Figure 1.
Regulation of C. elegans dauer development by cellular signaling pathways.
Dauer entry in C. elegans is regulated by four signaling pathways: a cyclic guanosine monophosphate (cGMP) signaling pathway, an insulin/IGF-1-like signaling (IIS) pathway, a dauer transforming growth factor β (TGFβ) signaling pathway, and a DAF-12 nuclear hormone receptor (NHR) regulated by dafachronic acid (DA) steroid ligands. Unfavorable conditions stimulate dauer entry (left panel) by down-regulating cGMP production, increasing expression of antagonistic insulin-like peptides that down-regulate IIS, decreasing expression of the dauer TGFβ ligand, and inhibiting production of DAs. When dauer larvae encounter favorable conditions, these pathways are hypothesized to act in reverse. Dotted lines signify down-regulated signaling through the pathway, while black lines signify up-regulated signaling through the pathway. Colored proteins are active; grayed out proteins are inactive. Grayed out ligands are absent. Adapted from [12], [32], [43], [91].
Table 1.
Transcript abundance of S. stercoralis homologs of C. elegans chemosensory 7TM GPCRs.
Table 2.
Identification of S. stercoralis heterotrimeric G protein orthologs.
Figure 2.
S. stercoralis L3i are activated by 8-bromo-cGMP.
The membrane-permeable cGMP analog, 8-bromo-cGMP, induced resumption of feeding, a hallmark of activation, in S. stercoralis L3i. Feeding was assessed by ingestion of a FITC dye after incubation at 37°C and 5% CO2 in air for 24 hours for all conditions. (A) At 200 µM, 8-bromo-cGMP dissolved in M9 buffer results in potent resumption of feeding in L3i, with 85.1% (±2.2, SD) of larvae feeding after 24 hours. In comparison, host-like cues consisting of DMEM, 10% canine serum (S), and 12.5 mM reduced glutathione (G), resulted in 43.9% (±2.6, SD) of L3i feeding, while the M9 buffer negative control resulted in 0.6% (±0.3, SD) of L3i feeding, after 24 hours. (B) Kinetics of activation were determined for both 200 µM 8-bromo-cGMP and host-like cues, consisting of DMEM, 10% canine serum, and 3.75 mM reduced glutathione, after incubation for 4, 6, 12, 18, or 24 hours. All conditions were incubated for a total of 24 hours at 37°C and 5% CO2 in air. Error bars represent ±1 standard deviation (SD).
Figure 3.
S. stercoralis L3i activation with Δ7-DA and inhibition with the PI3 kinase inhibitor LY294002.
The putative DAF-12 nuclear hormone receptor ligand Δ7-dafachronic acid (DA) induced resumption of feeding, a hallmark of activation, in S. stercoralis L3i in a dose-dependent manner. Feeding was assessed by ingestion of a FITC dye after incubation at 37°C and 5% CO2 in air for 24 hours for all conditions. Conditions included Δ7-DA at 800 nM, 400 nM, 200 nM, 100 nM, and 50 nM dissolved in M9 buffer. At 400 nM Δ7-DA, 93.3% (±1.1%, SD) of L3i resumed feeding in comparison to 1.2% (±0.4%) in the M9 buffer control. Additionally, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 kinase) inhibitor LY294002 was added to Δ7-DA to determine whether inhibition of PI3 kinases would block activation by Δ7-DA. At 100 µM, LY294002 dissolved in DMSO inhibited L3i activation in 400 nM Δ7-DA with 7.6% (±1.6%, SD) of L3i feeding in this condition; 0.28% (±0.2%) of L3i had resumed feeding in the M9 buffer with DMSO negative control. Error bars represent ±1 standard deviation (SD); *** p<0.01.
Figure 4.
S. stercoralis L3i activation with 8-bromo-cGMP, Δ7-DA, or DMEM modulates ILP ligand transcript levels.
Transcript levels of the insulin-like peptide (ILP) ligand-encoding genes Ss-ilp-1 through Ss-ilp-7 were quantified using RNAseq. Conditions included L3i that had no stimulation (only exposed to room temperature conditions in M9 buffer) as well as L3i incubated at 37°C and 5% CO2 in air for 24 hours in either M9 buffer, DMEM, 200 µM 8-bromo-cGMP in M9 buffer, or 400 nM Δ7-dafachronic acid (DA) in M9 buffer. (A) L3i feeding, a hallmark of activation, was assessed by ingestion of a FITC dye for worms incubated at 37°C and 5% CO2 in air for 24 hours. Error bars represent ±1 standard deviation. (B–H) Transcript abundance patterns for Ss-ilp-1 through -7 were determined by RNAseq for each condition. Transcript abundances were calculated as fragments per kilobase of coding exon per million fragments mapped (FPKM). Error bars represent ±95% confidence intervals. All statistically significant differences, with respect to the no stimulation condition, are marked with an asterisk.
Figure 5.
S. stercoralis ILP promoters are active in the nervous system and other tissues.
Transgenic S. stercoralis post-free-living larvae expressing enhanced green fluorescent protein (EGFP) under the control of three insulin-like peptide (ILP) promoters were assessed for tissue-specific expression. (A–D) Transgenic larvae carrying the Ss-ilp-1 promoter::egfp reporter construct; (A,C) DIC images and (B,D) fluorescent images. The Ss-ilp-1 promoter is active in the hypodermis/body wall and a single pair of head neurons (D, arrow). (E–H) Transgenic larvae carrying the Ss-ilp-6 promoter::egfp reporter construct; (E,G) DIC images and (F,H) fluorescent images. The Ss-ilp-6 promoter is active in the hypodermis/body wall and several head neurons. (I–L) Transgenic larvae carrying the Ss-ilp-7 promoter::egfp reporter construct; (I,K) DIC images and (J,L) fluorescent images. The Ss-ilp-7 promoter is active in the intestine and a single pair of head neurons, with a single process that extends dorsally almost to the anterior portion of the intestine (L, arrow), most consistent with the SIAV neurons in C. elegans.
Table 3.
Location of EGFP expression in transgenic S. stercoralis post-free-living larvae under the control of ILP promoters.