Due to its genetic accessibility and other advantages, the nematode Caenorhabditis elegans has been a powerful model system for the study of many mainstream biological problems, including those closely related to human diseases. In recent years, we have used this model system to address important questions.
Spatial and Temporal Control of Cell Differentiation
Cell-cell communication plays a key role in directing cells to differentiate during animal development. Vulval induction in C. elegans hermaphrodites is controlled by multiple cell signaling and regulatory pathways, including a conserved RTK/RAS/MAPK signal transduction pathway that induces three epidermal cells to differentiate into vulval tissues. By screening mutations that either enhance or suppress the phenotypes of existing mutations, we have identified many genes that encode key factors relaying the signals from RAS, factors positively or negatively modifying the activity of the pathway, and factors involved in regulating the timing of vulval development.
Vulval induction is also inhibited by the synthetic multivulva (SynMuv) genes that encode transcription and by chromatin-associated factors, including the ortholog of the mammalian tumor-suppressor RB. In collaboration with Iva Greenwald (HHMI, Columbia University College of Physicians and Surgeons) and Paul Sternberg (HHMI, California Institute of Technology), we have shown that the SynMuv A and SynMuv B gene classes are functionally redundant for transcriptional repression of the key target gene lin-3/EGF in the large epidermal syncytial cell hyp7. These results provide key insights into the mechanism of the SynMuv phenotype and underscore the importance of repressing gene expression in generating specific cell signaling events for developmental pattern formation.
Using a genome-wide screen, we have identified more than 30 genes that encode potential chromatin-modifying proteins that antagonize the functions of class B SynMuv genes in a variety of cellular and developmental processes, including vulval induction, germline-soma distinction, RNA interference (RNAi), and somatic transgene silencing. These results suggest that multiple chromatin-remodeling complexes are involved in regulating the expression of specific genes for proper developmental decisions.
Besides the positional cues provided by several signaling pathways, a developmental timing control mechanism mediated by genes in the so-called heterochronic pathway provides temporal regulation of vulval development. In the suppressor screens for factors involved in vulval differentiation, we have identified mutations in five genes that regulate the timing of postembryonic development. We have analyzed two of these genes, ain-1 and lin-66, extensively and revealed their roles in timing regulation. Our studies of lin-66 indicate that it represses the expression of lin-28, the master timing regulator. We also provide evidence that the stage-specific expression of lin-28 is regulated by multiple independent mechanisms, including inhibitory regulations by lin-66, nuclear receptor nhr-12, and multiple microRNAs (miRNAs). Analysis of genes defined by additional suppressors is under way. (A grant from the National Institutes of Health provided partial support for these projects.)
Mechanism of MicroRNA Functions
Our analysis of the ain-1 gene has connected the research in the laboratory to the analysis of miRNA functions. Our data indicate that AIN-1 is structurally similar to human protein GW182 and interacts with a protein complex containing an Argonaute protein, Dicer, and miRNAs. Characterizations using molecular methods suggest that AIN-1 regulates miRNA functions by interacting and localizing miRISCs (miRNA-induced silencing complexes) to processing bodies, facilitating degradation of translational inhibition of mRNA targets. We recently identified the AIN-2 protein as the C. elegans homolog of AIN-1. We found that AIN-1 and AIN-2 act redundantly in forming miRISC complexes and in regulating multiple developmental events. We are continuing to analyze the functions of these complexes. (A grant from the National Institutes of Health provided support for this project.)
Functions and Regulation of Branched Fatty Acids
Fatty Acids (FAs) are components of a large variety of lipid molecules that play essential roles in all biological processes. The maintenance of proper levels and ratios between different FA molecules is critical for their cellular functions. We have applied C. elegans genetics, gas chromatography, DNA microarray, and mass spectrometry to the study of FA biosynthesis and functions. Our efforts elucidated the roles of the little-known mono-methyl branched-chain fatty acids (mmBCFAs) through analysis of two elongases and other enzymes in mmBCFA biosynthesis. We demonstrated that mmBCFAs are essential for C. elegans growth and development. Our recent genetic analysis has indicated that at least some of the mmBCFA functions are regulatory. Expression analyses using microarrays have shown that the mmBCFA levels affect the expression of a number of genes, while some of these genes in turn regulate biosynthesis of mmBCFAs, revealing a regulatory circuit that controls the proper level of mmBCFAs.
Nuclear Positioning, Cell Migration, and Morphogenesis During Development
The position of the nucleus within a cell is important to the proper function of a wide variety of cell types. In collaboration with H. Robert Horvitz (HHMI, Massachusetts Institute of Technology), we previously established the function of UNC-84 and UNC-83 in nuclear positioning at the nuclear envelope and defined the SUN-domain protein family by identifying the mammalian SUN1 and SUN2 proteins as the homologs of UNC-84. Our studies of UNC-83 and ANC-1 established the role in nuclear migration and anchorage of proteins containing a KASH domain and the role of the SUN protein in anchoring the KASH protein at the nuclear envelope.
In collaboration with researchers at the Institute of Developmental Biology and Molecular Medicine (IDM, Fudan University) and Mark Grady and Joshua Sanes (Washington University), we also explored the physiological functions of the KASH-SUN interaction at the nuclear envelope in mice and Drosophila. We found that KASH-containing Syne proteins play critical roles in anchoring both synaptic and nonsynaptic nuclei in each skeleton muscle cell that contains many nuclei. In Drosophila, the homolog is essential in anchoring the nurse cell nuclei during cytoplasmic transfer during oogenesis. Furthermore, the students at IDM determined that SUN1 is required for telomere attachment to the nuclear envelope and gametogenesis in mice.
We have been carrying out genetic screens for genes involved in cell migration during morphogenesis and C. elegans development. We have identified a number of genes that interact with cell adhesion molecules for cell migration functions in embryos, and genes that regulate cell migration and cytokinesis in larval epidermal cells.
Genetic Redundancy and Tumor-Suppressor Functions
Genetic (or functional) redundancy by structurally unrelated genes is an extremely common biological phenomenon and an impediment for biologists seeking to determine gene functions through straightforward genetic approaches. We have designed and performed screens to isolate mutations that synthetically interact with null mutations in the C. elegans orthologs of two human tumor-suppressor genes, RB and Pten. By analyzing genes acting in concert with RB, we and David Fay (University of Wyoming) have revealed several roles of the worm RB in regulation of cell proliferation, organ morphogenesis, and larval growth, in addition to the known vulval repression function. We have used a genome-wide RNAi screen to show that at least 27 genes collaborate with the worm Pten gene for various functions previously concealed by genetic redundancy, including embryogenesis, cuticle turnover, egg laying, and oocyte maturation.