RNA interference

RNA interference (RNAi) refers to the silencing of gene expression by the overexpression of RNA molecules. This process is associated with a cellular and nuclear defense mechanism used to combat molecular parasites such as transposons and viruses. In addition, RNA interference has been shown to play a regulatory role in development. Work in C. elegans and other organisms have identified many key regulators and pathways necessary for this process. Specifically, RNAi has been adapted into a tool for the study of gene function; through the use of RNAi, the expression of a target gene can be inhibited by the reverse engineering of a corresponding dsRNA.

Gene expression

The production of a functional product from a given gene. Gene products can stem from protein coding or non-protein coding sequences. Non-protein coding genes include tRNAs, rRNAs, and microRNAs. The regulation of gene expression, (timing, location, and amount) is the basis for cell specification, differentiation, and morphogenesis during development and allows the animal to respond and adapt to environmental stimuli.

Wnt signaling pathway

Wnt glycoproteins are signaling molecules that control a wide range of developmental processes and is a conserved feature of metazoan development. In C. elegans Wnt signaling has been shown to play a role in cell fate specification and determination of cell polarity, cell migration, and axis determination during axon outgrowth. A 'canonical' Wnt signaling pathway has been elucidated in vertebrate and invertebrate model systems where Wnt binding leads to the stabilization of the transcription factor beta-catenin, which then enters the nucleus to regulate Wnt pathway target genes. Like other species, the C. elegans genome encodes multiple genes for Wnt ligands, EGL-20, LIN-44, MOM-2, CWN-1, CWN-2) and Wnt receptors (LIN-17, MOM-5, MIG-1, CFZ-2, LIN-18). Canonical Wnt signaling in C. elegans, utilizes the beta-catenin BAR-1 to convert POP-1 into an activator and controls the expression of several homeobox genes. However, unlike vertebrates or Drosophila, the C. elegans genome encodes multiple beta-catenin genes (HMP-2, BAR-1, SYS-1, WRM-1), which give rise to noncanonical Wnt signalling pathways: for example, the endoderm induction pathway requires the beta-catenin WRM-1 and parallel input from a mitogen-activated kinase (MAPK) pathway to downregulate POP-1.

RNA splicing

During pre-messenger RNA (pre-mrRNA) processing, cis-splicing reactions remove non-coding introns and joins coding exons. The majority of splicing is catalyzed by spliceosomes, large RNA-protein complexes composed of small nuclear ribonucleoproteins (snRNPs) that recognize splice donor and acceptor sites within the nascent RNA strand. Alternative mRNAs for a given gene can be created during the splicing reactions through varying the degree of cutting and splicing of introns and exons guided by alternative splice sites. C. elegans also exhibits trans-splicing where sequences from different primary RNA transcripts are used to make the mRNA. In C. elegans, ~70% of transcripts are trans-spliced with either a splice leader sequence SL1 or SL2 joined to the final transcript. Splice leaders are important

Programmed cell death

Programmed cell death (PCD), or apoptosis, is an integral component of C. elegans development. During development, 131 cells are fated to die by apoptosis. PCD is easily observed with Nomarski optics in the C. elegans embryo; the nucleus of the apoptotic cell becomes refractile, resembling a flat button, making C. elegans an easy model for following apoptotic cell death. PCD in C. elegans can be divided into three main phases (with participating genes): specification of which cells should live or die (ces-1, ces-2, in general); activation of the cell killing machinery (egl-1, ced-9, ced-4 and ced-3); and an execution phase where the dying cell is dismantled and removed through phagocytosis (ced-1,-2,-5,-6,-7,-10 and-12), which occurs in concert with apoptotic DNA degradation (cps-6, nuc-1). Molecular and biochemical studies in C. elegans revealed programmed cell death mechanisms conserved in humans. The regulatory pathway that controls cell death is composed of conserved cell death activators and inhibitors: EGL-1 and BH3-domain-only proteins, CED-9 and Bcl-2, CED-4 and Apaf-1, and CED-3 and caspases, in nematodes and mammals, respectively. Further the degradation of chromosomal DNA involves a mitochondrial proapoptotic endonuclease: endonuclease G (EndoG), and AIF in mammals and their orthologs CPS-6 and WAH-1 in worms.

Dauer development

Nematodes like C. elegans can survive the harshest of environmental conditions by adopting an alternative developmental program and entering into the dauer state. Dauer stage worms are reproductively immature. Entry into or exit from dauer in C. elegans is a carefully weighted decision taking into account factors such as food availability and population density, which is measured by pheromone levels in the population. Dauer development entails metabolic changes, such as increasing fat storage, and remodeling of tissues leading to the arrest of normal developmental programs, and a significant increase in lifespan. These metabolic and physiological changes are choreographed by changes in gene expression. One gene in particular, daf-16, a homolog of the human FKHR and AFX genes, appears to play a major transcriptional regulatory role in this process. A second key gene, a receptor tyrosine kinase and insulin/IGF receptor ortholog, daf-2, negatively regulates daf-16. Mutations in daf-2 result in a Dauer consititute phenotype and an extended lifespan. The C. elegans dauer stage larvae are often equated with the IJ infective juvenile stage larvae of parasitic nematodes.

EGF signaling pathway

The Epidermal Growth Factor (EGF) signaling pathway was initially identified by Nobel Prize winner Stanley Cohen who found EGF to be involved in numerous cellular signaling events in mouse development. The single C. elegans EGF ligand, lin-3 and its receptor, lin-28 were initially found in screens for vulval development mutants. Further analysis of similar mutants, led to the identification of the C. elegans core EGFR signaling pathway components, SEM-5/GRB2, SOS-1, LET-60/RAS, LIN-45/RAF, MEK-2/MEK, and MPK-1. Positive regulators of this pathway were subsequently found in suppressor screens, and include KSR-1, SUR-8/SOC-2, and SUR-6. Negative modulators were found in further forward and reverse genetic screens and include UNC-101, SLI-1, GAP-1, SUR-5 and ARK-1 (in conjunction with GRB-2). LIN-3/EGF acts as a signal from the single anchor cell to induce neighboring undifferentiated epithelial cells to adopt the vulval fate during larval development. Impairing this signaling event results in a failure of vulval fate specification, and eventually a vulvaless animal that cannot lay eggs (the "bag of worms" phenotype). As in the mouse, EGF signaling was found to be involved in a number of other processes, although perhaps most importantly it is required for larval survival; homozygous let-23 null worms die at the L1 stage. Further, EGF signaling has been shown to function in coordinating vulva to uterus communication during development, promoting male spicule development, inducing P12.p cell fate in the epithelia, at the same time acting as a physiological signal, promoting ovulatory contractions of the gonad sheath cells, inducing a reversible nervous system quiescence during each larval molt, and regulating protein and calcium homeostasis in the adult worm.

Insulin/IGF signaling pathway

The Insulin/IGF signaling (IIS) pathway is an evolutionarily conserved pathway that regulates fundamental biological processes such as development, metabolism, fecundity, cellular stress resistance and aging, in many organisms including fly and mammals. In C. elegans, IIS regulates dauer formation, longevity, fat metabolism, stress response, and innate immunity. This pathway is initiated through the Insulin-IGF receptor ortholog daf-2, which activates a phosphorylation cascade. The ultimate outcome of the signaling pathway is the phosphorylation of the forkhead transcription factor DAF-16. Phosphorylated DAF-16 is retained in the cytoplasm, functionally inhibiting the transcription of a number of DAF-16 regulated genes.

Corpse engulfment

Cell corpses are removed through phagocytosis as a final step of apoptosis. The removal of these corpses protects the organism from harmful contents that may be released from the dying cell. Cell corpse removal requires properly identifying, ingesting and degrading the dying cell. During this process the engulfing cell undergoes cytoskeletal reorganization to send a projection towards the apoptotic cell. Over a dozen genes have been identified in C. elegans that play a role in apoptosis, with many of them, including ced-12, ced-10, ced-5, and ced-2, being required for this final cell corpse engulfment step.

Response to osmotic stress

The increase or decrease in solute concentrations outside of an organism can result in a stress response, which can include changes in behavior or gene expression. As an animal that is hyperosmotic to its normal environment, C. elegans exhibits structures and mechanisms to assess and deal with changes in the osmolarity of its environment. On a behavioral level, C. elegans avoids regions of high osmolarity. On a physiological level, hypertonic stress results in the upregulation of glycerol 3-phosphate dehydrogenases, gpdh-1 (strong) and gpdh-2 (weak). These genes aid in adapting to hypertonic environments by adjusting glycerol concentrations within cells.