Regulation of signaling genes by TGFbeta during entry into dauer diapause in C. elegans

Abstract

Background: When resources are scant, C. elegans larvae arrest as long-lived dauers under the control of insulin/IGF- and TGFbeta-related signaling pathways. However, critical questions remain regarding the regulation of this developmental event. How do three dozen insulin-like proteins regulate one tyrosine kinase receptor to control complex events in dauer, metabolism and aging? How are signals from the TGFbeta and insulin/IGF pathways integrated? What gene expression programs do these pathways regulate, and how do they control complex downstream events?

Results: We have identified genes that show different levels of expression in a comparison of wild-type L2 or L3 larvae (non-dauer) to TGFbeta mutants at similar developmental stages undergoing dauer formation. Many insulin/IGF pathway and other known dauer regulatory genes have changes in expression that suggest strong positive feedback by the TGFbeta pathway. In addition, many insulin-like ligand and novel genes with similarity to the extracellular domain of insulin/IGF receptors have altered expression. We have identified a large group of regulated genes with putative binding sites for the FOXO transcription factor, DAF-16. Genes with DAF-16 sites upstream of the transcription start site tend to be upregulated, whereas genes with DAF-16 sites downstream of the coding region tend to be downregulated. Finally, we also see strong regulation of many novel hedgehog- and patched-related genes, hormone biosynthetic genes, cell cycle genes, and other regulatory genes.

Conclusions: The feedback regulation of insulin/IGF pathway and other dauer genes that we observe would be predicted to amplify signals from the TGFbeta pathway; this amplification may serve to ensure a decisive choice between "dauer" and "non-dauer", even if environmental cues are ambiguous. Up and down regulation of insulin-like ligands and novel genes with similarity to the extracellular domain of insulin/IGF receptors suggests opposing roles for several members of these large gene families. Unlike in adults, most genes with putative DAF-16 binding sites are upregulated during dauer entry, suggesting that DAF-16 has different activity in dauer versus adult metabolism and aging. However, our observation that the position of putative DAF-16 binding sites is correlated with the direction of regulation suggests a novel method of achieving gene-specific regulation from a single pathway. We see evidence of TGFbeta-mediated regulation of several other classes of regulatory genes, and we discuss possible functions of these genes in dauer formation.

Figure 1

Signal transduction pathways that regulate dauer. Relationships between genes are based on mutant phenotypes and genetic interactions, gene expression in mutants, and homology to pathways in other organisms.

Figure 2

Cluster diagram of gene expression responses. Each row represents an average of 3–4 independent experiments. Each gene on the array is represented as a line in each column. The color of the line represents the log2 of the expression ratio, as indicated by the scale bar.

Figure 3

Regulation of genes with DAF-16 binding sites. In all panels, the bars show groups defined by the location of DAF-16 sites, as shown on the X-axis. We used overlapping intervals to allow robust statistical analysis. Negative numbers are bp upstream of the initiation codon, and positive numbers are bp downstream of the stop codon. Asterisks indicate statistical significance (exact hypergeometric probability) of the proportion of strongly regulated genes in the group compared to all genes:*** is p < 0.001, ** is p < 0.01, * is p < 0.05. A. Strongly upregulated (>2.1 fold, p < 0.01) genes. B. Two fold upregulated genes, excluding those genes in panel A. C. Strongly upregulated genes with downstream DAF-16 sites. D. Strongly downregulated (<2.1 fold, p < 0.01) genes.

Figure 4

Model for gene regulatory events under the control of TGFβ signaling. Transcriptional regulatory events suggested by expression data in this paper are indicated in red. The TGFβ pathway is shown in green; DAF-3 and DAF-5 form a transcription factor complex that function in neurons to control dauer entry [9]. DAF-3 and DAF-5 are shown regulating DAF-9 and DAF-12 indirectly because these genes are likely to be regulated in non-neuronal tissues. Insulin genes are shown as directly regulated by DAF-3 and DAF-5, but it is equally likely that these genes are regulated by feedback from the insulin pathway or by DAF-12.