is a Conserved Transmembrane Protein That Enhances Bone Morphogenetic Protein Signaling
Bone morphogenetic protein (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf) animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.
Regulation of Genes Affecting Body Size and Innate Immunity by the DBL-1/BMP-like Pathway in Caenorhabditis Elegans
Bone morphogenetic proteins (BMPs) are members of the conserved transforming growth factor beta (TGFbeta superfamily, and play many developmental and homeostatic roles. In C. elegans, a BMP-like pathway, the DBL-1 pathway, controls body size and is involved in innate immunity. How these functions are carried out, though, and what most of the downstream targets of this pathway are, remain unknown.
