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First published online 3 July 2008
doi: 10.1242/dev.012435

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Notch signalling is required for both dauer maintenance and recovery in C. elegans

Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada.

View larger version (56K): [in this window] [in a new window]   Fig. 1. The DSL ligand lag-2 is expressed in the three pairs of IL2 neurons during the dauer stage. (A) A daf-7(e1372) dauer larva expressing the lag-2::GFP transgene and the corresponding DIC image overlaid with the GFP expression to show the position of the cells expressing the transgene. White arrowheads indicate the described expression of lag-2::GFP in the distal tip cells (DTC). Scale bar: 25 µm. (B) The head region of dauers expressing lag-2::GFP (qIs56) induced by either pheromone, or in various Daf-c mutants as indicated in the panels. (C) 3D reconstruction of a confocal stack of images of the IL2 neurons in dauer. White lines outline the pharynx of the dauer animal. Scale bars: 10 µm. (D) Diagram of the IL2 neurons indicating their position and their characteristic morphology (adapted with permission from wormatlas.org). (E) Merge of dauer expressing lag-2::GFP (green) in the IL2 neurons that were stained with the lipophilic dye DiI (red). As lag-2::GFP is expressed in the entire cell, whereas DiI only stains the membrane, colocalisation (yellow) is only observed at the membrane, giving a halo-like appearance. In all images, arrowheads indicate the IL2 neurons.

View larger version (21K): [in this window] [in a new window]   Fig. 2. A cluster of three forkhead-binding sites is sufficient for dauer-specific lag-2 expression in the IL2 neurons. (A) A 3-kb region upstream of the lag-2 translational start site was subjected to deletion analysis to determine the minimal fragment necessary for IL2/dauer-specific lag-2 expression in daf-7 animals. Enzyme sites used for the generation of the different promoter variants are indicated: Ns (NspI), A (AccI), E (EcoRI), Nc (NcoI) and H (HhaI). Solid lines represent fragments of the lag-2 promoter that were cloned upstream of the GFP-coding sequence; dashed lines represent deleted sequence. Asterisks represent the location of the predicted forkhead-binding sites in the lag-2 promoter. (B) Two potential forkhead-binding sites, named A and B, were identified in the minimal fragment required for IL2 neuron/dauer-specific expression. The consensus binding sites for the FoxC1 transcription factor are indicated in the grey box above the lag-2 sequence (A binding sites). Capital letters represent the core binding site and small letters indicate nucleotides required for efficient binding. (C) Smaller deletions of the 270 bp fragment were created to determine which forkhead-binding sites are required for IL2 neuron/dauer-specific expression. The white and grey boxes represent the identified forkhead-binding sites and the crosses indicate regions where the core binding site sequence was deleted. The relative intensity of GFP expression in the IL2 neurons is indicated as follows: +++, strong; ++, moderate; +, faint; -, no expression. For the consensus binding sites: w can be A or T; m can be A or C; and n can be A, T, C or G.

View larger version (39K): [in this window] [in a new window]   Fig. 3. The forkhead transcription factor UNC-130 is required for appropriate repression of lag-2 expression during reproductive development. (A) Expression in the head region of the various forkhead transcription factors predicted from the C. elegans genome database during the dauer stage in daf-7(e1372) mutants (Hope et al., 2004). (B) Confocal images depicting the expression of the UNC-130::RFP translational fusion protein in the daf-2(e1370); qIs56 (lag-2::GFP) background, and the merge of the two channels during the dauer stage. (C) The percentage of L3 larvae kept at sub-threshold temperature (20°C) that express lag-2::GFP in the IL2 neurons in the mutant background is indicated.

View larger version (128K): [in this window] [in a new window]   Fig. 4. GLP-1 is expressed in differentiated neurons in the head during dauer. (A) The glp-1p::GLP-1::YFP transgene is expressed in two head neurons during dauer in a daf-7(e1372) background. The cell bodies (indicated by arrow) are located in close proximity to the terminal bulb. The axon of these neurons (arrowheads) projects toward the anterior into the nerve ring, where they are within close proximity of the IL2 axons. Scale bar: 5 µm. (B,C) The same construct was injected in a rab-7(ok511)II/mIn1; daf-7(e1372) mutant, which disrupts the endocytic-mediated receptor recycling pathway. YFP-containing vesicles accumulate in the same neurons as in A. (C) Confocal micrograph depicting the accumulation of YFP vesicles along the length of the animal in neurons within the ventral cord. Scale bar: 20 µm. rol-6 was used as co-transformation marker, which accounts for the abnormal morphology of the ventral cord neuronal processes. Anterior is to the left in C.

View larger version (12K): [in this window] [in a new window]   Fig. 5. glp-1 and lin-12 regulate maintenance and recovery in wild-type dauer larvae. (A) A scatter plot representing the effects of Notch mutations on the frequency of dauer recovery in pheromone-induced dauers. Each X represents an independent trial of 50 animals, the mean of which is indicated by the solid line. *P<0.05, using Mann-Whitney test, compared with wild type. (B) Time course analysis of dauer recovery in wild-type dauers induced by starvation/crowding for both wild type (N2) and lin-12(n676n930lf) (see Materials and methods for details).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Proposed model for the requirement of the Notch signalling pathway during dauer development. Based on our observations, glp-1 and lin-12 play distinct and opposing roles during dauer development. We have shown that lag-2 in the IL2 neurons is downstream of all three known pathways and that this expression is regulated by UNC-130, which is required to repress lag-2 in the IL2 neurons during reproductive growth. LAG-2 expression in IL2 neurons will then activate GLP-1 in the adjacent AWC to block premature recovery and hence promote dauer maintenance. Signals that sense replete growth conditions somehow activate LIN-12, which, in cooperation with the insulin-like receptor DAF-2, promotes recovery from dauer. At present it is not clear whether insulin-like signalling functions in parallel or downstream of the lin-12 Notch signalling pathway. See Discussion for details.

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