Specific insulin-like peptides encode sensory information to regulate distinct developmental processes

Abstract

An insulin-like signaling pathway mediates the environmental influence on the switch between the C. elegans developmental programs of reproductive growth versus dauer arrest. However, the specific role of endogenous insulin-like peptide (ILP) ligands in mediating the switch between these programs remains unknown. C. elegans has 40 putative insulin-like genes, many of which are expressed in sensory neurons and interneurons, raising the intriguing possibility that ILPs encode different environmental information to regulate the entry into, and exit from, dauer arrest. These two developmental switches can have different regulatory requirements: here we show that the relative importance of three different ILPs varies between dauer entry and exit. Not only do we find that one ILP, ins-1, ensures dauer arrest under harsh environments and that two other ILPs, daf-28 and ins-6, ensure reproductive growth under good conditions, we also show that daf-28 and ins-6 have non-redundant functions in regulating these developmental switches. Notably, daf-28 plays a more primary role in inhibiting dauer entry, whereas ins-6 has a more significant role in promoting dauer exit. Moreover, the switch into dauer arrest surprisingly shifts ins-6 transcriptional expression from a set of dauer-inhibiting sensory neurons to a different set of neurons, where it promotes dauer exit. Together, our data suggest that specific ILPs generate precise responses to dauer-inducing cues, such as pheromones and low food levels, to control development through stimulus-regulated expression in different neurons.

Fig. 1.

daf-28 acts with ins-6 to inhibit dauer entry, whereas ins-1 promotes it. (A,B) The mean fractions of wild type and insulin-deletion C. elegans mutants that form dauers at 25°C (A) or 27°C (B). Each mean ± s.e.m. includes at least three independent trials of ~100 worms per trial. The detailed statistical comparisons between the dauer entry phenotypes of different genotypes under different conditions in this and subsequent figures can be found in Table S1 in the supplementary material. (C,D) The effect of different insulin deletions on the dauer entry of daf-2(e1368) mutants at 22.5°C (C) and 20°C (D). (E) The dauer entry of ins-6; daf-28 deletion mutants is suppressed by daf-16(mu86) at 25°C. *, P≤0.05; **, P≤0.01; ***, P≤0.001.

Fig. 2.

ins-6 acts with daf-28 to promote dauer exit, whereas ins-1 inhibits it. (A-D) The rates of dauer exit at 25°C of animals carrying different combinations of insulin deletions in a daf-2(e1368) mutant background. Each curve represents cumulative data from six independent trials. All curves are significantly different from that of the daf-2(e1368) control (P≤0.001, log-rank test). The complete statistical comparisons between the dauer exit phenotypes of the different groups of animals in this and subsequent figures are shown in Table S2 in the supplementary material.

Fig. 3.

Inhibition of dauer entry requires low levels of ins-6 and high levels of daf-28. (A,B) The mean fractions of dauers at 25°C (A) or 27°C (B) in ins-6; daf-28 mutants rescued with low (2 ng/μl, lo) or high (25 ng/μl, hi) levels of ins-6 are compared with those of wild-type C. elegans or with insulin-deletion mutants that carry the ofm-1p::gfp co-injection marker alone (jxEx18, jxEx21 or jxEx22). (C,D) The mean fractions of dauers at 25°C (C) or 27°C (D) in ins-6; daf-28 mutants rescued with low (2 ng/μl) or high (25 ng/μl) levels of daf-28 are compared with those of wild type carrying the co-injection marker alone. The daf-28 levels needed to fully rescue the dauer entry phenotype of the ins-6; daf-28 mutants are higher than 25 ng/μl. Error bars represent s.e.m.

Fig. 4.

Unlike dauer entry, higher ins-6 levels and lower daf-28 levels are required to promote dauer exit. (A,B) The rates of dauer exit of ins-6; daf-2(e1368) mutants that were rescued with low (A) or high (B) ins-6 levels are compared with those of daf-2(e1368) or ins-6; daf-2(e1368) mutants that carry the ofm-1p::gfp co-injection marker alone (jxEx18). Each curve represents the cumulative data from at least seven trials. The low-expressing ins-6 rescue lines are significantly different from the daf-2 control (P<0.0001), whereas the high-expressing ins-6 rescue lines behave the same as the daf-2 control. (C,D) The rates of dauer exit of daf-2(e1368); daf-28 mutants that were rescued with low (C) or high (D) daf-28 levels are compared with those of daf-2(e1368) or daf-2(e1368); daf-28 mutants that carry the jxEx22 co-injection marker. Each curve represents the cumulative data from three trials. See Table S2 in the supplementary material for a comparison of the rescue lines with other, additional control lines.

Fig. 5.

ins-6 transcription switches between ASI and ASJ in response to dauer pheromone and dauer arrest. (A) The twelve sensory neurons in the C. elegans amphid sensory organ (White et al., 1986). The ASI and ASJ neurons are indicated in black. Dauer pheromone inhibits ins-6 transcription in ASI, whereas both the pheromone and the dauer state activate ins-6 in ASJ. (B,D,F) ins-6p::mCherry is expressed in the ASI of well-fed L3 (B), L4 (D) and L1 (F) larvae. (C,E) ins-6p::mCherry becomes expressed in the ASJ of a dauer larva (C) and remains on in the ASJ of a post-dauer L4 larva (E). (G) ins-6p::mCherry is unaffected in ASI and is not activated in the ASJ of a starved L1 larva. All animals are oriented with their anterior to the lower left and their dorsal side up. Scale bars: 10 μm.

Fig. 6.

ins-6 acts primarily from ASJ to promote dauer exit. (A) The rates of dauer exit of daf-2(e1368) single and ins-6; daf-2(e1368) double mutants, in which ASJ is genetically ablated (jxEx100 and jxEx102). Control lines are daf-2(e1368) and ins-6; daf-2(e1368) mutants that carry the ofm-1p::gfp co-injection marker alone (jxEx18). The slight difference observed between the dauer exit phenotypes of daf-2 and ins-6; daf-2 mutants upon ASJ ablation might be due to the incomplete loss of ASJ in all animals, as this neuron can still sometimes be seen in some of the post-dauers (data not shown). (B) A model for daf-28, ins-6 and ins-1 function in dauer regulation. daf-28 and ins-6 inhibit dauer entry and promote dauer exit, whereas ins-1 promotes dauer entry and inhibits dauer exit. Whereas daf-28 plays a more prominent role in inhibiting dauer entry (depicted with daf-28 in larger font), ins-6 has a more primary role in promoting dauer exit (depicted with ins-6 in larger font). The sensory neurons in which daf-28 and/or ins-6 might function to regulate the different developmental switches are also shown.